For in-vitro Diagnostic Use. The BrightVision one step detection system Goat Anti-Rabbit IgG HRP and Goat anti-Mouse IgG AP, is intended for use in immunohistochemistry for the detection of mouse or rabbit antibodies.
The BrightVision detection system, peroxidase Goat Anti-Rabbit HRP and Goat anti-Mouse AP, is a Ready-to-Use system that has been manufactured to give an optimal staining, when using the protocol advised in this IFU. Prior to staining some routine fixed, paraffin-embedding tissue sections should be subjected to pre-treatment (HIER or digestive enzyme). The BrightVision detection system detects Rabbit and Mouse bound to an antigen in tissue sections. This polymer-complex is then visualized with a suitable substrate/chromogen. The clinical interpretation of any staining or its absence should be determined by a qualified pathologist and complemented by morphologic studies; controls should be evaluated within the context of the patients clinical history and/or other diagnostic tests.
Principle of method:
BrightVision, one step detection system Goat Anti- Rabbit HRP and Mouse AP (Ready-to-Use).
Reagents provided:
BrightVision, One step detection system Goat anti-Rabbit HRP / Goat anti-Mouse AP (Ready-to-use; 55 ml)
Storage and handling:
2-8°C and in the dark
Procedure:
1. Deparaffinize and rehydrate tissue section (slide/tissue peparing), 2. Wash Aqua dest (Wash; 2x 5 min), 3. If applicable, HIER or digestive enzyme (pre-treatment), 4. Wash buffer (PBS or TBS buffer; 2x 5 min), 5. H2O2 (conc3%) (Tissue preparing; 10 min), 6. Wash buffer (PBS or TBS buffer; 2x 5 min), 7. Primary mouse and/or rabbit antibody (Antibody; 30 min), 8. Wash buffer (PBS or TBS buffer; 2x 5 min), 9 Detection system, polymer Rabbit HRP/Mouse AP, (Labeled polymer; 30 min), 10. Wash buffer (TBS buffer; 2x 5 min), 11. Substrate (DAB; see applicble IFU), 12. Wash aqua dest (Wash; 2x 2 min), 13. Substrate (Fast Red / New Fuchsin; see applicable IFU), 14. Wash aqua dest (Wash; 2x 2 min), 15. Counterstain and coverslip with aqueous mounting medium.
Quality Control:
A positive control, negative control and reagent control are needed and processed in the same way as the unknow specimen slide to interpret staining results.
For in-vitro Diagnostic Use. The BrightVision one step detection system Goat Anti-Mouse IgG HRP and Goat anti-rabbit IgG AP, is intended for use in immunohistochemistry for the detection of mouse or rabbit antibodies.
The BrightVision detection system, peroxidase Goat Anti-Mouse HRP/Goat anti-Rabbit AP, is a Ready-to-Use system that has been manufactured to give an optimal staining, when using the protocol advised in this IFU. Prior to staining some routine fixed, paraffin-embedding tissue sections should be subjected to pre-treatment (HIER or digestive enzyme). The BrightVision detection system detects Rabbit and Mouse bound to an antigen in tissue sections. This polymer-complex is then visualized with a suitable substrate/chromogen. The clinical interpretation of any staining or its absence should be determined by a qualified pathologist and complemented by morphologic studies; controls should be evaluated within the context of the patients clinical history and/or other diagnostic tests.
Principle of method:
BrightVision, one step detection system Goat Anti- Mouse HRP and Rabbit AP (Ready-to-Use).
Reagents provided:
BrightVision, One step detection system Goat anti-Mouse HRP / Goat anti-Rabbit AP (Ready-to-use; 55 ml)
Storage and handling:
2-8°C and in the dark
Procedure:
1. Deparaffinize and rehydrate tissue section (slide/tissue peparing), 2. Wash Aqua dest (Wash; 2x 5 min), 3. If applicable, HIER or digestive enzyme (pre-treatment), 4. Wash buffer (PBS or TBS buffer; 2x 5 min), 5. H2O2 (conc3%) (Tissue preparing; 10 min), 6. Wash buffer (PBS or TBS buffer; 2x 5 min), 7. Primary mouse and/or rabbit antibody (Antibody; 30 min), 8. Wash buffer (PBS or TBS buffer; 2x 5 min), 9 Detection system, polymer Mouse HRP/Rabbit AP, (Labeled polymer; 30 min), 10. Wash buffer (TBS buffer; 2x 5 min), 11. Substrate (DAB; see applicble IFU), 12. Wash aqua dest (Wash; 2x 2 min), 13. Substrate (Fast Red / New Fuchsin; see applicable IFU), 14. Wash aqua dest (Wash; 2x 2 min), 15. Counterstain and coverslip with aqueous mounting medium.
Quality Control:
A positive control, negative control and reagent control are needed and processed in the same way as the unknow specimen slide to interpret staining results.
For in-vitro Diagnostic Use. The BrightVision one step detection system Goat Anti-Rabbit IgG AP, is intended for use in immunohistochemistry for the detection of rabbit antibodies.
The BrightVision detection system Goat Anti-Rabbit AP, is a Ready-to-Use system that has been manufactured to give an optimal staining, when using the protocol advised in this IFU. Prior to staining some routine fixed, paraffin-embedding tissue sections should be subjected to pre-treatment (HIER or digestive enzyme). The BrightVision detection system detects Rabbit bound to an antigen in tissue sections. This polymer-complex is then visualized with a suitable substrate/chromogen (not provided). Also available in 110 ml, 500 ml and 1000 ml.
Principle of method:
One step detection system goat anti-rabbit IgG AP
Reagents provided:
One step detection system Goat anti-Rabbit AP (Ready-to-use; 55 ml)
Storage and handling:
2-8°C and in the dark
Procedure:
1. Deparaffinize and rehydrate tissue section (slide/tissue peparing), 2. Wash Aqua dest (Wash; 2x 5 min), 3. If applicable, HIER or digestive enzyme (pre-treatment), 4. Wash buffer (PBS or TBS buffer; 2x 5 min), 5. H2O2 (conc3%) (Tissue preparing; 10 min), 6. Wash buffer (PBS or TBS buffer; 2x 5 min), 7. Primary rabbit antibody (Antibody; 30 min), 8. Wash buffer (TBS buffer; 2x 5 min), 9. Detection system, polymer Rabbit AP, (Labeled polymer; 30 min), 10. Wash buffer (TBS buffer; 2x 5 min), 11. Substrate (Fast Red / New Fuchsin; see applicable IFU), 12. Wash aqua dest (Wash; 2x 2 min), 12. Counterstain and coverslip with aqueous mounting medium.
Quality Control:
A positive control, negative control and reagent control are needed and processed in the same way as the unknow specimen slide to interpret staining results.
For in-vitro Diagnostic Use. The BrightVision one step detection system peroxidase Goat Anti-Mouse IgG AP, is intended for use in immunohistochemistry for the detection of mouse antibodies.
The BrightVision detection system Goat Anti- Mouse AP, is a Ready-to-Use system that has been manufactured to give an optimal staining, when using the protocol advised in this IFU. Prior to staining some routine fixed, paraffin-embedding tissue sections should be subjected to pre-treatment (HIER or digestive enzyme). The BrightVision detection system detects Mouse bound to an antigen in tissue sections. This polymer-complex is then visualized with a suitable substrate/chromogen (not provided). Also available in 110 ml, 500 ml and 1000 ml.
Principle of method:
One step detection system peroxidase goat anti-mouse IgG AP
Reagents provided:
One step detection system Goat anti-Mouse AP (Ready-to-use; 55 ml)
Storage and handling:
2-8°C and in the dark
Procedure:
1. Deparaffinize and rehydrate tissue section (slide/tissue peparing), 2. Wash Aqua dest (Wash; 2x 5 min), 3. If applicable, HIER or digestive enzyme (pre-treatment), 4. Wash buffer (PBS or TBS buffer; 2x 5 min), 5. H2O2 (conc3%) (Tissue preparing; 10 min), 6. Wash buffer (PBS or TBS buffer; 2x 5 min), 7. Primary mouse antibody (Antibody; 30 min), 8. Wash buffer (TBS buffer; 2x 5 min), 9. Detection system, polymer Mouse AP, (Labeled polymer; 30 min), 10. Wash buffer (TBS buffer; 2x 5 min), 11. Substrate (FAST Red / New Fuchsin; see applicable IFU), 12. Wash aqua dest (Wash; 2x 2 min), 13. Counterstain and coverslip with a aqueous mounting medium (Auxiliary)
Quality Control:
A positive control, negative control and reagent control are needed and processed in the same way as the unknow specimen slide to interpret staining results.
For in-vitro Diagnostic Use. The BrightVision one step detection system Goat Anti-Mouse/Rabbit IgG AP, is intended for use in immunohistochemistry for the detection of mouse or rabbit antibodies.
The BrightVision detection system Goat Anti- Mouse/Rabbit AP, is a Ready-to-Use system that has been manufactured to give an optimal staining, when using the protocol advised in this IFU. Prior to staining some routine fixed, paraffin-embedding tissue sections should be subjected to pre-treatment (HIER or digestive enzyme). The BrightVision detection system detects Mouse or Rabbit bound to an antigen in tissue sections. This polymer-complex is then visualized with a suitable substrate/chromogen (not provided). Also available in 110 ml, 500 ml and 1000 ml.
Principle of method:
One step detection system goat anti-mouse/rabbit IgG AP
Reagents provided:
One step detection system Goat anti-Mouse/Rabbit AP (Ready-to-use; 55 ml)
Storage and handling:
2-8°C and in the dark
Procedure:
1. Deparaffinize and rehydrate tissue section (slide/tissue peparing), 2. Wash Aqua dest (Wash; 2x 5 min), 3. If applicable, HIER or digestive enzyme (pre-treatment), 4. Wash buffer (PBS or TBS buffer; 2x 5 min), 5. H2O2 (conc3%) (Tissue preparing; 10 min), 6. Wash buffer (PBS or TBS buffer; 2x 5 min), 7. Primary mouse or rabbit antibody (Antibody; 30 min), 8. Wash buffer (PBS or TBS buffer; 2x 5 min), 9. Detection system, polymer Mouse/Rabbit AP, (Labeled polymer; 30 min), 10. Wash buffer (TBS buffer; 2x 5 min), 11. Substrate (Fast Red / New Fuchsin, see applicable IFU), 12. Wash aqua dest (Wash; 2x 2 min), 13. Counterstain and coverslip with aqueous mounting medium.
Quality Control:
A positive control, negative control and reagent control are needed and processed in the same way as the unknow specimen slide to interpret staining results.
The aqua mounting medium is a widely used for coverslipping from aqueous solutions and is non-fluorescing and has an antifade component to increase the viewing time of the specimen. Use Aqua-Poly/Mount with most fluorescent dyes and stains including DAB, Alkaline Phosphatase, Fast Red, AEC (aminoethylcarbazole) and a variety of other chromogens to enhance and retain fluorescent intensity. The mounting medium (water based) can be used for frozen sections, fat stains, chromogens for immunohistochemistry and in situ hybridization as well as other applications requiring a water-soluble mounting medium.
Principle of method:
Coverslipping from aqueous solutions
Reagents provided:
50 ml Mounting medium (water based), ready-to-use.
Storage and handling:
Store at room temperature
Procedure:
1. Prepare slides as required. 2. Prior to coverslipping rinse the slides in distilled or deionized water. 3. Place the bottle upside down with the end cap attached in a container before use. This will help clear of tiny bubbles. 4. Blot excess water from the slide without drying the tissue specimen. The tissue must be moist prior to mounting. 5. Apply enough drops of aqua mounting medium on the tissue section so that the tissue is completely covered. 6. Carefully lower the coverslip at an angle while gently applying pressure to force any excess medium and air bubbles away from the tissue and out from under the coverslip. 7. Slides can be dried at room temperature or at 4°C or in an oven but the temperature hight is depending what type of staining is used. 8. Allow the aqua mounting medium to dry before examine under the microscope.
Permanent HRP Green Kit is intended for immunohistochemical and in situ-hybridisation staining procedures with horse radish peroxidase (HRP). It results in the formation of a green precipitate at the location of the target antigen or target nucleic acid. The precipitate is insoluble in organic solvents and can be observed by light microscopy. The HRP Green precipitate shows a very good contrast to red chromogenic substrates used with alkaline phosphatase detection systems and is therefore especially recommended for double stains. Permanent HRP Green Kit is intended for research use only.
100 ml HRP Green Substrate Buffer 3 ml HRP Green Chromogen 1 Dilution Vial
Storage and handling:
The solutions should be stored at 2-8°C without fur ther dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. Do not use product after the expiry date. The working solution should be prepared freshly at the day of use. It is stable for at least 4 hours. Excess working solution should be disposed. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents please contact our technical support.
Reagent preparation:
1) Pipette 1 ml HRP Green Substrate Buffer into the provided dilution vial. 2) Add 1 drop (30 µl) of HRP Green Chromogen. Mix thoroughly. 3) The resulting working solution is stable for at least 4 hours. If you want to prepare other quantities of the working solution, please use the same ratio HRP Green Substrate and HRP Chromogen.
Procedure:
1) Rinse the slide with wash buffer after the previous incubation step. 2) Apply the Permanent HRP Green working solution onto the slide. Incubate for 2-10 minutes. 3) Rinse with distilled H2O. 4) Counterstain with haematoxylin for about 30 seconds up to 5 minutes (depending on the desired staining intensity). 5) Rinse with distilled H2O. 6) Blueing in tap water for 5 minutes. 7) Dehydrate through a graded series of ethanol and clear in xylene. Do not exceed incubation times of 30 sec per dehydration step. Use only high purity xylene. Mount with a permanent mounting medium. Note: The colour intensity can be intensified by increasing the chromogen concentration (up to 3 drops or 90 µl chromogen) in the working solution. Lithium carbonate may have a negative effect on the staining result. We recommend to only bluing in tap water. Occasionally precipitates may appear in the HRP Green Chromogen solution. This doesnt affect the staining result.
Expected results:
During the reaction of the substrate with horse radish peroxidase in presence of the Permanent HRP Green chromogen, a green precipitate is formed at the location of the target antigen or nucleic acid. The precipitate is insoluble in organic solvents and can be observed by light microscopy.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results please read these instructions carefully, contact our technical support. Also refer to the instructions of the detection systems for guidance on general troubleshooting.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining. Please refer to the instructions of the detection system for guidance on general quality control procedures.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Too long incubation steps at the final dehydration serious can diminish the staining intensity. Also low grade xylene and some forms of recycled alcohol can have a negative effect on the staining result. In double stain procedures we recommend to use Permanent HRP Green as the last chromogen. Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid contact of reagents or specimen with eye, skin or mucous membrane. In case of a reagent or specimen coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining may occur. Material safety data sheets (MSDS) are available upon request.
AEC Single Solution is a ready-to-use solution intended for immunohistochemical and in situ-hybridisation staining procedures with horse radish peroxidase (HRP). AEC (3-Amino-9-ethylcarbazol) leads to the formation of a red-brown precipitate at the location of the target antigen or target nucleic acid. The precipitate is insoluble in aqueous mounting media and can be observed by light microscopy. AEC Single Solution is especially useful when a high sensitivity is desired.
100 ml AEC Single Solution (ready-to-use) 2 Dropper Bottles
Storage and handling:
The solution should be stored at 2-8°C without furt her dilution. Please store the reagent in a dark place and do not freeze it. Under these conditions the solution is stable up to the expiry date indicated on the label. Do not use product after the expiry date. AEC Single Solution is a ready-to-use solution. Preparation of a working solution as in other chromogenic substrates is not necessary. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents please contact our technical support.
Reagent preparation:
The solution is ready-to-use. AEC Single Solution can be used directly from the refrigerator and should be stored again at 2-8°C aft er use. When using the small package (8 ml) please directly drop from the bottle. When using the 100 ml package (MON-APP209) please transfer up to 8 ml of the AEC Single Solution into one of the provided dropper bottles. The transferred solution is stable for many weeks if stored at 2-8°C. The volume required for several staining runs should be transferred so that the 100 ml stock bottle has to be opened only a few times. If you would like to pipette the solution use a clean vial from which you pipette. Remaining quantities should not be filled back into the bottle but disposed as hazardous material.
Procedure:
1) Rinse the slide with wash buffer after the previous incubation step. 2) Apply the AEC Single Solution to the slide. Incubate for 3-6 minutes. (Incubation time can be extended up to 30 minutes, if desired.) 3) Rinse with distilled H2O. 4) Counterstain with haematoxylin for about 30 seconds up to 5 minutes (depending on the desired staining intensity). 5) Rinse with distilled H2O. 6) Blueing in tap water for at least 5 minutes. 7) Mount with an aqueous mounting medium.
Expected results:
During the reaction of the substrate with horse radish peroxidase in presence of the chromogen AEC, a red-brown precipitate is formed at the location of the target antigen or nucleic acid. The precipitate is insoluble in aqueous solvents and can be observed by light microscopy.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results please read these instructions carefully, contact our technical support. Also refer to the instructions of the detection systems for guidance on general troubleshooting.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining. Please refer to the instructions of the detection system for guidance on general quality control procedures.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). In some tissues endogenous peroxidase activity may cause non-specific staining. The enzyme activity should be blocked by incubation with hydrogen peroxide solution (H2O2 solution). The step is carried out before incubation with primary antibody but after dewaxing and rehydration. Background staining due to endogenous biotin can be blocked through an avidin-biotin blocking step prior to the primary antibody incubation step. The coloured precipitate formed by AEC is soluble in organic solvents. The tissue sections therefore have to be counterstained with aqueous solutions (e. g. Gills or Mayers haematoxylin) and mounted with aqueous mounting media. The colour intensity of the reaction product can decrease with time, especially when exposed to light. The staining reaction itself can be influenced in the same way when carried out in strong light. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. AEC (3-Amino-9-ethylcarbazol) and the solvents used are considered hazardous materials. Material safety data sheets (MSDS) are available upon request. Wear protective clothing to avoid contact of reagent or specimen with eye, skin or mucous membrane. In case of reagent or specimen coming into contact with a sensitive area, wash the area with large amounts of water. Oxidising substances, e. g. metals, dust, bacteria or glass devices can influence the stability of AEC Single Solution. Such contaminations have to be avoided. Non-consumed solution needs to be discarded as dangerous substance.
The Fast AP One-Step Polymer anti-Mouse/Rabbit is designed for the qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. It was developed for use in combination with mono- and polyclonal primary antibodies and sera obtained from mice or rabbit. The kit can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE. It is intended for in vitro diagnostic use.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Fast AP One-Step Polymer anti-Mouse/Rabbit is a highly sensitive detection reagent intended for use in immunohistochemistry and immunocytochemistry. The enzyme polymer consists of several molecules of secondary antibodies covalently bound to several molecules of alkaline phosphatase (AP). Visualisation occurs via an enzymesubstrate reaction in the presence of a colorising reagent which permits microscopical analysis. The test system is suitable for the detection of mono- and polyclonal primary antibodies and sera obtained from mice or rabbit. Cross-reactivity with primary antibodies from rat has been observed. In contrast to other detection techniques, which often use the streptavidin-biotin system the Fast AP One-Step Polymer anti-Mouse/Rabbit avoids the problem of background staining caused by endogenous biotin in the tissue.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Background staining caused by unspecific binding of the primary antibody or the secondary antibody in the AP OneStep Polymer is minimized by incubation with a protein blocking solution. This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the AP One-Step Polymer is applied and incubated. Any excess of unbound polymer is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the alkaline phosphatase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen Permanent AP Red leads to the formation of a magentared product of reaction at the place of the target antigen.
Reagents provided:
100 ml Fast AP One-Step Polymer anti-Mouse/Rabbit (ready-to-use) Substrate systems recommended: Permanaent AP Red kit Materials required but not supplied Positive and negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O Reagents for enzyme digestion or heat pre-treatment Wash buffer PBS or TBS Blocking Solution (for protein blocking, optional) Pink PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solution should be stored at 2-8°C without further dilution. Please store the reagent in a dark place and do not freeze it. Under these conditions the solution is stable up to the expiry date. It should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagent, please contact our technical support
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. Tissue sections have to be completely covered with the different reagents in order to avoid drying out.
Procedure:
1. Blocking Solution (This step is optional.) 5 min. 2. Washing with wash buffer 1 x 2 min. 3. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 4. Washing with wash buffer 3 x 5 min. 5. AP One-Step Polymer anti-Mouse/Rabbit 30 Min. 6. Washing with wash buffer 3 x 2 min. 7. Permanent AP Red, 10-20 min. (Controlling the colour intensity via light microscope is recommended.) 8. Stopping the reaction with distilled H2O when the desired colour intensity is attained 9. Counterstaining and blueing 10. Mounting: permanent or aqueous with Permanent AP Red Kit
Expected results:
During the reaction of the substrate with alkaline phosphatase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support. No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from mouse or rabbit but from a different species. 6. The antigen/epitope was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. If you used heat pre-treatment or enzyme digestion it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. Incubation times were too short or primary antibody concentration too low. 7. Chromogenic substrate solution was too old. Nonspecific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme polymer or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use Blocking Solution or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high).
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear. ProClin 300 is used for stabilisation. A Material safety data sheet (MSDS) is available upon request.
The Fast AP One-Step Polymer anti-Mouse/Rabbit is designed for the qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. It was developed for use in combination with mono- and polyclonal primary antibodies and sera obtained from mice or rabbit. The kit can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE. It is intended for in vitro diagnostic use.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Fast AP One-Step Polymer anti-Mouse/Rabbit is a highly sensitive detection reagent intended for use in immunohistochemistry and immunocytochemistry. The enzyme polymer consists of several molecules of secondary antibodies covalently bound to several molecules of alkaline phosphatase (AP). Visualisation occurs via an enzymesubstrate reaction in the presence of a colorising reagent which permits microscopical analysis. The test system is suitable for the detection of mono- and polyclonal primary antibodies and sera obtained from mice or rabbit. Cross-reactivity with primary antibodies from rat has been observed. In contrast to other detection techniques, which often use the streptavidin-biotin system the Fast AP One-Step Polymer anti-Mouse/Rabbit avoids the problem of background staining caused by endogenous biotin in the tissue.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Background staining caused by unspecific binding of the primary antibody or the secondary antibody in the AP OneStep Polymer is minimized by incubation with a protein blocking solution. This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the AP One-Step Polymer is applied and incubated. Any excess of unbound polymer is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the alkaline phosphatase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen Permanent AP Red leads to the formation of a magentared product of reaction at the place of the target antigen.
Reagents provided:
6 ml Fast AP One-Step Polymer anti-Mouse/Rabbit (ready-to-use) Substrate systems recommended: Permanaent AP Red kit Materials required but not supplied Positive and negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O Reagents for enzyme digestion or heat pre-treatment Wash buffer PBS or TBS Blocking Solution (for protein blocking, optional) Pink PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solution should be stored at 2-8°C without further dilution. Please store the reagent in a dark place and do not freeze it. Under these conditions the solution is stable up to the expiry date. It should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagent, please contact our technical support
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. Tissue sections have to be completely covered with the different reagents in order to avoid drying out.
Procedure:
1. Blocking Solution (This step is optional.) 5 min. 2. Washing with wash buffer 1 x 2 min. 3. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 4. Washing with wash buffer 3 x 5 min. 5. AP One-Step Polymer anti-Mouse/Rabbit 30 Min. 6. Washing with wash buffer 3 x 2 min. 7. Permanent AP Red, 10-20 min. (Controlling the colour intensity via light microscope is recommended.) 8. Stopping the reaction with distilled H2O when the desired colour intensity is attained 9. Counterstaining and blueing 10. Mounting: permanent or aqueous with Permanent AP Red Kit
Expected results:
During the reaction of the substrate with alkaline phosphatase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support. No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from mouse or rabbit but from a different species. 6. The antigen/epitope was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. If you used heat pre-treatment or enzyme digestion it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. Incubation times were too short or primary antibody concentration too low. 7. Chromogenic substrate solution was too old. Nonspecific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme polymer or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use Blocking Solution or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high).
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear. ProClin 300 is used for stabilisation. A Material safety data sheet (MSDS) is available upon request.
The Plus AP Polymer anti-Mouse is designed for the qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. It is developed for use in combination with monoclonal primary antibodies obtained from mouse. The reagent can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus AP Polymer anti-Mouse is a highly sensitive detection reagent intended for use in immunohistochemistry and immunocytochemistry. The enzyme polymer in this kit consists of several molecules of secondary antibodies covalently bound to several molecules of alkaline phosphatase (AP). Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The reagent is suitable for the detection of mono- and polyclonal primary antibodies and sera obtained from mouse. In contrast to other detection techniques, which often use the streptavidin-biotin system the Plus AP Polymer anti-Mouse avoids the problem of background staining caused by endogenous biotin in the tissue.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Background staining caused by unspecific binding of the primary antibody or the secondary antibody in the AP polymer is minimized by incubation with a protein blocking solution (Blocking Solution). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the AP-polymer is applied and incubated. Any excess of unbound AP-polymer is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the alkaline phosphatase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen Fast Red leads to the formation of a magenta-red product of reaction at the place of the target antigen. Other suitable chromogens are Permanent AP Red (magenta-red), New Fuchsin (magenta-red) or NBT (blue-black) with its substrate BCIP.
Reagents provided:
100 ml AP-Polymer anti-Mouse (Ready-to-use) Substrate systems recommended: Permanent AP Red kit, Fast Red substrate kit, New Fuchsin kit. Materials required but not supplied: Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O Reagents for enzyme digestion or heat pre-treatment Wash buffer PBS or TBS PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solution should be stored at 2-8°C without furt her dilution. Please store the reagent in a dark place and do not freeze it. Under these conditions the solution is stable up to the expiry date indicated on the label. It should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support.
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. Tissue sections have to be completely covered with the different reagents in order to avoid drying out
Procedure:
1. Blocking Solution (protein block, this step is optional) 5 min. 2. Washing with wash buffer 1 x 2 min. 3. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 4. Washing with wash buffer 3 x 5 min. 5. AP-polymer anti Mouse 30 min. 6. Washing with wash buffer 3 x 2 min. 7. Fast Red, Permanent AP Red, NBT/BCIP or New Fuchsin 5-15 min. (Controlling the colour intensity via light microscope is recommended.) 8. Stopping the reaction with distilled H2O when the desired colour intensity is attained 9. Counterstaining and blueing 10. Mounting: aqueous with Fast Red, permanent with Permanent AP Red, NBT/BCIP or New Fuchsin
Expected results:
During the reaction of the substrate with alkaline phosphatase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support. No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from mouse, but from a different species. 6. The antigen/epitope was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. If you used heat pre-treatment or enzyme digestion it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. If you are using PBS-based wash buffer: the activity of alkaline phosphatase in the reagents is blocked if too much wash buffer remains on the slides. 7. Incubation times were too short or primary antibody concentration too low. 8. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme polymer or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use a Blocking Solution or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high).
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light.Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of the reagent coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagent must be avoided, since otherwise non-specific staining might appear. ProClin 950 is used for stabilisation. A Material safety data sheet (MSDS) is available upon request.
The Plus AP Polymer anti-Mouse is designed for the qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. It is developed for use in combination with monoclonal primary antibodies obtained from mouse. The reagent can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus AP Polymer anti-Mouse is a highly sensitive detection reagent intended for use in immunohistochemistry and immunocytochemistry. The enzyme polymer in this kit consists of several molecules of secondary antibodies covalently bound to several molecules of alkaline phosphatase (AP). Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The reagent is suitable for the detection of mono- and polyclonal primary antibodies and sera obtained from mouse. In contrast to other detection techniques, which often use the streptavidin-biotin system the Plus AP Polymer anti-Mouse avoids the problem of background staining caused by endogenous biotin in the tissue.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Background staining caused by unspecific binding of the primary antibody or the secondary antibody in the AP polymer is minimized by incubation with a protein blocking solution (Blocking Solution). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the AP-polymer is applied and incubated. Any excess of unbound AP-polymer is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the alkaline phosphatase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen Fast Red leads to the formation of a magenta-red product of reaction at the place of the target antigen. Other suitable chromogens are Permanent AP Red (magenta-red), New Fuchsin (magenta-red) or NBT (blue-black) with its substrate BCIP.
Reagents provided:
6 ml AP-Polymer anti-Mouse (Ready-to-use) Substrate systems recommended: Permanent AP Red kit, Fast Red substrate kit, New Fuchsin kit. Materials required but not supplied: Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O Reagents for enzyme digestion or heat pre-treatment Wash buffer PBS or TBS PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solution should be stored at 2-8°C without furt her dilution. Please store the reagent in a dark place and do not freeze it. Under these conditions the solution is stable up to the expiry date indicated on the label. It should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support.
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. Tissue sections have to be completely covered with the different reagents in order to avoid drying out
Procedure:
1. Blocking Solution (protein block, this step is optional) 5 min. 2. Washing with wash buffer 1 x 2 min. 3. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 4. Washing with wash buffer 3 x 5 min. 5. AP-polymer anti Mouse 30 min. 6. Washing with wash buffer 3 x 2 min. 7. Fast Red, Permanent AP Red, NBT/BCIP or New Fuchsin 5-15 min. (Controlling the colour intensity via light microscope is recommended.) 8. Stopping the reaction with distilled H2O when the desired colour intensity is attained 9. Counterstaining and blueing 10. Mounting: aqueous with Fast Red, permanent with Permanent AP Red, NBT/BCIP or New Fuchsin
Expected results:
During the reaction of the substrate with alkaline phosphatase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support. No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from mouse, but from a different species. 6. The antigen/epitope was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. If you used heat pre-treatment or enzyme digestion it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. If you are using PBS-based wash buffer: the activity of alkaline phosphatase in the reagents is blocked if too much wash buffer remains on the slides. 7. Incubation times were too short or primary antibody concentration too low. 8. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme polymer or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use a Blocking Solution or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high).
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of the reagent coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagent must be avoided, since otherwise non-specific staining might appear. ProClin 950 is used for stabilisation. A Material safety data sheet (MSDS) is available upon request.
The Plus HRP Polymer anti-Rabbit is designed for the qualitative detection of antigens in fixed paraffinembedded tissue sections, in frozen tissue sections, and in cytological samples. It is developed for use in combination with mono- and polyclonal primary antibodies and sera obtained from rabbits. The reagent can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus HRP Polymer anti-Rabbit is a highly sensitive detection reagent intended for use in immunohistochemistry and immunocytochemistry. The enzyme polymer in this kit consists of several molecules of secondary antibodies covalently bound to several molecules of horse radish peroxidase (HRP). Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The reagent is suitable for the detection of mono- and polyclonal primary antibodies and sera obtained from rabbits. In contrast to other detection techniques, which often use the streptavidin-biotin system the Plus HRP Polymer anti-Rabbit avoids the problem of background staining caused by endogenous biotin in the tissue.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Endogenous peroxidase activity in the tissue may cause non-specific staining. This enzyme activity can be blocked by incubation with 3% H2O2-solution (Peroxide block). Background staining caused by unspecific binding of the primary antibody or the secondary antibody in the HRP polymer is minimized by incubation with a protein blocking solution (Blocking Solution). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the HRP-polymer is applied and incubated. Any excess of unbound HRP-polymer is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the horse radish peroxidase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen AEC leads to the formation of a red-brown product of reaction at the place of the target antigen. The chromogen DAB forms a dark brown precipitate.
Reagents provided:
100 ml HRP-Polymer anti-Rabbit (Ready-to-use) Substrate systems recommended: Permanent AEC kit, AEC Single Solution, AEC Substrate kit, DAB Substrate kit, DAB High contrast kit Materials required but not supplied: Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O 3% H2O2 solution Reagents for enzyme digestion or heat pre-treatment Wash buffer PBS or TBS PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solution should be stored at 2-8°C without further dilution. Please store the reagent in a dark place and do not freeze it. Under these conditions the solution is stable up to the expiry date indicated on the label. They should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. Tissue sections have to be completely covered with the different reagents in order to avoid drying out.
Procedure:
1. Peroxide blocking (3 % H2O2 solution) 10 min. 2. Washing with wash buffer 1 x 2 min. 3. Blocking Solution (This step is optional.) 5 min. 4. Washing with wash buffer 1 x 2 min. 5. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 6. Washing with wash buffer 3 x 5 min. 7. HRP-polymer anti-Rabbit 30 min. 8. Washing with wash buffer 3 x 2 min. 9. AEC or DAB (Controlling the colour intensity via light microscope is recommended.) 5-15 min. 10. Stopping the reaction with distilled H2O when the desired colour intensity is attained 11. Counterstaining and blueing 12. Mounting: aqueous with AEC, permanent with DAB or Permanent AEC
Expected results:
During the reaction of the substrate with horse radish peroxidase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support. No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from rabbit, but from a different species. 6. The antigen/epitope was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. If you used heat pre-treatment or enzyme digestion it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. Incubation times were too short or primary antibody concentration too low. 7. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme polymer or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use a Blocking Solution or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous horse radish peroxidase in the tissue. Maybe the hydrogen peroxide solution used for blocking was inactivated
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous peroxidase or pseudoperoxidase activity may cause non-specific staining. The enzyme activity is blocked by incubation with hydrogen peroxide solution. Tissues containing Hepatitis B Surface Antigen (HBsAg) may give false positive results with HRP (horse radish peroxidase) detection systems (Omata et al, 1980). Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagent. In case of the reagent coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagent must be avoided, since otherwise non-specific staining might appear. ProClin is used for stabilisation. A Material safety data sheet (MSDS) is available upon request.
The Plus HRP Polymer anti-Rabbit is designed for the qualitative detection of antigens in fixed paraffinembedded tissue sections, in frozen tissue sections, and in cytological samples. It is developed for use in combination with mono- and polyclonal primary antibodies and sera obtained from rabbits. The reagent can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus HRP Polymer anti-Rabbit is a highly sensitive detection reagent intended for use in immunohistochemistry and immunocytochemistry. The enzyme polymer in this kit consists of several molecules of secondary antibodies covalently bound to several molecules of horse radish peroxidase (HRP). Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The reagent is suitable for the detection of mono- and polyclonal primary antibodies and sera obtained from rabbits. In contrast to other detection techniques, which often use the streptavidin-biotin system the Plus HRP Polymer anti-Rabbit avoids the problem of background staining caused by endogenous biotin in the tissue.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Endogenous peroxidase activity in the tissue may cause non-specific staining. This enzyme activity can be blocked by incubation with 3% H2O2-solution (Peroxide block). Background staining caused by unspecific binding of the primary antibody or the secondary antibody in the HRP polymer is minimized by incubation with a protein blocking solution (Blocking Solution). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the HRP-polymer is applied and incubated. Any excess of unbound HRP-polymer is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the horse radish peroxidase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen AEC leads to the formation of a red-brown product of reaction at the place of the target antigen. The chromogen DAB forms a dark brown precipitate.
Reagents provided:
6 ml HRP-Polymer anti-Rabbit (Ready-to-use) Substrate systems recommended: Permanent AEC kit, AEC Single Solution, AEC Substrate kit, DAB Substrate kit, DAB High contrast kit Materials required but not supplied: Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O 3% H2O2 solution Reagents for enzyme digestion or heat pre-treatment Wash buffer PBS or TBS PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solution should be stored at 2-8°C without further dilution. Please store the reagent in a dark place and do not freeze it. Under these conditions the solution is stable up to the expiry date indicated on the label. They should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. Tissue sections have to be completely covered with the different reagents in order to avoid drying out.
Procedure:
1. Peroxide blocking (3 % H2O2 solution) 10 min. 2. Washing with wash buffer 1 x 2 min. 3. Blocking Solution (This step is optional.) 5 min. 4. Washing with wash buffer 1 x 2 min. 5. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 6. Washing with wash buffer 3 x 5 min. 7. HRP-polymer anti-Rabbit 30 min. 8. Washing with wash buffer 3 x 2 min. 9. AEC or DAB (Controlling the colour intensity via light microscope is recommended.) 5-15 min. 10. Stopping the reaction with distilled H2O when the desired colour intensity is attained 11. Counterstaining and blueing 12. Mounting: aqueous with AEC, permanent with DAB or Permanent AEC
Expected results:
During the reaction of the substrate with horse radish peroxidase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support. No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from rabbit, but from a different species. 6. The antigen/epitope was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. If you used heat pre-treatment or enzyme digestion it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. Incubation times were too short or primary antibody concentration too low. 7. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme polymer or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use a Blocking Solution or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous horse radish peroxidase in the tissue. Maybe the hydrogen peroxide solution used for blocking was inactivated
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous peroxidase or pseudoperoxidase activity may cause non-specific staining. The enzyme activity is blocked by incubation with hydrogen peroxide solution. Tissues containing Hepatitis B Surface Antigen (HBsAg) may give false positive results with HRP (horse radish peroxidase) detection systems (Omata et al, 1980). Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagent. In case of the reagent coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagent must be avoided, since otherwise non-specific staining might appear. ProClin is used for stabilisation. A Material safety data sheet (MSDS) is available upon request.
The Plus AP Polymer anti-Rabbit is designed for the qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. It is developed for use in combination with monoand polyclonal primary antibodies and sera obtained from rabbits. The reagent can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus AP Polymer anti-Rabbit is a highly sensitive detection reagent intended for use in immunohistochemistry and immunocytochemistry. The enzyme polymer in this kit consists of several molecules of secondary antibodies covalently bound to several molecules of alkaline phosphatase (AP). Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The reagent is suitable for the detection of mono- and polyclonal primary antibodies and sera obtained from rabbits. In contrast to other detection techniques, which often use the streptavidin-biotin system the Plus AP Polymer anti-Rabbit avoids the problem of background staining caused by endogenous biotin in the tissue.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Background staining caused by unspecific binding of the primary antibody or the secondary antibody in the AP polymer is minimized by incubation with a protein blocking solution (Blocking Solution). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the AP-polymer is applied and incubated. Any excess of unbound AP-polymer is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the alkaline phosphatase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen Permanent AP Red leads to the formation of a magentared product of reaction at the place of the target antigen.
Reagents provided:
6 ml AP-Polymer anti-Rabbit (Ready-to-use) Substrate systems recommended: Permanent AP Red kit. Materials required but not supplied: Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O Reagents for enzyme digestion or heat pre-treatment Wash buffer PBS or TBS PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solutions should be stored at 2-8°C without further dilution. Please store the reagent in a dark place and do not freeze it. Under these conditions the solutions are stable up to the expiry date indicated on the label. They should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support
Reagent preparation:
Reagents should be at room temperature when used. ? Deparaffinise and rehydrate paraffin-embedded tissue sections. ? Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. ? Tissue sections have to be completely covered with the different reagents in order to avoid drying out
Procedure:
1. Blocking Solution (protein block, this step is optional) 5 min. 2. Washing with wash buffer 1 x 2 min. 3. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 4. Washing with wash buffer 3 x 5 min. 5. AP-polymer anti Rabbit 30 min. 6. Washing with wash buffer 3 x 2 min. 7. Permanent AP Red 5-15 min. (Controlling the colour intensity via light microscope is recommended.) 8. Stopping the reaction with distilled H2O when the desired colour intensity is attained 9. Counterstaining and blueing 10. Mounting: permanent or aqueous with Permanent AP Red
Expected results:
During the reaction of the substrate with alkaline phosphatase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support . No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from rabbit, but from a different species. 6. The antigen/epitope was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. If you used heat pre-treatment or enzyme digestion it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. If you are using PBS-based wash buffer: the activity of alkaline phosphatase in the reagents is blocked if too much wash buffer remains on the slides. 7. Incubation times were too short or primary antibody concentration too low. 8. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme polymer or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use a Blocking Solution or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high).
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of the reagent coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagent must be avoided, since otherwise non-specific staining might appear. ProClin 950 is used for stabilisation. A Material safety data sheet (MSDS) is available upon request.
Aqueous Mount is an aqueous mounting medium for histological and cytological preparations. It is especially suitable for mounting of tissue sections stained with alcohol-soluble chromogenic substrates such as Fast Red or AEC. Even sections stained with alcohol-resistant chromogenic substrates can be mounted with Aqueous Mount if they were not previously dehydrated. Aqueous Mount is intended for research use only.
Aqueous Mount in the following formats: 60 ml Ready-to-use
Storage and handling:
The solution should be stored at room temperature in the original container without further dilution. Please store the reagent in a dark place and do not freeze it. Under these conditions the solution is stable up to the expiry date indicated on the label. Do not use product after the expiry date. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents please contact our technical support.
Reagent preparation:
The solution is ready-to-use
Procedure:
Apply a sufficient amount of Aqueous Mount onto the stained histological or cytological section; depending on the size of section 2 to 5 drops. Cover with a coverslip. Heating of the mounted section for hardening is not necessary. Coverslips can be removed by soaking the section over night in water.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid contact of reagents or specimen with eye, skin or mucous membrane. In case of a reagent or specimen coming into contact with a sensitive area, wash the area with large amounts of water. In case of swallowing rinse the mouth excessively with large amounts of water and turn to a doctor. A material safety data sheet (MSDS) is available upon request.
Aqueous Mount is an aqueous mounting medium for histological and cytological preparations. It is especially suitable for mounting of tissue sections stained with alcohol-soluble chromogenic substrates such as Fast Red or AEC. Even sections stained with alcohol-resistant chromogenic substrates can be mounted with Aqueous Mount if they were not previously dehydrated. Aqueous Mount is intended for research use only.
Aqueous Mount in the following formats: 30 ml Ready-to-use
Storage and handling:
The solution should be stored at room temperature in the original container without further dilution. Please store the reagent in a dark place and do not freeze it. Under these conditions the solution is stable up to the expiry date indicated on the label. Do not use product after the expiry date. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents please contact our technical support.
Reagent preparation:
The solution is ready-to-use
Procedure:
Apply a sufficient amount of Aqueous Mount onto the stained histological or cytological section; depending on the size of section 2 to 5 drops. Cover with a coverslip. Heating of the mounted section for hardening is not necessary. Coverslips can be removed by soaking the section over night in water.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid contact of reagents or specimen with eye, skin or mucous membrane. In case of a reagent or specimen coming into contact with a sensitive area, wash the area with large amounts of water. In case of swallowing rinse the mouth excessively with large amounts of water and turn to a doctor. A material safety data sheet (MSDS) is available upon request.
Permanent AP Red Kit is developed for immunohistochemical and in situ-hybridisation staining procedures with alkaline phosphatase. Permanent AP Red leads to the formation of a magenta-red precipitate at the location of the target antigen or target nucleic acid. The precipitate is insoluble in aqueous and organic solvents and can be observed by light or fluorescence microscopy.
500 ml Permanent AP Red Buffer 8 ml Permanent AP Red Chromogen 1 Dilution Vial
Storage and handling:
The solutions should be stored at 2-8°C without further dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. Do not use product after the expiry date. The working solution prepared is stable for about 60 minutes and should therefore be used directly after preparation. Excess working solution should be discarded. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents please contact our technical support.
Reagent preparation:
Reagent preparation (Preparation of the working solution) 1) Pipette 2.5 ml AP Red Buffer into the provided dilution vial and let it come to room temperature. The chromogen should still be kept cool. 2) Directly prior to use add 1 drop of Permanent AP Red Chromogen into the buffer. Mix thoroughly. 3) The solution is stable for about 60 minutes. Preparation should be done directly before use. Make sure to pipet the chromogen/substrate mix on the last slide of the staining run within 40 min after mixing. If you want to prepare other quantities of the working solution, please use same ratio AP Red Buffer and Chromogen
Procedure:
1) Rinse the slide with wash buffer after the previous incubation step. 2) Apply freshly prepared Permanent AP Red working solution onto the slide. Incubate for 10 minutes. 3) Rinse with distilled H2O. 4) Counterstain with haematoxylin for about 30 seconds up to 5 minutes (depending on the desired staining intensity). 5) Rinse with distilled H2O. 6) Blueing in tap water for at least 5 minutes. 7) Dehydrate through a graded series of ethanol and clear in xylene. Mount with a permanent mounting medium. Note: It is also possible to mount Permanent AP Red with aqueous mounting media.
Expected results:
During the reaction of the substrate with alkaline phosphatase in presence of the chromogen Permanent AP Red, a magenta-red precipitate is formed at the location of the target antigen or nucleic acid. The precipitate is insoluble in aqueous and organic solvents and can be observed by light or fluorescence microscopy (Texas Red filter).
Trouble shooting:
If you observe unusual staining or other deviations from the expected results please read these instructions carefully, or contact our technical support. Also refer to the instructions of the detection systems for guidance on general troubleshooting.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining. Please refer to the instructions of the detection system for guidance on general quality control procedures.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). In some tissues endogenous alkaline phosphatase activity may cause non-specific staining. However, neither intestinal nor placental alkaline phosphatase can be blocked with levamisole. Therefore, tissues of this origin should be stained with peroxidase detection systems. A higher sensitivity can be obtained when a second chromogenic substrate step is used (i. e. 2 x 10 min Permanent AP Red). Background staining due to endogenous biotin can be blocked through an avidin-biotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. A longer exposure to absolute ethanol can result in decreasing staining intensity. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid contact of reagents or specimen with eye, skin or mucous membrane. In case of a reagent or specimen coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear. A material safety data sheet (MSDS) is available upon request.
Permanent AP Red Kit is developed for immunohistochemical and in situ-hybridisation staining procedures with alkaline phosphatase. Permanent AP Red leads to the formation of a magenta-red precipitate at the location of the target antigen or target nucleic acid. The precipitate is insoluble in aqueous and organic solvents and can be observed by light or fluorescence microscopy.
125 ml Permanent AP Red Buffer 2 ml Permanent AP Red Chromogen 1 Dilution Vial
Storage and handling:
The solutions should be stored at 2-8°C without further dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. Do not use product after the expiry date. The working solution prepared is stable for about 60 minutes and should therefore be used directly after preparation. Excess working solution should be discarded. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents please contact our technical support.
Reagent preparation:
Reagent preparation (Preparation of the working solution) 1) Pipette 2.5 ml AP Red Buffer into the provided dilution vial and let it come to room temperature. The chromogen should still be kept cool. 2) Directly prior to use add 1 drop of Permanent AP Red Chromogen into the buffer. Mix thoroughly. 3) The solution is stable for about 60 minutes. Preparation should be done directly before use. Make sure to pipet the chromogen/substrate mix on the last slide of the staining run within 40 min after mixing. If you want to prepare other quantities of the working solution, please use same ratio AP Red Buffer and Chromogen
Procedure:
1) Rinse the slide with wash buffer after the previous incubation step. 2) Apply freshly prepared Permanent AP Red working solution onto the slide. Incubate for 10 minutes. 3) Rinse with distilled H2O. 4) Counterstain with haematoxylin for about 30 seconds up to 5 minutes (depending on the desired staining intensity). 5) Rinse with distilled H2O. 6) Blueing in tap water for at least 5 minutes. 7) Dehydrate through a graded series of ethanol and clear in xylene. Mount with a permanent mounting medium. Note: It is also possible to mount Permanent AP Red with aqueous mounting media.
Expected results:
During the reaction of the substrate with alkaline phosphatase in presence of the chromogen Permanent AP Red, a magenta-red precipitate is formed at the location of the target antigen or nucleic acid. The precipitate is insoluble in aqueous and organic solvents and can be observed by light or fluorescence microscopy (Texas Red filter).
Trouble shooting:
If you observe unusual staining or other deviations from the expected results please read these instructions carefully, or contact our technical support. Also refer to the instructions of the detection systems for guidance on general troubleshooting.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining. Please refer to the instructions of the detection system for guidance on general quality control procedures.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). In some tissues endogenous alkaline phosphatase activity may cause non-specific staining. However, neither intestinal nor placental alkaline phosphatase can be blocked with levamisole. Therefore, tissues of this origin should be stained with peroxidase detection systems. A higher sensitivity can be obtained when a second chromogenic substrate step is used (i. e. 2 x 10 min Permanent AP Red). Background staining due to endogenous biotin can be blocked through an avidin-biotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. A longer exposure to absolute ethanol can result in decreasing staining intensity. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid contact of reagents or specimen with eye, skin or mucous membrane. In case of a reagent or specimen coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear. A material safety data sheet (MSDS) is available upon request.
DAB Substrate kit is intended for immunohistochemical and in situ-hybridisation staining procedures with horse radish peroxidase (HRP). DAB (3,3-Diaminobenzidine) leads to the formation of a brown precipitate at the location of the target antigen or target nucleic acid. The precipitate is insoluble in aqueous and organic solvents and can be observed by light microscopy.
30 ml DAB Chromogen (liquid DAB concentrate) 500 ml DAB Substrate Buffer
Storage and handling:
The solutions should be stored at 2-8°C without fur ther dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. Do not use product after the expiry date. The working solution should be prepared freshly at the day of use. Once the two reagents are combined, the resulting solution can be used for up to six hours. Excess working solution needs to be disposed as hazardous substance. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents please contact our technical support.
Reagent preparation:
Add 50 µl DAB Chromogen (DAB concentrate) to 1 ml of DAB Substrate Buffer and mix thoroughly. Note: Typical working concentrations are 50 µl (0.9 mg) DAB per ml substrate buffer. The colour intensity can be adjusted by decreasing or increasing the DAB concentration in the working solution. Maximum sensitivity in immunohistochemical staining can be achieved by working concentrations of about 80 µl (1.5 mg) DAB per ml substrate buffer.
Procedure:
1) Rinse the slide with wash buffer after the previous incubation step. 2) Apply the DAB working solution onto the slide. Incubate for 5-15 minutes. 3) Rinse with distilled H2O. 4) Counterstain with haematoxylin for about 30 seconds up to 5 minutes (depending on the desired staining intensity). 5) Rinse with distilled H2O. 6) Blueing in tap water for at least 5 minutes. 7) Dehydrate through a graded series of ethanol and clear in xylene. Mount with a permanent mounting medium. Note: It is also possible to mount DAB with aqueous mounting media.
Expected results:
During the reaction of the substrate with horse radish peroxidase in presence of the chromogen DAB, a brown precipitate is formed at the location of the target antigen or nucleic acid. The precipitate is insoluble in aqueous and organic solvents and can be observed by light microscopy.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results please read these instructions carefully, or contact our technical support. Also refer to the instructions of the detection systems for guidance on general troubleshooting.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining. Please refer to the instructions of the detection system for guidance on general quality control procedures.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). In some tissues endogenous peroxidase activity may cause non-specific staining. The enzyme activity should be blocked by incubation with hydrogen peroxide solution (H2O2 solution). The step is carried out before incubation with primary antibody but after dewaxing and rehydration. Background staining due to endogenous biotin can be blocked through an avidin-biotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. The DAB chromogen is hazardous to your health. Wear protective clothing to avoid contact of reagents or specimen with eye, skin or mucous membrane. In case of a reagent or specimen coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining may occur. Material safety data sheets (MSDS) are available upon request.
DAB Substrate kit is intended for immunohistochemical and in situ-hybridisation staining procedures with horse radish peroxidase (HRP). DAB (3,3-Diaminobenzidine) leads to the formation of a brown precipitate at the location of the target antigen or target nucleic acid. The precipitate is insoluble in aqueous and organic solvents and can be observed by light microscopy.
3 ml DAB Chromogen (liquid DAB concentrate) 11 x 5 ml DAB Substrate Buffer
Storage and handling:
The solutions should be stored at 2-8°C without fur ther dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. Do not use product after the expiry date. The working solution should be prepared freshly at the day of use. Once the two reagents are combined, the resulting solution can be used for up to six hours. Excess working solution needs to be disposed as hazardous substance. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents please contact our technical support.
Reagent preparation:
Add 4 drops of DAB Chromogen (DAB concentrate) to one bottle of DAB Substrate Buffer and mix thoroughly.
Procedure:
1) Rinse the slide with wash buffer after the previous incubation step. 2) Apply the DAB working solution onto the slide. Incubate for 5-15 minutes. 3) Rinse with distilled H2O. 4) Counterstain with haematoxylin for about 30 seconds up to 5 minutes (depending on the desired staining intensity). 5) Rinse with distilled H2O. 6) Blueing in tap water for at least 5 minutes. 7) Dehydrate through a graded series of ethanol and clear in xylene. Mount with a permanent mounting medium. Note: It is also possible to mount DAB with aqueous mounting media.
Expected results:
During the reaction of the substrate with horse radish peroxidase in presence of the chromogen DAB, a brown precipitate is formed at the location of the target antigen or nucleic acid. The precipitate is insoluble in aqueous and organic solvents and can be observed by light microscopy.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results please read these instructions carefully, or contact our technical support. Also refer to the instructions of the detection systems for guidance on general troubleshooting.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining. Please refer to the instructions of the detection system for guidance on general quality control procedures.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). In some tissues endogenous peroxidase activity may cause non-specific staining. The enzyme activity should be blocked by incubation with hydrogen peroxide solution (H2O2 solution). The step is carried out before incubation with primary antibody but after dewaxing and rehydration. Background staining due to endogenous biotin can be blocked through an avidin-biotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. The DAB chromogen is hazardous to your health. Wear protective clothing to avoid contact of reagents or specimen with eye, skin or mucous membrane. In case of a reagent or specimen coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining may occur. Material safety data sheets (MSDS) are available upon request.
DAB Substrate kit High Contrast is developed for immunohistochemical and in situ-hybridisation staining procedures with horse radish peroxidase (HRP). DAB (3,3-Diaminobenzidine) leads to the formation of a brown precipitate at the location of the target antigen or target nucleic acid. The precipitate is insoluble in aqueous and organic solvents and can be observed by light microscopy. The kit is especially useful when a high contrast between chromogen and counter stain is desired. Compared to standard DAB staining systems the DAB Substrate High Contrast kit gives a darker brown colour and a higher sensitivity.
30 ml DAB Chromogen (liquid DAB concentrate) 500 ml DAB Substrate Buffer High Contrast
Storage and handling:
The solutions should be stored at 2-8°C without fur ther dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. Do not use product after the expiry date. The working solution should be prepared freshly at the day of use. Once the two reagents are combined, the resulting solution is stable for up to six hours. Excess working solution should be disposed as hazardous substance. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents please contact our technical support.
Reagent preparation:
Add 50 µl of DAB Chromogen (DAB concentrate) to 1 ml of DAB Substrate Buffer High Contrast and mix thoroughly. Note: Typical working concentrations are 50 µl (0.9 mg) DAB per ml substrate buffer. The colour intensity can be adjusted by decreasing or increasing the DAB concentration in the working solution. Maximum sensitivity in immunohistochemical staining can be achieved by working concentrations of about 80 µl (1.5 mg) DAB per ml substrate buffer.
Procedure:
1) Rinse the slide with wash buffer after the previous incubation step. 2) Apply the DAB High contrast working solution to the slide. Incubate for 5-15 minutes. 3) Rinse with distilled H2O. 4) Counterstain with haematoxylin for about 30 seconds up to 5 minutes (depending on the desired staining intensity). 5) Rinse with distilled H2O. 6) Blueing in tap water for at least 5 minutes. 7) Dehydrate through a graded series of ethanol and clear in xylene. Mount with a permanent mounting medium. Note: It is also possible to mount DAB High Contrast with aqueous mounting media.
Expected results:
During the reaction of the substrate with horse radish peroxidase in presence of the chromogen DAB, a brown precipitate is formed at the location of the target antigen or nucleic acid. The precipitate is insoluble in aqueous and organic solvents and can be observed by light microscopy.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results please read these instructions carefully, or contact our technical support. Also refer to the instructions of the detection systems for guidance on general troubleshooting.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining. Please refer to the instructions of the detection system for guidance on general quality control procedures.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). In some tissues endogenous peroxidase activity may cause non-specific staining. The enzyme activity should be blocked by incubation with hydrogen peroxide solution (H2O2 solution). The step is carried out before incubation with primary antibody but after dewaxing and rehydration. Background staining due to endogenous biotin can be blocked through an avidin-biotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. The DAB chromogen is hazardous to your health. Wear protective clothing to avoid contact of reagents or specimen with eye, skin or mucous membrane. In case of a reagent or specimen coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining may occur. Material safety data sheets (MSDS) are available upon request.
DAB Substrate kit High Contrast is developed for immunohistochemical and in situ-hybridisation staining procedures with horse radish peroxidase (HRP). DAB (3,3-Diaminobenzidine) leads to the formation of a brown precipitate at the location of the target antigen or target nucleic acid. The precipitate is insoluble in aqueous and organic solvents and can be observed by light microscopy. The kit is especially useful when a high contrast between chromogen and counter stain is desired. Compared to standard DAB staining systems the DAB Substrate High Contrast kit gives a darker brown colour and a higher sensitivity.
3 ml DAB Chromogen (liquid DAB concentrate) 11 x 5 ml DAB Substrate Buffer High Contrast
Storage and handling:
The solutions should be stored at 2-8°C without fur ther dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. Do not use product after the expiry date. The working solution should be prepared freshly at the day of use. Once the two reagents are combined, the resulting solution is stable for up to six hours. Excess working solution should be disposed as hazardous substance. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents please contact our technical support.
Reagent preparation:
Add 5 drops of DAB Chromogen (DAB concentrate) to one bottle of DAB Substrate Buffer High Contrast and mix thoroughly.
Procedure:
1) Rinse the slide with wash buffer after the previous incubation step. 2) Apply the DAB High contrast working solution to the slide. Incubate for 5-15 minutes. 3) Rinse with distilled H2O. 4) Counterstain with haematoxylin for about 30 seconds up to 5 minutes (depending on the desired staining intensity). 5) Rinse with distilled H2O. 6) Blueing in tap water for at least 5 minutes. 7) Dehydrate through a graded series of ethanol and clear in xylene. Mount with a permanent mounting medium. Note: It is also possible to mount DAB High Contrast with aqueous mounting media.
Expected results:
During the reaction of the substrate with horse radish peroxidase in presence of the chromogen DAB, a brown precipitate is formed at the location of the target antigen or nucleic acid. The precipitate is insoluble in aqueous and organic solvents and can be observed by light microscopy.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results please read these instructions carefully, or contact our technical support. Also refer to the instructions of the detection systems for guidance on general troubleshooting.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining. Please refer to the instructions of the detection system for guidance on general quality control procedures.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). In some tissues endogenous peroxidase activity may cause non-specific staining. The enzyme activity should be blocked by incubation with hydrogen peroxide solution (H2O2 solution). The step is carried out before incubation with primary antibody but after dewaxing and rehydration. Background staining due to endogenous biotin can be blocked through an avidin-biotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. The DAB chromogen is hazardous to your health. Wear protective clothing to avoid contact of reagents or specimen with eye, skin or mucous membrane. In case of a reagent or specimen coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining may occur. Material safety data sheets (MSDS) are available upon request.
Permanent AEC Kit is intended for immunohistochemical and in situ-hybridisation staining procedures with horse radish peroxidase (HRP). AEC (3-Amino-9-ethylcarbazol) leads to the formation of a red-brown precipitate at the location of the target antigen or target nucleic acid. The precipitate is insoluble in aqueous and organic solvents and can be observed by light microscopy. Permanent AEC Kit is intended for research use only.
5.5 ml Reagent 1 3 ml Reagent 2 3 ml Reagent 3 (Chromogen) 4.5 ml Reagent 4 (H2O2) 1 Dilution Vial
Storage and handling:
The solutions should be stored at 2-8°C without fur ther dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. Do not use product after the expiry date. The working solution should be prepared freshly at the day of use. Excess working solution should be disposed. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents please contact our technical support.
Reagent preparation:
1) Pipette 5 ml distilled or deionised water into the provided dilution vial. 2) Add 3 drops buffer concentrate (Reagent 1). Mix thoroughly. 3) Add 2 drops Reagent 2. Mix thoroughly. 4) Add 2 drops AEC chromogen (Reagent 3). Mix thoroughly. 5) Add 2 drops H2O2 substrate (Reagent 4). Mix thoroughly. This working solution is stable for at least 16 hours if stored at 2-8°C in a dark place.
Procedure:
1) Apply the Permanent AEC working solution onto the slide. Incubate for 5-15 minutes. (Incubation time can be extended, if desired.) 2) Rinse with distilled or deionised H2O. 3) Counterstain with haematoxylin for about 30 seconds up to 5 minutes (depending on the desired staining intensity). 4) Rinse with distilled or deionised H2O. 5) Blueing in tap water for at least 5 minutes. 6) Dehydrate through a graded series of ethanol and clear in xylene. Mount with a permanent mounting medium.
Expected results:
During the reaction of the substrate with horse radish peroxidase in presence of the chromogen AEC, a red-brown precipitate is formed at the location of the target antigen or nucleic acid. The precipitate is insoluble in aqueous and organic solvents and can be observed by light microscopy.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results please read these instructions carefully, or contact our technical support. Also refer to the instructions of the detection systems for guidance on general troubleshooting.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining. Please refer to the instructions of the detection system for guidance on general quality control procedures.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). In some tissues endogenous peroxidase activity may cause non-specific staining. The enzyme activity should be blocked by incubation with hydrogen peroxide solution (H2O2 solution). The step is carried out before incubation with primary antibody but after dewaxing and rehydration. Background staining due to endogenous biotin can be blocked through an avidin-biotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. A longer exposure to absolute ethanol can result in decreasing staining intensity. Use of recycled alcohol to dehydrate tissue slides after staining is not recommended. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid contact of reagents or specimen with eye, skin or mucous membrane. In case of a reagent or specimen coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining may occur. A material safety data sheet (MSDS) is available upon request.
AEC Substrate kit is intended for immunohistochemical and in situ-hybridisation staining procedures with horse radish peroxidase (HRP). AEC (3-Amino-9-ethylcarbazol) leads to the formation of a red-brown precipitate at the location of the target antigen or target nucleic acid. The precipitate is insoluble in aqueous mounting media and can be observed by light microscopy.
15 ml AEC Chromogen (liquid AEC concentrate) 500 ml AEC Substrate Buffer
Storage and handling:
The solutions should be stored at 2-8°C without fur ther dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. Do not use product after the expiry date. The working solution should be prepared freshly at the day of use. Once the two reagents are combined, the resulting solution is stable for up to three hours. Excess working solution needs to be disposed as hazardous substance. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents please contact our technical support.
Reagent preparation:
Add 20 µl AEC Chromogen (AEC concentrate) to 1 ml of AEC Substrate Buffer and mix thoroughly. Note: The colour intensity can be adjusted by decreasing or increasing the AEC concentration in the working solution.
Procedure:
1) Rinse the slide with wash buffer after the previous incubation step. 2) Apply the AEC working solution onto the slide. Incubate for 5-20 minutes. 3) Rinse with distilled H2O. 4) Counterstain with haematoxylin for about 30 seconds up to 5 minutes (depending on the desired staining intensity). 5) Rinse with distilled H2O. 6) Blueing in tap water for at least 5 minutes. 7) Mount with an aqueous mounting medium.
Expected results:
During the reaction of the substrate with horse radish peroxidase in presence of the chromogen AEC, a red-brown precipitate is formed at the location of the target antigen or nucleic acid. The precipitate is insoluble in aqueous solvents and can be observed by light microscopy.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results please read these instructions carefully, or contact our technical support. Also refer to the instructions of the detection systems for guidance on general troubleshooting.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining. Please refer to the instructions of the detection system for guidance on general quality control procedures.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). In some tissues endogenous peroxidase activity may cause non-specific staining. The enzyme activity should be blocked by incubation with hydrogen peroxide solution (H2O2 solution). The step is carried out before incubation with primary antibody but after dewaxing and rehydration. Background staining due to endogenous biotin can be blocked through an avidin-biotin blocking step prior to the primary antibody incubation step. The coloured precipitate formed by AEC is soluble in organic solvents. The tissue sections therefore have to be counterstained with aqueous solutions (e. g. Gills or Mayers haematoxylin) and mounted with aqueous mounting media. The colour intensity of the reaction product can decrease with time, especially when exposed to light. The staining reaction itself can be influenced in the same way when carried out in strong light. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided
Precautions:
Use by qualified personnel only. Some of the reagents used in this kit are hazardous to your health. Wear protective clothing to avoid contact of reagents or specimen with eye, skin or mucous membrane. In case of a reagent or specimen coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining may occur. Material safety data sheets (MSDS) are available upon request.
AEC Substrate kit is intended for immunohistochemical and in situ-hybridisation staining procedures with horse radish peroxidase (HRP). AEC (3-Amino-9-ethylcarbazol) leads to the formation of a red-brown precipitate at the location of the target antigen or target nucleic acid. The precipitate is insoluble in aqueous mounting media and can be observed by light microscopy.
3 ml AEC Chromogen (liquid AEC concentrate) 11 x 5 ml AEC Substrate Buffer
Storage and handling:
The solutions should be stored at 2-8°C without fur ther dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. Do not use product after the expiry date. The working solution should be prepared freshly at the day of use. Once the two reagents are combined, the resulting solution is stable for up to three hours. Excess working solution needs to be disposed as hazardous substance. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents please contact our technical support.
Reagent preparation:
Add 2 drops of AEC Chromogen (AEC concentrate) to one bottle of AEC Substrate Buffer and mix thoroughly.
Procedure:
1) Rinse the slide with wash buffer after the previous incubation step. 2) Apply the AEC working solution onto the slide. Incubate for 5-20 minutes. 3) Rinse with distilled H2O. 4) Counterstain with haematoxylin for about 30 seconds up to 5 minutes (depending on the desired staining intensity). 5) Rinse with distilled H2O. 6) Blueing in tap water for at least 5 minutes. 7) Mount with an aqueous mounting medium.
Expected results:
During the reaction of the substrate with horse radish peroxidase in presence of the chromogen AEC, a red-brown precipitate is formed at the location of the target antigen or nucleic acid. The precipitate is insoluble in aqueous solvents and can be observed by light microscopy.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results please read these instructions carefully, or contact our technical support. Also refer to the instructions of the detection systems for guidance on general troubleshooting.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining. Please refer to the instructions of the detection system for guidance on general quality control procedures.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). In some tissues endogenous peroxidase activity may cause non-specific staining. The enzyme activity should be blocked by incubation with hydrogen peroxide solution (H2O2 solution). The step is carried out before incubation with primary antibody but after dewaxing and rehydration. Background staining due to endogenous biotin can be blocked through an avidin-biotin blocking step prior to the primary antibody incubation step. The coloured precipitate formed by AEC is soluble in organic solvents. The tissue sections therefore have to be counterstained with aqueous solutions (e. g. Gills or Mayers haematoxylin) and mounted with aqueous mounting media. The colour intensity of the reaction product can decrease with time, especially when exposed to light. The staining reaction itself can be influenced in the same way when carried out in strong light. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided
Precautions:
Use by qualified personnel only. Some of the reagents used in this kit are hazardous to your health. Wear protective clothing to avoid contact of reagents or specimen with eye, skin or mucous membrane. In case of a reagent or specimen coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining may occur. Material safety data sheets (MSDS) are available upon request.
AEC Single Solution is a ready-to-use solution intended for immunohistochemical and in situ-hybridisation staining procedures with horse radish peroxidase (HRP). AEC (3-Amino-9-ethylcarbazol) leads to the formation of a red-brown precipitate at the location of the target antigen or target nucleic acid. The precipitate is insoluble in aqueous mounting media and can be observed by light microscopy. AEC Single Solution is especially useful when a high sensitivity is desired.
The solution should be stored at 2-8°C without furt her dilution. Please store the reagent in a dark place and do not freeze it. Under these conditions the solution is stable up to the expiry date indicated on the label. Do not use product after the expiry date. AEC Single Solution is a ready-to-use solution. Preparation of a working solution as in other chromogenic substrates is not necessary. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents please contact our technical support.
Reagent preparation:
The solution is ready-to-use. AEC Single Solution can be used directly from the refrigerator and should be stored again at 2-8°C aft er use. When using the small package (8 ml MON-APP169) please directly drop from the bottle. When using the 100 ml package (MON_APP?) please transfer up to 8 ml of the AEC Single Solution into one of the provided dropper bottles. The transferred solution is stable for many weeks if stored at 2-8°C. The vo lume required for several staining runs should be transferred so that the 100 ml stock bottle has to be opened only a few times. If you would like to pipette the solution use a clean vial from which you pipette. Remaining quantities should not be filled back into the bottle but disposed as hazardous material.
Procedure:
1) Rinse the slide with wash buffer after the previous incubation step. 2) Apply the AEC Single Solution to the slide. Incubate for 3-6 minutes. (Incubation time can be extended up to 30 minutes, if desired.) 3) Rinse with distilled H2O. 4) Counterstain with haematoxylin for about 30 seconds up to 5 minutes (depending on the desired staining intensity). 5) Rinse with distilled H2O. 6) Blueing in tap water for at least 5 minutes. 7) Mount with an aqueous mounting medium.
Expected results:
During the reaction of the substrate with horse radish peroxidase in presence of the chromogen AEC, a red-brown precipitate is formed at the location of the target antigen or nucleic acid. The precipitate is insoluble in aqueous solvents and can be observed by light microscopy.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results please read these instructions carefully, or contact our technical support. Also refer to the instructions of the detection systems for guidance on general troubleshooting.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining. Please refer to the instructions of the detection system for guidance on general quality control procedures.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). In some tissues endogenous peroxidase activity may cause non-specific staining. The enzyme activity should be blocked by incubation with hydrogen peroxide solution (H2O2 solution). The step is carried out before incubation with primary antibody but after dewaxing and rehydration. Background staining due to endogenous biotin can be blocked through an avidin-biotin blocking step prior to the primary antibody incubation step. The coloured precipitate formed by AEC is soluble in organic solvents. The tissue sections therefore have to be counterstained with aqueous solutions (e. g. Gills or Mayers haematoxylin) and mounted with aqueous mounting media. The colour intensity of the reaction product can decrease with time, especially when exposed to light. The staining reaction itself can be influenced in the same way when carried out in strong light. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided
Precautions:
Use by qualified personnel only. AEC (3-Amino-9-ethylcarbazol) and the solvents used are considered hazardous materials. Material safety data sheets (MSDS) are available upon request. Wear protective clothing to avoid contact of reagent or specimen with eye, skin or mucous membrane. In case of reagent or specimen coming into contact with a sensitive area, wash the area with large amounts of water. Oxidising substances, e. g. metals, dust, bacteria or glass devices can influence the stability of AEC Single Solution. Such contaminations have to be avoided. Non-consumed solution needs to be discarded as dangerous substance.
The Plus HRP Kit, Rabbit is based on the streptavidin-biotin system. It is designed for qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. The kit is developed for use in combination with mono- and polyclonal primary antibodies and sera obtained from rabbit. The Plus HRP Kit, Rabbit can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus HRP Kit, Rabbit is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The method is based on the streptavidin-biotin system which means that a biotinylated secondary antibody binds to several molecules of a conjugate composed of streptavidin and horse radish peroxidase. Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The biotinylated secondary antibody in the Plus HRP Kit, Rabbit binds to rabbit primary antibodies. Therefore this kit can detect mono- and polyclonal primary antibodies and sera obtained from rabbit.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Endogenous peroxidase activity in the tissue may cause non-specific staining. This enzyme activity can be blocked by incubation with 3% H2O2-solution (Peroxide Block). Background staining caused by unspecific binding of the primary or secondary antibody is minimized by incubation with a protein blocking solution (Blocking Solution). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the biotinylated secondary antibody is applied and incubated. This antibody functions as a link between primary antibody and the streptavidin-horse radish peroxidase-conjugate (Streptavidin-HRP-Conjugate). A second washing is followed by the application of this conjugate. It binds to the biotin at the secondary antibody. Any excess of unbound streptavidin-HRPconjugate is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the horse radish peroxidase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen AEC (included only in kit MON-APP132) leads to the formation of a red-brown product of reaction at the place of the target antigen. The chromogen DAB (included only in kit MON-APP133) forms a dark brown precipitate.
Reagents provided:
4x 15 ml Blocking Solution Reagent 1 (ready-to-use) 4x 15 ml Biotinylated Secondary Antibody, Rabbit Reagent 2 (ready-to-use) 4x 15 ml Streptavidin-HRP-Conjugate Reagent 3 (ready-to-use) Substrate systems recommended (if not included in the kit): Permanent AEC kit, AEC Single Solution, AEC substrate kit, DAB Substrate kit, DAB High Contrast kit Materials required but not supplied: Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O 3% H2O2 solution Reagents for enzyme digestion or heat pre-treatment Wash buffer Pink PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solutions should be stored at 2-8°C without fur ther dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. They should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support
Procedure:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. The tissue sections have to be completely covered with the different reagents in order to avoid drying out. Preparation of the chromogenic substrate AEC working solution (with MON-APP132 only): Add 2 drops (100 µl) of AEC Concentrate to one bottle of AEC Substrate Buffer and mix thoroughly. Preparation of the chromogenic substrate DAB working solution (with MON-APP133 only): Add 4 drops (200 µl) of DAB Concentrate to one bottle of DAB Substrate Buffer and mix thoroughly.
Expected results:
During the reaction of the substrate with horse radish peroxidase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support. No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pretreatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from rabbit. 6. The antigen was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pretreatment or enzyme digestion. If you used a pre-treatment it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. Incubation times were too short or primary antibody concentration too low. 7. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme conjugate or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous horse radish peroxidase. Maybe the hydrogen peroxide solution used for blocking was inactivated. 9. Non-specific binding of the secondary antibody to endogenous biotin in the tissue section. Carry out an avidin-biotin block before incubation
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous peroxidase or pseudoperoxidase activity or the endogenous biotin content may cause non-specific staining. The enzyme activity can be blocked by incubation with 3% H2O2 solution. Tissues containing Hepatitis B Surface Antigen (HBsAg) may give false positive results with HRP (horse radish peroxidase) detection systems (Omata et al, 1980). Background staining due to endogenous biotin can be blocked through an avidinbiotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the Blocking Solution instead of just draining it away as in other procedures. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear. ProClin 300 and sodium azide (NaN3) are used for stabilisation. Sodium azide deposits in drainage pipes made of lead or copper can result in the formation of highly explosive metallic azides. To avoid such deposits in drainage pipes, sodium azide should be discarded in a large volume of running water. Material safety data sheets (MSDS) are available upon request.
The Plus HRP Kit, Rabbit is based on the streptavidin-biotin system. It is designed for qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. The kit is developed for use in combination with mono- and polyclonal primary antibodies and sera obtained from rabbit. The Plus HRP Kit, Rabbit can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus HRP Kit, Rabbit is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The method is based on the streptavidin-biotin system which means that a biotinylated secondary antibody binds to several molecules of a conjugate composed of streptavidin and horse radish peroxidase. Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The biotinylated secondary antibody in the Plus HRP Kit, Rabbit binds to rabbit primary antibodies. Therefore this kit can detect mono- and polyclonal primary antibodies and sera obtained from rabbit.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Endogenous peroxidase activity in the tissue may cause non-specific staining. This enzyme activity can be blocked by incubation with 3% H2O2-solution (Peroxide Block). Background staining caused by unspecific binding of the primary or secondary antibody is minimized by incubation with a protein blocking solution (Blocking Solution). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the biotinylated secondary antibody is applied and incubated. This antibody functions as a link between primary antibody and the streptavidin-horse radish peroxidase-conjugate (Streptavidin-HRP-Conjugate). A second washing is followed by the application of this conjugate. It binds to the biotin at the secondary antibody. Any excess of unbound streptavidin-HRPconjugate is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the horse radish peroxidase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen AEC (included only in kit MON-APP132) leads to the formation of a red-brown product of reaction at the place of the target antigen. The chromogen DAB (included only in kit MON-APP133) forms a dark brown precipitate.
Reagents provided:
500 ml Blocking Solution Reagent 1 (ready-to-use) 500 ml Biotinylated Secondary Antibody, Rabbit Reagent 2 (ready-to-use) 500 ml Streptavidin-HRP-Conjugate Reagent 3 (ready-to-use) Substrate systems recommended (if not included in the kit): Permanent AEC kit, AEC Single Solution, AEC substrate kit, DAB Substrate kit, DAB High Contrast kit Materials required but not supplied: Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O 3% H2O2 solution Reagents for enzyme digestion or heat pre-treatment Wash buffer Pink PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solutions should be stored at 2-8°C without fur ther dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. They should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support
Procedure:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. The tissue sections have to be completely covered with the different reagents in order to avoid drying out. Preparation of the chromogenic substrate AEC working solution (with MON-APP132 only): Add 2 drops (100 µl) of AEC Concentrate to one bottle of AEC Substrate Buffer and mix thoroughly. Preparation of the chromogenic substrate DAB working solution (with MON-APP133 only): Add 4 drops (200 µl) of DAB Concentrate to one bottle of DAB Substrate Buffer and mix thoroughly.
Expected results:
During the reaction of the substrate with horse radish peroxidase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support. No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pretreatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from rabbit. 6. The antigen was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pretreatment or enzyme digestion. If you used a pre-treatment it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. Incubation times were too short or primary antibody concentration too low. 7. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme conjugate or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous horse radish peroxidase. Maybe the hydrogen peroxide solution used for blocking was inactivated. 9. Non-specific binding of the secondary antibody to endogenous biotin in the tissue section. Carry out an avidin-biotin block before incubation
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous peroxidase or pseudoperoxidase activity or the endogenous biotin content may cause non-specific staining. The enzyme activity can be blocked by incubation with 3% H2O2 solution. Tissues containing Hepatitis B Surface Antigen (HBsAg) may give false positive results with HRP (horse radish peroxidase) detection systems (Omata et al, 1980). Background staining due to endogenous biotin can be blocked through an avidinbiotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the Blocking Solution instead of just draining it away as in other procedures. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear. ProClin 300 and sodium azide (NaN3) are used for stabilisation. Sodium azide deposits in drainage pipes made of lead or copper can result in the formation of highly explosive metallic azides. To avoid such deposits in drainage pipes, sodium azide should be discarded in a large volume of running water. Material safety data sheets (MSDS) are available upon request.
The Plus HRP Kit, Rabbit is based on the streptavidin-biotin system. It is designed for qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. The kit is developed for use in combination with mono- and polyclonal primary antibodies and sera obtained from rabbit. The Plus HRP Kit, Rabbit can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus HRP Kit, Rabbit is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The method is based on the streptavidin-biotin system which means that a biotinylated secondary antibody binds to several molecules of a conjugate composed of streptavidin and horse radish peroxidase. Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The biotinylated secondary antibody in the Plus HRP Kit, Rabbit binds to rabbit primary antibodies. Therefore this kit can detect mono- and polyclonal primary antibodies and sera obtained from rabbit.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Endogenous peroxidase activity in the tissue may cause non-specific staining. This enzyme activity can be blocked by incubation with 3% H2O2-solution (Peroxide Block). Background staining caused by unspecific binding of the primary or secondary antibody is minimized by incubation with a protein blocking solution (Blocking Solution). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the biotinylated secondary antibody is applied and incubated. This antibody functions as a link between primary antibody and the streptavidin-horse radish peroxidase-conjugate (Streptavidin-HRP-Conjugate). A second washing is followed by the application of this conjugate. It binds to the biotin at the secondary antibody. Any excess of unbound streptavidin-HRPconjugate is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the horse radish peroxidase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen AEC (included only in kit MON-APP132) leads to the formation of a red-brown product of reaction at the place of the target antigen. The chromogen DAB (included only in kit MON-APP133) forms a dark brown precipitate.
Reagents provided:
125 ml Blocking Solution Reagent 1 (ready-to-use) 125 ml Biotinylated Secondary Antibody, Rabbit Reagent 2 (ready-to-use) 125 ml Streptavidin-HRP-Conjugate Reagent 3 (ready-to-use) Substrate systems recommended (if not included in the kit): Permanent AEC kit, AEC Single Solution, AEC substrate kit, DAB Substrate kit, DAB High Contrast kit Materials required but not supplied: Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O 3% H2O2 solution Reagents for enzyme digestion or heat pre-treatment Wash buffer Pink PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solutions should be stored at 2-8°C without fur ther dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. They should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support
Procedure:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. The tissue sections have to be completely covered with the different reagents in order to avoid drying out. Preparation of the chromogenic substrate AEC working solution (with MON-APP132 only): Add 2 drops (100 µl) of AEC Concentrate to one bottle of AEC Substrate Buffer and mix thoroughly. Preparation of the chromogenic substrate DAB working solution (with MON-APP133 only): Add 4 drops (200 µl) of DAB Concentrate to one bottle of DAB Substrate Buffer and mix thoroughly.
Expected results:
During the reaction of the substrate with horse radish peroxidase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support. No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pretreatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from rabbit. 6. The antigen was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pretreatment or enzyme digestion. If you used a pre-treatment it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. Incubation times were too short or primary antibody concentration too low. 7. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme conjugate or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous horse radish peroxidase. Maybe the hydrogen peroxide solution used for blocking was inactivated. 9. Non-specific binding of the secondary antibody to endogenous biotin in the tissue section. Carry out an avidin-biotin block before incubation
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous peroxidase or pseudoperoxidase activity or the endogenous biotin content may cause non-specific staining. The enzyme activity can be blocked by incubation with 3% H2O2 solution. Tissues containing Hepatitis B Surface Antigen (HBsAg) may give false positive results with HRP (horse radish peroxidase) detection systems (Omata et al, 1980). Background staining due to endogenous biotin can be blocked through an avidinbiotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the Blocking Solution instead of just draining it away as in other procedures. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear. ProClin 300 and sodium azide (NaN3) are used for stabilisation. Sodium azide deposits in drainage pipes made of lead or copper can result in the formation of highly explosive metallic azides. To avoid such deposits in drainage pipes, sodium azide should be discarded in a large volume of running water. Material safety data sheets (MSDS) are available upon request.
The Plus HRP Kit, Rabbit is based on the streptavidin-biotin system. It is designed for qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. The kit is developed for use in combination with mono- and polyclonal primary antibodies and sera obtained from rabbit. The Plus HRP Kit, Rabbit can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus HRP Kit, Rabbit is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The method is based on the streptavidin-biotin system which means that a biotinylated secondary antibody binds to several molecules of a conjugate composed of streptavidin and horse radish peroxidase. Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The biotinylated secondary antibody in the Plus HRP Kit, Rabbit binds to rabbit primary antibodies. Therefore this kit can detect mono- and polyclonal primary antibodies and sera obtained from rabbit.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Endogenous peroxidase activity in the tissue may cause non-specific staining. This enzyme activity can be blocked by incubation with 3% H2O2-solution (Peroxide Block). Background staining caused by unspecific binding of the primary or secondary antibody is minimized by incubation with a protein blocking solution (Blocking Solution). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the biotinylated secondary antibody is applied and incubated. This antibody functions as a link between primary antibody and the streptavidin-horse radish peroxidase-conjugate (Streptavidin-HRP-Conjugate). A second washing is followed by the application of this conjugate. It binds to the biotin at the secondary antibody. Any excess of unbound streptavidin-HRPconjugate is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the horse radish peroxidase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen AEC (included only in kit MON-APP132) leads to the formation of a red-brown product of reaction at the place of the target antigen. The chromogen DAB (included only in kit MON-APP133) forms a dark brown precipitate.
Reagents provided:
8 ml Peroxide Block (ready-to-use) 8 ml Blocking Solution Reagent 1 (ready-to-use) 8 ml Biotinylated Secondary Antibody, Rabbit Reagent 2 (ready-to-use) 8 ml Streptavidin-HRP-Conjugate Reagent 3 (ready-to-use) 7 x 5 ml DAB Substrate Buffer 3 ml DAB Concentrate (Chromogen) Substrate systems recommended (if not included in the kit): Permanent AEC kit, AEC Single Solution, AEC substrate kit, DAB Substrate kit, DAB High Contrast kit Materials required but not supplied: Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O 3% H2O2 solution Reagents for enzyme digestion or heat pre-treatment Wash buffer Pink PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solutions should be stored at 2-8°C without fur ther dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. They should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support
Procedure:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. The tissue sections have to be completely covered with the different reagents in order to avoid drying out. Preparation of the chromogenic substrate AEC working solution (with MON-APP132 only): Add 2 drops (100 µl) of AEC Concentrate to one bottle of AEC Substrate Buffer and mix thoroughly. Preparation of the chromogenic substrate DAB working solution (with MON-APP133 only): Add 4 drops (200 µl) of DAB Concentrate to one bottle of DAB Substrate Buffer and mix thoroughly.
Expected results:
During the reaction of the substrate with horse radish peroxidase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support. No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pretreatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from rabbit. 6. The antigen was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pretreatment or enzyme digestion. If you used a pre-treatment it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. Incubation times were too short or primary antibody concentration too low. 7. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme conjugate or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous horse radish peroxidase. Maybe the hydrogen peroxide solution used for blocking was inactivated. 9. Non-specific binding of the secondary antibody to endogenous biotin in the tissue section. Carry out an avidin-biotin block before incubation
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous peroxidase or pseudoperoxidase activity or the endogenous biotin content may cause non-specific staining. The enzyme activity can be blocked by incubation with 3% H2O2 solution. Tissues containing Hepatitis B Surface Antigen (HBsAg) may give false positive results with HRP (horse radish peroxidase) detection systems (Omata et al, 1980). Background staining due to endogenous biotin can be blocked through an avidinbiotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the Blocking Solution instead of just draining it away as in other procedures. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear. ProClin 300 and sodium azide (NaN3) are used for stabilisation. Sodium azide deposits in drainage pipes made of lead or copper can result in the formation of highly explosive metallic azides. To avoid such deposits in drainage pipes, sodium azide should be discarded in a large volume of running water. Material safety data sheets (MSDS) are available upon request.
The Plus HRP Kit, Rabbit is based on the streptavidin-biotin system. It is designed for qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. The kit is developed for use in combination with mono- and polyclonal primary antibodies and sera obtained from rabbit. The Plus HRP Kit, Rabbit can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus HRP Kit, Rabbit is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The method is based on the streptavidin-biotin system which means that a biotinylated secondary antibody binds to several molecules of a conjugate composed of streptavidin and horse radish peroxidase. Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The biotinylated secondary antibody in the Plus HRP Kit, Rabbit binds to rabbit primary antibodies. Therefore this kit can detect mono- and polyclonal primary antibodies and sera obtained from rabbit.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Endogenous peroxidase activity in the tissue may cause non-specific staining. This enzyme activity can be blocked by incubation with 3% H2O2-solution (Peroxide Block). Background staining caused by unspecific binding of the primary or secondary antibody is minimized by incubation with a protein blocking solution (Blocking Solution). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the biotinylated secondary antibody is applied and incubated. This antibody functions as a link between primary antibody and the streptavidin-horse radish peroxidase-conjugate (Streptavidin-HRP-Conjugate). A second washing is followed by the application of this conjugate. It binds to the biotin at the secondary antibody. Any excess of unbound streptavidin-HRPconjugate is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the horse radish peroxidase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen AEC (included only in kit MON-APP132) leads to the formation of a red-brown product of reaction at the place of the target antigen. The chromogen DAB (included only in kit MON-APP133) forms a dark brown precipitate.
Reagents provided:
8 ml Peroxide Block (ready-to-use) 8 ml Blocking Solution Reagent 1 (ready-to-use) 8 ml Biotinylated Secondary Antibody, Rabbit Reagent 2 (ready-to-use) 8 ml Streptavidin-HRP-Conjugate Reagent 3 (ready-to-use) 7 x 5 ml AEC Substrate Buffer 3 ml AEC Concentrate (Chromogen) Substrate systems recommended (if not included in the kit): Permanent AEC kit, AEC Single Solution, AEC substrate kit, DAB Substrate kit, DAB High Contrast kit Materials required but not supplied: Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O 3% H2O2 solution Reagents for enzyme digestion or heat pre-treatment Wash buffer Pink PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solutions should be stored at 2-8°C without fur ther dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. They should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support
Procedure:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. The tissue sections have to be completely covered with the different reagents in order to avoid drying out. Preparation of the chromogenic substrate AEC working solution (with MON-APP132 only): Add 2 drops (100 µl) of AEC Concentrate to one bottle of AEC Substrate Buffer and mix thoroughly. Preparation of the chromogenic substrate DAB working solution (with MON-APP133 only): Add 4 drops (200 µl) of DAB Concentrate to one bottle of DAB Substrate Buffer and mix thoroughly.
Expected results:
During the reaction of the substrate with horse radish peroxidase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support. No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pretreatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from rabbit. 6. The antigen was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pretreatment or enzyme digestion. If you used a pre-treatment it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. Incubation times were too short or primary antibody concentration too low. 7. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme conjugate or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous horse radish peroxidase. Maybe the hydrogen peroxide solution used for blocking was inactivated. 9. Non-specific binding of the secondary antibody to endogenous biotin in the tissue section. Carry out an avidin-biotin block before incubation
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous peroxidase or pseudoperoxidase activity or the endogenous biotin content may cause non-specific staining. The enzyme activity can be blocked by incubation with 3% H2O2 solution. Tissues containing Hepatitis B Surface Antigen (HBsAg) may give false positive results with HRP (horse radish peroxidase) detection systems (Omata et al, 1980). Background staining due to endogenous biotin can be blocked through an avidinbiotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the Blocking Solution instead of just draining it away as in other procedures. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear. ProClin 300 and sodium azide (NaN3) are used for stabilisation. Sodium azide deposits in drainage pipes made of lead or copper can result in the formation of highly explosive metallic azides. To avoid such deposits in drainage pipes, sodium azide should be discarded in a large volume of running water. Material safety data sheets (MSDS) are available upon request.
The Plus AP Kit, Rabbit is based on the streptavidin-biotin system. It is designed for the qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. The kit is developed for use in combination with mono- and polyclonal primary antibodies and sera obtained from rabbit. The Plus AP Kit, Rabbit can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus AP Kit, Rabbit is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The method based on the streptavidin-biotin system which means that a biotinylated secondary antibody binds to several molecules of a conjugate composed of streptavidin and alkaline phosphatase. Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The biotinylated secondary antibody in the Plus AP Kit, Rabbit binds to rabbit primary antibodies. Therefore this kit can detect mono- and polyclonal primary antibodies and sera obtained from rabbit.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Background staining caused by unspecific binding of the primary or secondary antibody is minimized by incubation with a protein blocking solution (Blocking Solution provided with the kit). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the biotinylated secondary antibody is applied and incubated. This antibody functions as a link between primary antibody and streptavidin-alkaline phosphatase-conjugate (Streptavidin-AP-Conjugate). A second washing is followed by the application of this conjugate. It binds to the biotin at the secondary antibody. Any excess of unbound streptavidin-AP-conjugate is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the alkaline phosphatase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen Permanent Red (included only in kit MON-APP128) leads to the formation of a magenta-red product of reaction at the place of the target antigen. Other suitable chromogens are Permanent AP Red (magenta-red) or NBT (blue-black) with its substrate BCIP.
Reagents provided:
4x 15 ml Blocking Solution Reagent 1 (ready-to-use) 4x 15 ml Biotinylated Secondary Antibody, Rabbit Reagent 2 (ready-to-use) 4x 15 ml Streptavidin-AP-Conjugate Reagent 3 (ready-to-use) Substrate systems recommended (if not included in the kit) Permanent AP Red kit, BCIP/NBT Materials required but not supplied: Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O Reagents for enzyme digestion or heat pre-treatment Wash buffer PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solutions should be stored at 2-8°C without further dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. They should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. Tissue sections have to be completely covered with the different reagents in order to avoid drying out. Preparation of the chromogenic substrate working solution (with MON-APP128 only): Add 2 drops (60 µl) of Permanent Red Concentrate to one bottle of Permanent Red Buffer (Substrate Buffer) and mix. This solution should be used directly after preparation.
Procedure:
1. Blocking Solution (protein block, Reagent 1) (This step is optional.) 5 min. 2. Washing with wash buffer 1 x 2 min. 3. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 4. Washing with wash buffer 3 x 2 min. 5. Biotinylated Secondary Antibody, Rabbit (Reagent 2, yellow) 10-15 min. 6. Washing with wash buffer 3 x 2 min. 7. Streptavidin-AP-Conjugate (Reagent 3, red) 10-15 min. 8. Washing with wash buffer 3 x 2 min. 9. Permanent Red substrate-chromogen solution (with AP008RED-RB) 5 min. 10. Wash with distilled H2O 1 min. 11. Permanent Red substrate-chromogen solution (with AP008RED-RB) 5 min. 12. Wash with destilled water 3 x 1 min. 13. Counterstaining and blueing 14. Mounting: aqueous or permanent after dehydration * The incubation times should be adjusted, when using other substrate-chromogen systems
Expected results:
During the reaction of the substrate with alkaline phosphatase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support. No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from rabbit, but from a different species. 6. The antigen/epitope was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. If you used heat pre-treatment or enzyme digestion it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. If you are using PBS-based wash buffer: the activity of alkaline phosphatase in the reagents is blocked if too much wash buffer remains on the slides. 7. Incubation times were too short or primary antibody concentration too low. 8. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme conjugate or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous alkaline phosphatase in the tissue. This undesired activity can often be suppressed using levamisole (see section Limitations of the Procedure). 9. Non-specific binding of the secondary antibody to endogenous biotin in the tissue section. Carry out an avidin-biotin block before incubation with the primary antibody.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific binding.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous alkaline phosphatase activity or the endogenous biotin content may cause non-specific staining. The enzyme activity can be blocked by incubation with levamisole. However, neither intestinal nor placental alkaline phosphatase can be blocked with levamisole. Background staining due to endogenous biotin can be blocked through an avidin-biotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the Blocking Solution instead of just draining it away as in other procedures. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. ProClin 300 and sodium azide (NaN3) are used for stabilisation. Sodium azide deposits in drainage pipes made of lead or copper can result in the formation of highly explosive metallic azides. To avoid such deposits in drainage pipes, sodium azide should be discarded in a large volume of running water. Material safety data sheets (MSDS) for the pure substances are available upon request. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear.
The Plus AP Kit, Rabbit is based on the streptavidin-biotin system. It is designed for the qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. The kit is developed for use in combination with mono- and polyclonal primary antibodies and sera obtained from rabbit. The Plus AP Kit, Rabbit can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus AP Kit, Rabbit is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The method based on the streptavidin-biotin system which means that a biotinylated secondary antibody binds to several molecules of a conjugate composed of streptavidin and alkaline phosphatase. Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The biotinylated secondary antibody in the Plus AP Kit, Rabbit binds to rabbit primary antibodies. Therefore this kit can detect mono- and polyclonal primary antibodies and sera obtained from rabbit.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Background staining caused by unspecific binding of the primary or secondary antibody is minimized by incubation with a protein blocking solution (Blocking Solution provided with the kit). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the biotinylated secondary antibody is applied and incubated. This antibody functions as a link between primary antibody and streptavidin-alkaline phosphatase-conjugate (Streptavidin-AP-Conjugate). A second washing is followed by the application of this conjugate. It binds to the biotin at the secondary antibody. Any excess of unbound streptavidin-AP-conjugate is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the alkaline phosphatase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen Permanent Red (included only in kit MON-APP128) leads to the formation of a magenta-red product of reaction at the place of the target antigen. Other suitable chromogens are Permanent AP Red (magenta-red) or NBT (blue-black) with its substrate BCIP.
Reagents provided:
500 ml Blocking Solution Reagent 1 (ready-to-use) 500 ml Biotinylated Secondary Antibody, Rabbit Reagent 2 (ready-to-use) 500 ml Streptavidin-AP-Conjugate Reagent 3 (ready-to-use) Substrate systems recommended (if not included in the kit) Permanent AP Red kit, BCIP/NBT Materials required but not supplied: Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O Reagents for enzyme digestion or heat pre-treatment Wash buffer PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solutions should be stored at 2-8°C without further dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. They should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. Tissue sections have to be completely covered with the different reagents in order to avoid drying out. Preparation of the chromogenic substrate working solution (with MON-APP128 only): Add 2 drops (60 µl) of Permanent Red Concentrate to one bottle of Permanent Red Buffer (Substrate Buffer) and mix. This solution should be used directly after preparation.
Procedure:
1. Blocking Solution (protein block, Reagent 1) (This step is optional.) 5 min. 2. Washing with wash buffer 1 x 2 min. 3. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 4. Washing with wash buffer 3 x 2 min. 5. Biotinylated Secondary Antibody, Rabbit (Reagent 2, yellow) 10-15 min. 6. Washing with wash buffer 3 x 2 min. 7. Streptavidin-AP-Conjugate (Reagent 3, red) 10-15 min. 8. Washing with wash buffer 3 x 2 min. 9. Permanent Red substrate-chromogen solution (with AP008RED-RB) 5 min. 10. Wash with distilled H2O 1 min. 11. Permanent Red substrate-chromogen solution (with AP008RED-RB) 5 min. 12. Wash with destilled water 3 x 1 min. 13. Counterstaining and blueing 14. Mounting: aqueous or permanent after dehydration * The incubation times should be adjusted, when using other substrate-chromogen systems
Expected results:
During the reaction of the substrate with alkaline phosphatase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support. No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from rabbit, but from a different species. 6. The antigen/epitope was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. If you used heat pre-treatment or enzyme digestion it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. If you are using PBS-based wash buffer: the activity of alkaline phosphatase in the reagents is blocked if too much wash buffer remains on the slides. 7. Incubation times were too short or primary antibody concentration too low. 8. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme conjugate or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous alkaline phosphatase in the tissue. This undesired activity can often be suppressed using levamisole (see section Limitations of the Procedure). 9. Non-specific binding of the secondary antibody to endogenous biotin in the tissue section. Carry out an avidin-biotin block before incubation with the primary antibody.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific binding.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous alkaline phosphatase activity or the endogenous biotin content may cause non-specific staining. The enzyme activity can be blocked by incubation with levamisole. However, neither intestinal nor placental alkaline phosphatase can be blocked with levamisole. Background staining due to endogenous biotin can be blocked through an avidin-biotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the Blocking Solution instead of just draining it away as in other procedures. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. ProClin 300 and sodium azide (NaN3) are used for stabilisation. Sodium azide deposits in drainage pipes made of lead or copper can result in the formation of highly explosive metallic azides. To avoid such deposits in drainage pipes, sodium azide should be discarded in a large volume of running water. Material safety data sheets (MSDS) for the pure substances are available upon request. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear.
The Plus AP Kit, Rabbit is based on the streptavidin-biotin system. It is designed for the qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. The kit is developed for use in combination with mono- and polyclonal primary antibodies and sera obtained from rabbit. The Plus AP Kit, Rabbit can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus AP Kit, Rabbit is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The method based on the streptavidin-biotin system which means that a biotinylated secondary antibody binds to several molecules of a conjugate composed of streptavidin and alkaline phosphatase. Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The biotinylated secondary antibody in the Plus AP Kit, Rabbit binds to rabbit primary antibodies. Therefore this kit can detect mono- and polyclonal primary antibodies and sera obtained from rabbit.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Background staining caused by unspecific binding of the primary or secondary antibody is minimized by incubation with a protein blocking solution (Blocking Solution provided with the kit). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the biotinylated secondary antibody is applied and incubated. This antibody functions as a link between primary antibody and streptavidin-alkaline phosphatase-conjugate (Streptavidin-AP-Conjugate). A second washing is followed by the application of this conjugate. It binds to the biotin at the secondary antibody. Any excess of unbound streptavidin-AP-conjugate is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the alkaline phosphatase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen Permanent Red (included only in kit MON-APP128) leads to the formation of a magenta-red product of reaction at the place of the target antigen. Other suitable chromogens are Permanent AP Red (magenta-red) or NBT (blue-black) with its substrate BCIP.
Reagents provided:
125 ml Blocking Solution Reagent 1 (ready-to-use) 125 ml Biotinylated Secondary Antibody, Rabbit Reagent 2 (ready-to-use) 125 ml Streptavidin-AP-Conjugate Reagent 3 (ready-to-use) Substrate systems recommended (if not included in the kit) Permanent AP Red kit, BCIP/NBT Materials required but not supplied: Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O Reagents for enzyme digestion or heat pre-treatment Wash buffer PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solutions should be stored at 2-8°C without further dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. They should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. Tissue sections have to be completely covered with the different reagents in order to avoid drying out. Preparation of the chromogenic substrate working solution (with MON-APP128 only): Add 2 drops (60 µl) of Permanent Red Concentrate to one bottle of Permanent Red Buffer (Substrate Buffer) and mix. This solution should be used directly after preparation.
Procedure:
1. Blocking Solution (protein block, Reagent 1) (This step is optional.) 5 min. 2. Washing with wash buffer 1 x 2 min. 3. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 4. Washing with wash buffer 3 x 2 min. 5. Biotinylated Secondary Antibody, Rabbit (Reagent 2, yellow) 10-15 min. 6. Washing with wash buffer 3 x 2 min. 7. Streptavidin-AP-Conjugate (Reagent 3, red) 10-15 min. 8. Washing with wash buffer 3 x 2 min. 9. Permanent Red substrate-chromogen solution (with AP008RED-RB) 5 min. 10. Wash with distilled H2O 1 min. 11. Permanent Red substrate-chromogen solution (with AP008RED-RB) 5 min. 12. Wash with destilled water 3 x 1 min. 13. Counterstaining and blueing 14. Mounting: aqueous or permanent after dehydration * The incubation times should be adjusted, when using other substrate-chromogen systems
Expected results:
During the reaction of the substrate with alkaline phosphatase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support. No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from rabbit, but from a different species. 6. The antigen/epitope was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. If you used heat pre-treatment or enzyme digestion it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. If you are using PBS-based wash buffer: the activity of alkaline phosphatase in the reagents is blocked if too much wash buffer remains on the slides. 7. Incubation times were too short or primary antibody concentration too low. 8. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme conjugate or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous alkaline phosphatase in the tissue. This undesired activity can often be suppressed using levamisole (see section Limitations of the Procedure). 9. Non-specific binding of the secondary antibody to endogenous biotin in the tissue section. Carry out an avidin-biotin block before incubation with the primary antibody.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific binding.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous alkaline phosphatase activity or the endogenous biotin content may cause non-specific staining. The enzyme activity can be blocked by incubation with levamisole. However, neither intestinal nor placental alkaline phosphatase can be blocked with levamisole. Background staining due to endogenous biotin can be blocked through an avidin-biotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the Blocking Solution instead of just draining it away as in other procedures. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. ProClin 300 and sodium azide (NaN3) are used for stabilisation. Sodium azide deposits in drainage pipes made of lead or copper can result in the formation of highly explosive metallic azides. To avoid such deposits in drainage pipes, sodium azide should be discarded in a large volume of running water. Material safety data sheets (MSDS) for the pure substances are available upon request. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear.
The Plus AP Kit, Rabbit is based on the streptavidin-biotin system. It is designed for the qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. The kit is developed for use in combination with mono- and polyclonal primary antibodies and sera obtained from rabbit. The Plus AP Kit, Rabbit can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus AP Kit, Rabbit is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The method based on the streptavidin-biotin system which means that a biotinylated secondary antibody binds to several molecules of a conjugate composed of streptavidin and alkaline phosphatase. Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The biotinylated secondary antibody in the Plus AP Kit, Rabbit binds to rabbit primary antibodies. Therefore this kit can detect mono- and polyclonal primary antibodies and sera obtained from rabbit.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Background staining caused by unspecific binding of the primary or secondary antibody is minimized by incubation with a protein blocking solution (Blocking Solution provided with the kit). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the biotinylated secondary antibody is applied and incubated. This antibody functions as a link between primary antibody and streptavidin-alkaline phosphatase-conjugate (Streptavidin-AP-Conjugate). A second washing is followed by the application of this conjugate. It binds to the biotin at the secondary antibody. Any excess of unbound streptavidin-AP-conjugate is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the alkaline phosphatase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen Permanent Red (included only in kit MON-APP128) leads to the formation of a magenta-red product of reaction at the place of the target antigen. Other suitable chromogens are Permanent AP Red (magenta-red) or NBT (blue-black) with its substrate BCIP.
Reagents provided:
8 ml Blocking Solution Reagent 1 (ready-to-use) 8 ml Biotinylated Secondary Antibody, Rabbit Reagent 2 (ready-to-use) 8 ml Streptavidin-AP-Conjugate Reagent 3 (ready-to-use) 8 x 5 ml Permanent Red Buffer (Substrate Buffer) 2 ml Permanent Red Concentrate (Chromogen) Substrate systems recommended (if not included in the kit) Permanent AP Red kit, BCIP/NBT Materials required but not supplied: Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O Reagents for enzyme digestion or heat pre-treatment Wash buffer PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solutions should be stored at 2-8°C without further dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. They should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. Tissue sections have to be completely covered with the different reagents in order to avoid drying out. Preparation of the chromogenic substrate working solution (with MON-APP128 only): Add 2 drops (60 µl) of Permanent Red Concentrate to one bottle of Permanent Red Buffer (Substrate Buffer) and mix. This solution should be used directly after preparation.
Procedure:
1. Blocking Solution (protein block, Reagent 1) (This step is optional.) 5 min. 2. Washing with wash buffer 1 x 2 min. 3. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 4. Washing with wash buffer 3 x 2 min. 5. Biotinylated Secondary Antibody, Rabbit (Reagent 2, yellow) 10-15 min. 6. Washing with wash buffer 3 x 2 min. 7. Streptavidin-AP-Conjugate (Reagent 3, red) 10-15 min. 8. Washing with wash buffer 3 x 2 min. 9. Permanent Red substrate-chromogen solution (with AP008RED-RB) 5 min. 10. Wash with distilled H2O 1 min. 11. Permanent Red substrate-chromogen solution (with AP008RED-RB) 5 min. 12. Wash with destilled water 3 x 1 min. 13. Counterstaining and blueing 14. Mounting: aqueous or permanent after dehydration * The incubation times should be adjusted, when using other substrate-chromogen systems
Expected results:
During the reaction of the substrate with alkaline phosphatase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support. No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from rabbit, but from a different species. 6. The antigen/epitope was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. If you used heat pre-treatment or enzyme digestion it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. If you are using PBS-based wash buffer: the activity of alkaline phosphatase in the reagents is blocked if too much wash buffer remains on the slides. 7. Incubation times were too short or primary antibody concentration too low. 8. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme conjugate or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous alkaline phosphatase in the tissue. This undesired activity can often be suppressed using levamisole (see section Limitations of the Procedure). 9. Non-specific binding of the secondary antibody to endogenous biotin in the tissue section. Carry out an avidin-biotin block before incubation with the primary antibody.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific binding.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous alkaline phosphatase activity or the endogenous biotin content may cause non-specific staining. The enzyme activity can be blocked by incubation with levamisole. However, neither intestinal nor placental alkaline phosphatase can be blocked with levamisole. Background staining due to endogenous biotin can be blocked through an avidin-biotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the Blocking Solution instead of just draining it away as in other procedures. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. ProClin 300 and sodium azide (NaN3) are used for stabilisation. Sodium azide deposits in drainage pipes made of lead or copper can result in the formation of highly explosive metallic azides. To avoid such deposits in drainage pipes, sodium azide should be discarded in a large volume of running water. Material safety data sheets (MSDS) for the pure substances are available upon request. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear.
The Plus HRP Kits, Mouse is based on the streptavidin-biotin system. It is designed for qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. The kit is developed for use in combination with monoclonal primary antibodies and sera obtained from mice. The Plus HRP Kit, Mouse can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus HRP Kit, Mouse is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The method is based on the streptavidin-biotin system which means that a biotinylated secondary antibody binds to several molecules of a conjugate composed of streptavidin and horse radish peroxidase. Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The biotinylated secondary antibody in the Plus HRP Kit, Mouse binds to mouse primary antibodies. Therefore this kit can detect monoclonal primary antibodies and sera obtained from mice.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Endogenous peroxidase activity in the tissue may cause non-specific staining. This enzyme activity can be blocked by incubation with 3% H2O2-solution (Peroxide Block). Background staining caused by unspecific binding of the primary or secondary antibody is minimized by incubation with a protein blocking solution (Blocking Solution). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the biotinylated secondary antibody is applied and incubated. This secondary antibody functions as a link between primary antibody and the streptavidin-horse radish peroxidase-conjugate (Streptavidin-HRP-Conjugate). A second washing is followed by the application of this conjugate. It binds to the biotin at the secondary antibody. Any excess of unbound streptavidin-HRP-conjugate is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the horse radish peroxidase which leads to colour precipitation where the primary antibody is bound. The colour can be observed via a light microscope. The chromogen used determines the colour. The chromogen AEC (included only in kit MON-APP123) leads to the formation of a red-brown product of reaction at the place of the target antigen. The chromogen DAB (included only in kit MON-APP124) forms a dark brown precipitate.
Reagents provided:
4x 15 ml Blocking Solution Reagent 1 (ready-to-use) 4x 15 ml Biotinylated Secondary Antibody, Mouse Reagent 2 (ready-to-use) 4x 15 ml Streptavidin-HRP-Conjugate Reagent 3 (ready-to-use) Substrate systems recommended (if not included in the kit): Permanent AEC kit, AEC single solution, AEC substrate kit, DAB substrate kit, DAB high contrast kit. Materials required but not supplied Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O 3% H2O2 solution Reagents for enzyme digestion or heat pre-treatment Wash buffer Pink PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solutions should be stored at 2-8°C without further dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. They should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. The tissue sections have to be completely covered with the different reagents in order to avoid drying out. Preparation of the chromogenic substrate AEC working solution (with MON-APP123 only): Add 2 drops (100 µl) of AEC Concentrate to one bottle of AEC Substrate Buffer and mix thoroughly. Preparation of the chromogenic substrate DAB working solution (with MON-APP124 only): Add 4 drops (200 µl) of DAB Concentrate to one bottle of DAB Substrate Buffer and mix thoroughly
Procedure:
1. Peroxide Block (3% H2O2 solution) 10 min. 2. Washing with wash buffer 1 x 2 min. 3. Blocking Solution (protein block, Reagent 1) (This step is optional.) 5 min. 4. Washing with wash buffer 1 x 2 min. 5. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 6. Washing with wash buffer 3 x 2 min. 7. Biotinylated Secondary Antibody, Mouse (Reagent 2, yellow) 10-15 min. 8. Washing with wash buffer 3 x 2 min. 9. Streptavidin-HRP-Conjugate (Reagent 3, red) 10-15 min. 10. Washing with wash buffer 3 x 2 min. 11. AEC or DAB (Controlling the colour intensity via light microscope is recommended.) 5-15 min. 12. Stopping the reaction with distilled H2O when the desired colour intensity is attained 13. Counterstaining and blueing 14. Mounting: aqueous with AEC, permanent with DAB or Permanent AEC
Expected results:
During the reaction of the substrate with horse radish peroxidase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support. No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pretreatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from mouse. 6. The antigen was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pretreatment or enzyme digestion. If you used a pre-treatment it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. Incubation times were too short or primary antibody concentration too low. 7. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme conjugate or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous horse radish peroxidase. Maybe the hydrogen peroxide solution used for blocking was inactivated. 9. Non-specific binding of the secondary antibody to endogenous biotin in the tissue section. Carry out an avidin-biotin block before incubation with the primary antibody.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous peroxidase or pseudoperoxidase activity or the endogenous biotin content may cause non-specific staining. The enzyme activity can be blocked by incubation with 3% H2O2 solution. Tissues containing Hepatitis B Surface Antigen (HBsAg) may give false positive results with HRP (horse radish peroxidase) detection systems (Omata et al, 1980). Background staining due to endogenous biotin can be blocked through an avidinbiotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the Blocking Solution instead of just draining it away as in other procedures. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear. ProClin 300 and sodium azide (NaN3) are used for stabilisation. Sodium azide deposits in drainage pipes made of lead or copper can result in the formation of highly explosive metallic azides. To avoid such deposits in drainage pipes, sodium azide should be discarded in a large volume of running water. Material safety data sheets (MSDS) are available upon request.
The Plus HRP Kits, Mouse is based on the streptavidin-biotin system. It is designed for qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. The kit is developed for use in combination with monoclonal primary antibodies and sera obtained from mice. The Plus HRP Kit, Mouse can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus HRP Kit, Mouse is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The method is based on the streptavidin-biotin system which means that a biotinylated secondary antibody binds to several molecules of a conjugate composed of streptavidin and horse radish peroxidase. Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The biotinylated secondary antibody in the Plus HRP Kit, Mouse binds to mouse primary antibodies. Therefore this kit can detect monoclonal primary antibodies and sera obtained from mice.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Endogenous peroxidase activity in the tissue may cause non-specific staining. This enzyme activity can be blocked by incubation with 3% H2O2-solution (Peroxide Block). Background staining caused by unspecific binding of the primary or secondary antibody is minimized by incubation with a protein blocking solution (Blocking Solution). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the biotinylated secondary antibody is applied and incubated. This secondary antibody functions as a link between primary antibody and the streptavidin-horse radish peroxidase-conjugate (Streptavidin-HRP-Conjugate). A second washing is followed by the application of this conjugate. It binds to the biotin at the secondary antibody. Any excess of unbound streptavidin-HRP-conjugate is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the horse radish peroxidase which leads to colour precipitation where the primary antibody is bound. The colour can be observed via a light microscope. The chromogen used determines the colour. The chromogen AEC (included only in kit MON-APP123) leads to the formation of a red-brown product of reaction at the place of the target antigen. The chromogen DAB (included only in kit MON-APP124) forms a dark brown precipitate.
Reagents provided:
500 ml Blocking Solution Reagent 1 (ready-to-use) 500 ml Biotinylated Secondary Antibody, Mouse Reagent 2 (ready-to-use) 500 ml Streptavidin-HRP-Conjugate Reagent 3 (ready-to-use) Substrate systems recommended (if not included in the kit): Permanent AEC kit, AEC single solution, AEC substrate kit, DAB substrate kit, DAB high contrast kit. Materials required but not supplied Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O 3% H2O2 solution Reagents for enzyme digestion or heat pre-treatment Wash buffer Pink PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solutions should be stored at 2-8°C without further dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. They should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. The tissue sections have to be completely covered with the different reagents in order to avoid drying out. Preparation of the chromogenic substrate AEC working solution (with MON-APP123 only): Add 2 drops (100 µl) of AEC Concentrate to one bottle of AEC Substrate Buffer and mix thoroughly. Preparation of the chromogenic substrate DAB working solution (with MON-APP124 only): Add 4 drops (200 µl) of DAB Concentrate to one bottle of DAB Substrate Buffer and mix thoroughly
Procedure:
1. Peroxide Block (3% H2O2 solution) 10 min. 2. Washing with wash buffer 1 x 2 min. 3. Blocking Solution (protein block, Reagent 1) (This step is optional.) 5 min. 4. Washing with wash buffer 1 x 2 min. 5. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 6. Washing with wash buffer 3 x 2 min. 7. Biotinylated Secondary Antibody, Mouse (Reagent 2, yellow) 10-15 min. 8. Washing with wash buffer 3 x 2 min. 9. Streptavidin-HRP-Conjugate (Reagent 3, red) 10-15 min. 10. Washing with wash buffer 3 x 2 min. 11. AEC or DAB (Controlling the colour intensity via light microscope is recommended.) 5-15 min. 12. Stopping the reaction with distilled H2O when the desired colour intensity is attained 13. Counterstaining and blueing 14. Mounting: aqueous with AEC, permanent with DAB or Permanent AEC
Expected results:
During the reaction of the substrate with horse radish peroxidase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support. No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pretreatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from mouse. 6. The antigen was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pretreatment or enzyme digestion. If you used a pre-treatment it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. Incubation times were too short or primary antibody concentration too low. 7. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme conjugate or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous horse radish peroxidase. Maybe the hydrogen peroxide solution used for blocking was inactivated. 9. Non-specific binding of the secondary antibody to endogenous biotin in the tissue section. Carry out an avidin-biotin block before incubation with the primary antibody.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous peroxidase or pseudoperoxidase activity or the endogenous biotin content may cause non-specific staining. The enzyme activity can be blocked by incubation with 3% H2O2 solution. Tissues containing Hepatitis B Surface Antigen (HBsAg) may give false positive results with HRP (horse radish peroxidase) detection systems (Omata et al, 1980). Background staining due to endogenous biotin can be blocked through an avidinbiotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the Blocking Solution instead of just draining it away as in other procedures. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear. ProClin 300 and sodium azide (NaN3) are used for stabilisation. Sodium azide deposits in drainage pipes made of lead or copper can result in the formation of highly explosive metallic azides. To avoid such deposits in drainage pipes, sodium azide should be discarded in a large volume of running water. Material safety data sheets (MSDS) are available upon request.
The Plus HRP Kits, Mouse is based on the streptavidin-biotin system. It is designed for qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. The kit is developed for use in combination with monoclonal primary antibodies and sera obtained from mice. The Plus HRP Kit, Mouse can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus HRP Kit, Mouse is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The method is based on the streptavidin-biotin system which means that a biotinylated secondary antibody binds to several molecules of a conjugate composed of streptavidin and horse radish peroxidase. Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The biotinylated secondary antibody in the Plus HRP Kit, Mouse binds to mouse primary antibodies. Therefore this kit can detect monoclonal primary antibodies and sera obtained from mice.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Endogenous peroxidase activity in the tissue may cause non-specific staining. This enzyme activity can be blocked by incubation with 3% H2O2-solution (Peroxide Block). Background staining caused by unspecific binding of the primary or secondary antibody is minimized by incubation with a protein blocking solution (Blocking Solution). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the biotinylated secondary antibody is applied and incubated. This secondary antibody functions as a link between primary antibody and the streptavidin-horse radish peroxidase-conjugate (Streptavidin-HRP-Conjugate). A second washing is followed by the application of this conjugate. It binds to the biotin at the secondary antibody. Any excess of unbound streptavidin-HRP-conjugate is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the horse radish peroxidase which leads to colour precipitation where the primary antibody is bound. The colour can be observed via a light microscope. The chromogen used determines the colour. The chromogen AEC (included only in kit MON-APP123) leads to the formation of a red-brown product of reaction at the place of the target antigen. The chromogen DAB (included only in kit MON-APP124) forms a dark brown precipitate.
Reagents provided:
125 ml Blocking Solution Reagent 1 (ready-to-use) 125 ml Biotinylated Secondary Antibody, Mouse Reagent 2 (ready-to-use) 125 ml Streptavidin-HRP-Conjugate Reagent 3 (ready-to-use) Substrate systems recommended (if not included in the kit): Permanent AEC kit, AEC single solution, AEC substrate kit, DAB substrate kit, DAB high contrast kit. Materials required but not supplied Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O 3% H2O2 solution Reagents for enzyme digestion or heat pre-treatment Wash buffer Pink PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solutions should be stored at 2-8°C without further dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. They should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. The tissue sections have to be completely covered with the different reagents in order to avoid drying out. Preparation of the chromogenic substrate AEC working solution (with MON-APP123 only): Add 2 drops (100 µl) of AEC Concentrate to one bottle of AEC Substrate Buffer and mix thoroughly. Preparation of the chromogenic substrate DAB working solution (with MON-APP124 only): Add 4 drops (200 µl) of DAB Concentrate to one bottle of DAB Substrate Buffer and mix thoroughly
Procedure:
1. Peroxide Block (3% H2O2 solution) 10 min. 2. Washing with wash buffer 1 x 2 min. 3. Blocking Solution (protein block, Reagent 1) (This step is optional.) 5 min. 4. Washing with wash buffer 1 x 2 min. 5. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 6. Washing with wash buffer 3 x 2 min. 7. Biotinylated Secondary Antibody, Mouse (Reagent 2, yellow) 10-15 min. 8. Washing with wash buffer 3 x 2 min. 9. Streptavidin-HRP-Conjugate (Reagent 3, red) 10-15 min. 10. Washing with wash buffer 3 x 2 min. 11. AEC or DAB (Controlling the colour intensity via light microscope is recommended.) 5-15 min. 12. Stopping the reaction with distilled H2O when the desired colour intensity is attained 13. Counterstaining and blueing 14. Mounting: aqueous with AEC, permanent with DAB or Permanent AEC
Expected results:
During the reaction of the substrate with horse radish peroxidase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support. No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pretreatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from mouse. 6. The antigen was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pretreatment or enzyme digestion. If you used a pre-treatment it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. Incubation times were too short or primary antibody concentration too low. 7. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme conjugate or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous horse radish peroxidase. Maybe the hydrogen peroxide solution used for blocking was inactivated. 9. Non-specific binding of the secondary antibody to endogenous biotin in the tissue section. Carry out an avidin-biotin block before incubation with the primary antibody.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous peroxidase or pseudoperoxidase activity or the endogenous biotin content may cause non-specific staining. The enzyme activity can be blocked by incubation with 3% H2O2 solution. Tissues containing Hepatitis B Surface Antigen (HBsAg) may give false positive results with HRP (horse radish peroxidase) detection systems (Omata et al, 1980). Background staining due to endogenous biotin can be blocked through an avidinbiotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the Blocking Solution instead of just draining it away as in other procedures. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear. ProClin 300 and sodium azide (NaN3) are used for stabilisation. Sodium azide deposits in drainage pipes made of lead or copper can result in the formation of highly explosive metallic azides. To avoid such deposits in drainage pipes, sodium azide should be discarded in a large volume of running water. Material safety data sheets (MSDS) are available upon request.
The Plus HRP Kits, Mouse is based on the streptavidin-biotin system. It is designed for qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. The kit is developed for use in combination with monoclonal primary antibodies and sera obtained from mice. The Plus HRP Kit, Mouse can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus HRP Kit, Mouse is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The method is based on the streptavidin-biotin system which means that a biotinylated secondary antibody binds to several molecules of a conjugate composed of streptavidin and horse radish peroxidase. Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The biotinylated secondary antibody in the Plus HRP Kit, Mouse binds to mouse primary antibodies. Therefore this kit can detect monoclonal primary antibodies and sera obtained from mice.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Endogenous peroxidase activity in the tissue may cause non-specific staining. This enzyme activity can be blocked by incubation with 3% H2O2-solution (Peroxide Block). Background staining caused by unspecific binding of the primary or secondary antibody is minimized by incubation with a protein blocking solution (Blocking Solution). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the biotinylated secondary antibody is applied and incubated. This secondary antibody functions as a link between primary antibody and the streptavidin-horse radish peroxidase-conjugate (Streptavidin-HRP-Conjugate). A second washing is followed by the application of this conjugate. It binds to the biotin at the secondary antibody. Any excess of unbound streptavidin-HRP-conjugate is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the horse radish peroxidase which leads to colour precipitation where the primary antibody is bound. The colour can be observed via a light microscope. The chromogen used determines the colour. The chromogen AEC (included only in kit MON-APP123) leads to the formation of a red-brown product of reaction at the place of the target antigen. The chromogen DAB (included only in kit MON-APP124) forms a dark brown precipitate.
Reagents provided:
8 ml Peroxide Block (ready-to-use) 8 ml Blocking Solution Reagent 1 (ready-to-use) 8 ml Biotinylated Secondary Antibody, Mouse Reagent 2 (ready-to-use) 8 ml Streptavidin-HRP-Conjugate Reagent 3 (ready-to-use) 7 x 5 ml DAB Substrate Buffer 3 ml DAB Concentrate (Chromogen) Substrate systems recommended (if not included in the kit): Permanent AEC kit, AEC single solution, AEC substrate kit, DAB substrate kit, DAB high contrast kit. Materials required but not supplied Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O 3% H2O2 solution Reagents for enzyme digestion or heat pre-treatment Wash buffer Pink PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solutions should be stored at 2-8°C without further dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. They should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. The tissue sections have to be completely covered with the different reagents in order to avoid drying out. Preparation of the chromogenic substrate AEC working solution (with MON-APP123 only): Add 2 drops (100 µl) of AEC Concentrate to one bottle of AEC Substrate Buffer and mix thoroughly. Preparation of the chromogenic substrate DAB working solution (with MON-APP124 only): Add 4 drops (200 µl) of DAB Concentrate to one bottle of DAB Substrate Buffer and mix thoroughly
Procedure:
1. Peroxide Block (3% H2O2 solution) 10 min. 2. Washing with wash buffer 1 x 2 min. 3. Blocking Solution (protein block, Reagent 1) (This step is optional.) 5 min. 4. Washing with wash buffer 1 x 2 min. 5. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 6. Washing with wash buffer 3 x 2 min. 7. Biotinylated Secondary Antibody, Mouse (Reagent 2, yellow) 10-15 min. 8. Washing with wash buffer 3 x 2 min. 9. Streptavidin-HRP-Conjugate (Reagent 3, red) 10-15 min. 10. Washing with wash buffer 3 x 2 min. 11. AEC or DAB (Controlling the colour intensity via light microscope is recommended.) 5-15 min. 12. Stopping the reaction with distilled H2O when the desired colour intensity is attained 13. Counterstaining and blueing 14. Mounting: aqueous with AEC, permanent with DAB or Permanent AEC
Expected results:
During the reaction of the substrate with horse radish peroxidase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support. No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pretreatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from mouse. 6. The antigen was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pretreatment or enzyme digestion. If you used a pre-treatment it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. Incubation times were too short or primary antibody concentration too low. 7. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme conjugate or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous horse radish peroxidase. Maybe the hydrogen peroxide solution used for blocking was inactivated. 9. Non-specific binding of the secondary antibody to endogenous biotin in the tissue section. Carry out an avidin-biotin block before incubation with the primary antibody.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous peroxidase or pseudoperoxidase activity or the endogenous biotin content may cause non-specific staining. The enzyme activity can be blocked by incubation with 3% H2O2 solution. Tissues containing Hepatitis B Surface Antigen (HBsAg) may give false positive results with HRP (horse radish peroxidase) detection systems (Omata et al, 1980). Background staining due to endogenous biotin can be blocked through an avidinbiotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the Blocking Solution instead of just draining it away as in other procedures. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear. ProClin 300 and sodium azide (NaN3) are used for stabilisation. Sodium azide deposits in drainage pipes made of lead or copper can result in the formation of highly explosive metallic azides. To avoid such deposits in drainage pipes, sodium azide should be discarded in a large volume of running water. Material safety data sheets (MSDS) are available upon request.
The Plus HRP Kits, Mouse is based on the streptavidin-biotin system. It is designed for qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. The kit is developed for use in combination with monoclonal primary antibodies and sera obtained from mice. The Plus HRP Kit, Mouse can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus HRP Kit, Mouse is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The method is based on the streptavidin-biotin system which means that a biotinylated secondary antibody binds to several molecules of a conjugate composed of streptavidin and horse radish peroxidase. Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The biotinylated secondary antibody in the Plus HRP Kit, Mouse binds to mouse primary antibodies. Therefore this kit can detect monoclonal primary antibodies and sera obtained from mice.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Endogenous peroxidase activity in the tissue may cause non-specific staining. This enzyme activity can be blocked by incubation with 3% H2O2-solution (Peroxide Block). Background staining caused by unspecific binding of the primary or secondary antibody is minimized by incubation with a protein blocking solution (Blocking Solution). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the biotinylated secondary antibody is applied and incubated. This secondary antibody functions as a link between primary antibody and the streptavidin-horse radish peroxidase-conjugate (Streptavidin-HRP-Conjugate). A second washing is followed by the application of this conjugate. It binds to the biotin at the secondary antibody. Any excess of unbound streptavidin-HRP-conjugate is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the horse radish peroxidase which leads to colour precipitation where the primary antibody is bound. The colour can be observed via a light microscope. The chromogen used determines the colour. The chromogen AEC (included only in kit MON-APP123) leads to the formation of a red-brown product of reaction at the place of the target antigen. The chromogen DAB (included only in kit MON-APP124) forms a dark brown precipitate.
Reagents provided:
8 ml Peroxide Block (ready-to-use) 8 ml Blocking Solution Reagent 1 (ready-to-use) 8 ml Biotinylated Secondary Antibody, Mouse Reagent 2 (ready-to-use) 8 ml Streptavidin-HRP-Conjugate Reagent 3 (ready-to-use) 7 x 5 ml AEC Substrate Buffer 3 ml AEC Concentrate (Chromogen) Substrate systems recommended (if not included in the kit): Permanent AEC kit, AEC single solution, AEC substrate kit, DAB substrate kit, DAB high contrast kit. Materials required but not supplied Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O 3% H2O2 solution Reagents for enzyme digestion or heat pre-treatment Wash buffer Pink PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solutions should be stored at 2-8°C without further dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. They should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. The tissue sections have to be completely covered with the different reagents in order to avoid drying out. Preparation of the chromogenic substrate AEC working solution (with MON-APP123 only): Add 2 drops (100 µl) of AEC Concentrate to one bottle of AEC Substrate Buffer and mix thoroughly. Preparation of the chromogenic substrate DAB working solution (with MON-APP124 only): Add 4 drops (200 µl) of DAB Concentrate to one bottle of DAB Substrate Buffer and mix thoroughly
Procedure:
1. Peroxide Block (3% H2O2 solution) 10 min. 2. Washing with wash buffer 1 x 2 min. 3. Blocking Solution (protein block, Reagent 1) (This step is optional.) 5 min. 4. Washing with wash buffer 1 x 2 min. 5. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 6. Washing with wash buffer 3 x 2 min. 7. Biotinylated Secondary Antibody, Mouse (Reagent 2, yellow) 10-15 min. 8. Washing with wash buffer 3 x 2 min. 9. Streptavidin-HRP-Conjugate (Reagent 3, red) 10-15 min. 10. Washing with wash buffer 3 x 2 min. 11. AEC or DAB (Controlling the colour intensity via light microscope is recommended.) 5-15 min. 12. Stopping the reaction with distilled H2O when the desired colour intensity is attained 13. Counterstaining and blueing 14. Mounting: aqueous with AEC, permanent with DAB or Permanent AEC
Expected results:
During the reaction of the substrate with horse radish peroxidase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support. No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pretreatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from mouse. 6. The antigen was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pretreatment or enzyme digestion. If you used a pre-treatment it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. Incubation times were too short or primary antibody concentration too low. 7. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme conjugate or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous horse radish peroxidase. Maybe the hydrogen peroxide solution used for blocking was inactivated. 9. Non-specific binding of the secondary antibody to endogenous biotin in the tissue section. Carry out an avidin-biotin block before incubation with the primary antibody.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous peroxidase or pseudoperoxidase activity or the endogenous biotin content may cause non-specific staining. The enzyme activity can be blocked by incubation with 3% H2O2 solution. Tissues containing Hepatitis B Surface Antigen (HBsAg) may give false positive results with HRP (horse radish peroxidase) detection systems (Omata et al, 1980). Background staining due to endogenous biotin can be blocked through an avidinbiotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the Blocking Solution instead of just draining it away as in other procedures. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear. ProClin 300 and sodium azide (NaN3) are used for stabilisation. Sodium azide deposits in drainage pipes made of lead or copper can result in the formation of highly explosive metallic azides. To avoid such deposits in drainage pipes, sodium azide should be discarded in a large volume of running water. Material safety data sheets (MSDS) are available upon request.
The Plus AP Kit, Mouse is based on the streptavidin-biotin system. It is designed for the qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. The kit is developed for use in combination with monoclonal primary antibodies and sera obtained from mice. The Plus AP Kit, Mouse can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of immunohistochemical staining is to make tissue and cell antigens visible. The Plus AP Kit, Mouse is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The method is based on the streptavidin-biotin system which means that a biotinylated secondary antibody binds to several molecules of a conjugate composed of streptavidin and alkaline phosphatase. Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The biotinylated secondary antibody in the Plus AP Kit, Mouse binds to mouse primary antibodies. Therefore this kit can detect monoclonal primary antibodies and sera obtained from mice.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Background staining caused by unspecific binding of the primary or secondary antibody is minimized by incubation with a protein blocking solution (Blocking Solution provided with the kit). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the biotinylated secondary antibody is applied and incubated. This secondary antibody functions as a link between primary antibody and the streptavidinalkaline phosphatase-conjugate (Streptavidin-AP-Conjugate). A second washing is followed by the application of this conjugate. It binds to the biotin at the secondary antibody. Any excess of unbound streptavidin-AP-conjugate is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the alkaline phosphatase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen Permanent Red (included only in kit MON-APP119) leads to a magenta-red product of reaction at the place of the target antigen. Other suitable chromogens are Permanent AP Red (magenta-red) or NBT (blue-black) with its substrate BCIP.
Reagents provided:
4x 15 ml Blocking Solution Reagent 1 (ready-to-use) 4x 15 ml Biotinylated Secondary Antibody, Mouse Reagent 2 (ready-to-use) 4x 15 ml Streptavidin-AP-Conjugate Reagent 3 (ready-to-use) Substrate systems recommended (if not included in the kit): Permanent AP Red Kit, BCIP/NBT Materials required but not supplied Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O Reagents for enzyme digestion or heat pre-treatment Wash buffer PBS or TBS PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solutions should be stored at 2-8°C without further dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. They should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. Tissue sections have to be completely covered with the different reagents in order to avoid drying out. Preparation of the chromogenic substrate working solution (with MON-APP119 only): Add 2 drops (60 µl) of Permanent Red Concentrate to one bottle of Permanent Red Buffer (Substrate Buffer) and mix. This solution should be used directly after preparation.
Procedure:
1. Blocking Solution (protein block, Reagent 1) (This step is optional.) 5 min. 2. Washing with wash buffer 1 x 2 min. 3. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 4. Washing with wash buffer 3 x 2 min. 5. Biotinylated Secondary Antibody, Mouse (Reagent 2, yellow) 10-15 min. 6. Washing with wash buffer 3 x 2 min. 7. Streptavidin-AP-Conjugate (Reagent 3, red) 10-15 min. 8. Washing with wash buffer 3 x 2 min. 9. Permanent Red substrate-chromogen solution (with MON-APP119) 5 min. 10. Wash with distilled H2O 1 min. 11. Permanent Red substrate-chromogen solution (with MON-APP119) 5 min. 12. Wash with destilled water 3 x 1 min. 13. Counterstaining and blueing 14. Mounting: aqueous or permanent after dehydration * The incubation times should be adjusted, when using other substrate-chromogen systems.
Expected results:
During the reaction of the substrate with alkaline phosphatase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support . No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from mouse, but from a different species. 6. The antigen/epitope was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. If you used heat pre-treatment or enzyme digestion it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. If you are using PBS-based wash buffer: the activity of alkaline phosphatase in the reagents is blocked if too much wash buffer remains on the slides. 7. Incubation times were too short or primary antibody concentration too low. 8. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme conjugate or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous alkaline phosphatase in the tissue. This undesired activity can often be suppressed using levamisole (see section Limitations of the Procedure). 9. Non-specific binding of the secondary antibody to endogenous biotin in the tissue section. Carry out an avidin-biotin block before incubation with the primary antibody.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous alkaline phosphatase activity or the endogenous biotin content may cause non-specific staining. The enzyme activity can be blocked by incubation with levamisole. However, neither intestinal nor placental alkaline phosphatase can be blocked with levamisole. Background staining due to endogenous biotin can be blocked through an avidin-biotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the Blocking Solution instead of just draining it away as in other procedures. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. ProClin 300 and sodium azide (NaN3) are used for stabilisation. Sodium azide deposits in drainage pipes made of lead or copper can result in the formation of highly explosive metallic azides. To avoid such deposits in drainage pipes, sodium azide should be discarded in a large volume of running water. Material safety data sheets (MSDS) for the pure substances are available upon request. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear
The Plus AP Kit, Mouse is based on the streptavidin-biotin system. It is designed for the qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. The kit is developed for use in combination with monoclonal primary antibodies and sera obtained from mice. The Plus AP Kit, Mouse can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of immunohistochemical staining is to make tissue and cell antigens visible. The Plus AP Kit, Mouse is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The method is based on the streptavidin-biotin system which means that a biotinylated secondary antibody binds to several molecules of a conjugate composed of streptavidin and alkaline phosphatase. Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The biotinylated secondary antibody in the Plus AP Kit, Mouse binds to mouse primary antibodies. Therefore this kit can detect monoclonal primary antibodies and sera obtained from mice.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Background staining caused by unspecific binding of the primary or secondary antibody is minimized by incubation with a protein blocking solution (Blocking Solution provided with the kit). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the biotinylated secondary antibody is applied and incubated. This secondary antibody functions as a link between primary antibody and the streptavidinalkaline phosphatase-conjugate (Streptavidin-AP-Conjugate). A second washing is followed by the application of this conjugate. It binds to the biotin at the secondary antibody. Any excess of unbound streptavidin-AP-conjugate is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the alkaline phosphatase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen Permanent Red (included only in kit MON-APP119) leads to a magenta-red product of reaction at the place of the target antigen. Other suitable chromogens are Permanent AP Red (magenta-red) or NBT (blue-black) with its substrate BCIP.
Reagents provided:
500 ml Blocking Solution Reagent 1 (ready-to-use) 500 ml Biotinylated Secondary Antibody, Mouse Reagent 2 (ready-to-use) 500 ml Streptavidin-AP-Conjugate Reagent 3 (ready-to-use) Substrate systems recommended (if not included in the kit): Permanent AP Red Kit, BCIP/NBT Materials required but not supplied Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O Reagents for enzyme digestion or heat pre-treatment Wash buffer PBS or TBS PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solutions should be stored at 2-8°C without further dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. They should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. Tissue sections have to be completely covered with the different reagents in order to avoid drying out. Preparation of the chromogenic substrate working solution (with MON-APP119 only): Add 2 drops (60 µl) of Permanent Red Concentrate to one bottle of Permanent Red Buffer (Substrate Buffer) and mix. This solution should be used directly after preparation.
Procedure:
1. Blocking Solution (protein block, Reagent 1) (This step is optional.) 5 min. 2. Washing with wash buffer 1 x 2 min. 3. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 4. Washing with wash buffer 3 x 2 min. 5. Biotinylated Secondary Antibody, Mouse (Reagent 2, yellow) 10-15 min. 6. Washing with wash buffer 3 x 2 min. 7. Streptavidin-AP-Conjugate (Reagent 3, red) 10-15 min. 8. Washing with wash buffer 3 x 2 min. 9. Permanent Red substrate-chromogen solution (with MON-APP119) 5 min. 10. Wash with distilled H2O 1 min. 11. Permanent Red substrate-chromogen solution (with MON-APP119) 5 min. 12. Wash with destilled water 3 x 1 min. 13. Counterstaining and blueing 14. Mounting: aqueous or permanent after dehydration * The incubation times should be adjusted, when using other substrate-chromogen systems.
Expected results:
During the reaction of the substrate with alkaline phosphatase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support . No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from mouse, but from a different species. 6. The antigen/epitope was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. If you used heat pre-treatment or enzyme digestion it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. If you are using PBS-based wash buffer: the activity of alkaline phosphatase in the reagents is blocked if too much wash buffer remains on the slides. 7. Incubation times were too short or primary antibody concentration too low. 8. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme conjugate or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous alkaline phosphatase in the tissue. This undesired activity can often be suppressed using levamisole (see section Limitations of the Procedure). 9. Non-specific binding of the secondary antibody to endogenous biotin in the tissue section. Carry out an avidin-biotin block before incubation with the primary antibody.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous alkaline phosphatase activity or the endogenous biotin content may cause non-specific staining. The enzyme activity can be blocked by incubation with levamisole. However, neither intestinal nor placental alkaline phosphatase can be blocked with levamisole. Background staining due to endogenous biotin can be blocked through an avidin-biotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the Blocking Solution instead of just draining it away as in other procedures. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. ProClin 300 and sodium azide (NaN3) are used for stabilisation. Sodium azide deposits in drainage pipes made of lead or copper can result in the formation of highly explosive metallic azides. To avoid such deposits in drainage pipes, sodium azide should be discarded in a large volume of running water. Material safety data sheets (MSDS) for the pure substances are available upon request. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear
The Plus AP Kit, Mouse is based on the streptavidin-biotin system. It is designed for the qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. The kit is developed for use in combination with monoclonal primary antibodies and sera obtained from mice. The Plus AP Kit, Mouse can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of immunohistochemical staining is to make tissue and cell antigens visible. The Plus AP Kit, Mouse is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The method is based on the streptavidin-biotin system which means that a biotinylated secondary antibody binds to several molecules of a conjugate composed of streptavidin and alkaline phosphatase. Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The biotinylated secondary antibody in the Plus AP Kit, Mouse binds to mouse primary antibodies. Therefore this kit can detect monoclonal primary antibodies and sera obtained from mice.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Background staining caused by unspecific binding of the primary or secondary antibody is minimized by incubation with a protein blocking solution (Blocking Solution provided with the kit). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the biotinylated secondary antibody is applied and incubated. This secondary antibody functions as a link between primary antibody and the streptavidinalkaline phosphatase-conjugate (Streptavidin-AP-Conjugate). A second washing is followed by the application of this conjugate. It binds to the biotin at the secondary antibody. Any excess of unbound streptavidin-AP-conjugate is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the alkaline phosphatase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen Permanent Red (included only in kit MON-APP119) leads to a magenta-red product of reaction at the place of the target antigen. Other suitable chromogens are Permanent AP Red (magenta-red) or NBT (blue-black) with its substrate BCIP.
Reagents provided:
125 ml Blocking Solution Reagent 1 (ready-to-use) 125 ml Biotinylated Secondary Antibody, Mouse Reagent 2 (ready-to-use) 125 ml Streptavidin-AP-Conjugate Reagent 3 (ready-to-use) Substrate systems recommended (if not included in the kit): Permanent AP Red Kit, BCIP/NBT Materials required but not supplied Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O Reagents for enzyme digestion or heat pre-treatment Wash buffer PBS or TBS PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solutions should be stored at 2-8°C without further dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. They should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. Tissue sections have to be completely covered with the different reagents in order to avoid drying out. Preparation of the chromogenic substrate working solution (with MON-APP119 only): Add 2 drops (60 µl) of Permanent Red Concentrate to one bottle of Permanent Red Buffer (Substrate Buffer) and mix. This solution should be used directly after preparation.
Procedure:
1. Blocking Solution (protein block, Reagent 1) (This step is optional.) 5 min. 2. Washing with wash buffer 1 x 2 min. 3. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 4. Washing with wash buffer 3 x 2 min. 5. Biotinylated Secondary Antibody, Mouse (Reagent 2, yellow) 10-15 min. 6. Washing with wash buffer 3 x 2 min. 7. Streptavidin-AP-Conjugate (Reagent 3, red) 10-15 min. 8. Washing with wash buffer 3 x 2 min. 9. Permanent Red substrate-chromogen solution (with MON-APP119) 5 min. 10. Wash with distilled H2O 1 min. 11. Permanent Red substrate-chromogen solution (with MON-APP119) 5 min. 12. Wash with destilled water 3 x 1 min. 13. Counterstaining and blueing 14. Mounting: aqueous or permanent after dehydration * The incubation times should be adjusted, when using other substrate-chromogen systems.
Expected results:
During the reaction of the substrate with alkaline phosphatase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support . No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from mouse, but from a different species. 6. The antigen/epitope was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. If you used heat pre-treatment or enzyme digestion it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. If you are using PBS-based wash buffer: the activity of alkaline phosphatase in the reagents is blocked if too much wash buffer remains on the slides. 7. Incubation times were too short or primary antibody concentration too low. 8. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme conjugate or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous alkaline phosphatase in the tissue. This undesired activity can often be suppressed using levamisole (see section Limitations of the Procedure). 9. Non-specific binding of the secondary antibody to endogenous biotin in the tissue section. Carry out an avidin-biotin block before incubation with the primary antibody.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous alkaline phosphatase activity or the endogenous biotin content may cause non-specific staining. The enzyme activity can be blocked by incubation with levamisole. However, neither intestinal nor placental alkaline phosphatase can be blocked with levamisole. Background staining due to endogenous biotin can be blocked through an avidin-biotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the Blocking Solution instead of just draining it away as in other procedures. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. ProClin 300 and sodium azide (NaN3) are used for stabilisation. Sodium azide deposits in drainage pipes made of lead or copper can result in the formation of highly explosive metallic azides. To avoid such deposits in drainage pipes, sodium azide should be discarded in a large volume of running water. Material safety data sheets (MSDS) for the pure substances are available upon request. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear
The Plus AP Kit, Mouse is based on the streptavidin-biotin system. It is designed for the qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. The kit is developed for use in combination with monoclonal primary antibodies and sera obtained from mice. The Plus AP Kit, Mouse can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of immunohistochemical staining is to make tissue and cell antigens visible. The Plus AP Kit, Mouse is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The method is based on the streptavidin-biotin system which means that a biotinylated secondary antibody binds to several molecules of a conjugate composed of streptavidin and alkaline phosphatase. Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The biotinylated secondary antibody in the Plus AP Kit, Mouse binds to mouse primary antibodies. Therefore this kit can detect monoclonal primary antibodies and sera obtained from mice.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Background staining caused by unspecific binding of the primary or secondary antibody is minimized by incubation with a protein blocking solution (Blocking Solution provided with the kit). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the biotinylated secondary antibody is applied and incubated. This secondary antibody functions as a link between primary antibody and the streptavidinalkaline phosphatase-conjugate (Streptavidin-AP-Conjugate). A second washing is followed by the application of this conjugate. It binds to the biotin at the secondary antibody. Any excess of unbound streptavidin-AP-conjugate is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the alkaline phosphatase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen Permanent Red (included only in kit MON-APP119) leads to a magenta-red product of reaction at the place of the target antigen. Other suitable chromogens are Permanent AP Red (magenta-red) or NBT (blue-black) with its substrate BCIP.
Reagents provided:
8 ml Blocking Solution Reagent 1 (ready-to-use) 8 ml Biotinylated Secondary Antibody, Mouse Reagent 2 (ready-to-use) 8 ml Streptavidin-AP-Conjugate Reagent 3 (ready-to-use) 8 x 5 ml Permanent Red Buffer (Substrate Buffer) 2 ml Permanent Red Concentrate (Chromogen) Substrate systems recommended (if not included in the kit): Permanent AP Red Kit, BCIP/NBT Materials required but not supplied Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O Reagents for enzyme digestion or heat pre-treatment Wash buffer PBS or TBS PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solutions should be stored at 2-8°C without further dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. They should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. Tissue sections have to be completely covered with the different reagents in order to avoid drying out. Preparation of the chromogenic substrate working solution (with MON-APP119 only): Add 2 drops (60 µl) of Permanent Red Concentrate to one bottle of Permanent Red Buffer (Substrate Buffer) and mix. This solution should be used directly after preparation.
Procedure:
1. Blocking Solution (protein block, Reagent 1) (This step is optional.) 5 min. 2. Washing with wash buffer 1 x 2 min. 3. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 4. Washing with wash buffer 3 x 2 min. 5. Biotinylated Secondary Antibody, Mouse (Reagent 2, yellow) 10-15 min. 6. Washing with wash buffer 3 x 2 min. 7. Streptavidin-AP-Conjugate (Reagent 3, red) 10-15 min. 8. Washing with wash buffer 3 x 2 min. 9. Permanent Red substrate-chromogen solution (with MON-APP119) 5 min. 10. Wash with distilled H2O 1 min. 11. Permanent Red substrate-chromogen solution (with MON-APP119) 5 min. 12. Wash with destilled water 3 x 1 min. 13. Counterstaining and blueing 14. Mounting: aqueous or permanent after dehydration * The incubation times should be adjusted, when using other substrate-chromogen systems.
Expected results:
During the reaction of the substrate with alkaline phosphatase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support . No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from mouse, but from a different species. 6. The antigen/epitope was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. If you used heat pre-treatment or enzyme digestion it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. If you are using PBS-based wash buffer: the activity of alkaline phosphatase in the reagents is blocked if too much wash buffer remains on the slides. 7. Incubation times were too short or primary antibody concentration too low. 8. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme conjugate or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous alkaline phosphatase in the tissue. This undesired activity can often be suppressed using levamisole (see section Limitations of the Procedure). 9. Non-specific binding of the secondary antibody to endogenous biotin in the tissue section. Carry out an avidin-biotin block before incubation with the primary antibody.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous alkaline phosphatase activity or the endogenous biotin content may cause non-specific staining. The enzyme activity can be blocked by incubation with levamisole. However, neither intestinal nor placental alkaline phosphatase can be blocked with levamisole. Background staining due to endogenous biotin can be blocked through an avidin-biotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the Blocking Solution instead of just draining it away as in other procedures. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. ProClin 300 and sodium azide (NaN3) are used for stabilisation. Sodium azide deposits in drainage pipes made of lead or copper can result in the formation of highly explosive metallic azides. To avoid such deposits in drainage pipes, sodium azide should be discarded in a large volume of running water. Material safety data sheets (MSDS) for the pure substances are available upon request. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear
The Plus HRP Kit, Broad Spectrum is based on the streptavidin-biotin system. It is designed for the qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. The kit is developed for use in combination with mono- and polyclonal primary antibodies and sera obtained from mouse, rabbit, rat, and guinea pig. The Plus HRP Kit, Broad Spectrum can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of immunohistochemical staining is to make tissue and cell antigens visible. The Plus HRP Kit, Broad Spectrum is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The method is based on the streptavidin-biotin system which means that a biotinylated secondary antibody binds to several molecules of a conjugate composed of streptavidin and horse radish peroxidase. Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The biotinylated secondary antibody in the Plus HRP Kit, Broad Spectrum is polyvalent. Therefore this kit can detect mono- and polyclonal primary antibodies and sera obtained from mouse, rabbit, rat, and guinea pig.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Endogenous peroxidase activity in the tissue may cause non-specific staining. This enzyme activity can be blocked by incubation with 3% H2O2-solution (Peroxide Block). Background staining caused by unspecific binding of the primary or secondary antibody is minimized by incubation with a protein blocking solution (Blocking Solution). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the biotinylated secondary antibody is applied and incubated. This secondary antibody functions as a link between primary antibody and the streptavidin-horse radish peroxidase-conjugate (Streptavidin-HRP-Conjugate). A second washing is followed by the application of this conjugate. It binds to the biotin at the secondary antibody. Any excess of unbound streptavidin-HRP-conjugate is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the horse radish peroxidase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen AEC (included only in kit MON-APP114) leads to the formation of a red-brown product of reaction at the place of the target antigen. The chromogen DAB (included only in kit MON-APP115) forms a dark brown precipitate.
Reagents provided:
8 ml Peroxide Block (ready-to-use) 8 ml Blocking Solution Reagent 1 (ready-to-use) 8 ml Biotinylated Secondary Antibody, polyvalent Reagent 2 (ready-to-use) 8 ml Streptavidin-HRP-Conjugate Reagent 3 (ready-to-use) 7 x 5 ml DAB Substrate Buffer 3 ml DAB Concentrate (Chromogen) Substrate systems recommended (if not included in the kit): Permanent AEC kit, AEC single solution, AEC substrate kit, DAB substrate kit, DAB high contrast kit. Materials required but not supplied Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O 3% H2O2 solution Reagents for enzyme digestion or heat pre-treatment Wash buffer Pink PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solutions should be stored at 2-8°C without fur ther dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. They should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support.
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. The tissue sections have to be completely covered with the different reagents in order to avoid drying out. Preparation of the chromogenic substrate AEC working solution (with MON-APP114 only): Add 2 drops (100 µl) of AEC Concentrate to one bottle of AEC Substrate Buffer and mix thoroughly. Preparation of the chromogenic substrate DAB working solution (with MON-APP115 only): Add 4 drops (200 µl) of DAB Concentrate to one bottle of DAB Substrate Buffer and mix thoroughly.
Procedure:
1. Peroxide Block (3% H2O2 solution) 10 min. 2. Washing with wash buffer 1 x 2 min. 3. Blocking Solution (protein block, Reagent 1) (This step is optional.) 5 min. 4. Washing with wash buffer 1 x 2 min. 5. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 6. Washing with wash buffer 3 x 2 min. 7. Biotinylated Secondary Antibody, polyvalent (Reagent 2, yellow) 10-15 min. 8. Washing with wash buffer 3 x 2 min. 9. Streptavidin-HRP-Conjugate (Reagent 3, red) 10-15 min. 10. Washing with wash buffer 3 x 2 min. 11. AEC or DAB (Controlling the colour intensity via light microscope is recommended.) 5-15 min. 12. Stopping the reaction with distilled H2O when the desired colour intensity is attained 13. Counterstaining and blueing 14. Mounting: aqueous with AEC, permanent with DAB or Permanent AEC
Expected results:
During the reaction of the substrate with horse radish peroxidase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support . No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pretreatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from mouse, rabbit, rat or guinea pig. 6. The antigen was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pretreatment or enzyme digestion. If you used a pre-treatment it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. Incubation times were too short or primary antibody concentration too low. 7. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme conjugate or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous horse radish peroxidase. Maybe the hydrogen peroxide solution used for blocking was inactivated. 9. Non-specific binding of the secondary antibody to endogenous biotin in the tissue section. Carry out an avidin-biotin block before incubation with the primary antibody.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous peroxidase or pseudoperoxidase activity or the endogenous biotin content may cause non-specific staining. The enzyme activity can be blocked by incubation with 3% H2O2 solution. Tissues containing Hepatitis B Surface Antigen (HBsAg) may give false positive results with HRP (horse radish peroxidase) detection systems (Omata et al, 1980). Background staining due to endogenous biotin can be blocked through an avidinbiotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the Blocking Solution instead of just draining it away as in other procedures. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear. ProClin 300 and sodium azide (NaN3) are used for stabilisation. Sodium azide deposits in drainage pipes made of lead or copper can result in the formation of highly explosive metallic azides. To avoid such deposits in drainage pipes, sodium azide should be discarded in a large volume of running water. Material safety data sheets (MSDS) are available upon request.
The Plus HRP Kit, Broad Spectrum is based on the streptavidin-biotin system. It is designed for the qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. The kit is developed for use in combination with mono- and polyclonal primary antibodies and sera obtained from mouse, rabbit, rat, and guinea pig. The Plus HRP Kit, Broad Spectrum can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of immunohistochemical staining is to make tissue and cell antigens visible. The Plus HRP Kit, Broad Spectrum is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The method is based on the streptavidin-biotin system which means that a biotinylated secondary antibody binds to several molecules of a conjugate composed of streptavidin and horse radish peroxidase. Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The biotinylated secondary antibody in the Plus HRP Kit, Broad Spectrum is polyvalent. Therefore this kit can detect mono- and polyclonal primary antibodies and sera obtained from mouse, rabbit, rat, and guinea pig.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Endogenous peroxidase activity in the tissue may cause non-specific staining. This enzyme activity can be blocked by incubation with 3% H2O2-solution (Peroxide Block). Background staining caused by unspecific binding of the primary or secondary antibody is minimized by incubation with a protein blocking solution (Blocking Solution). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the biotinylated secondary antibody is applied and incubated. This secondary antibody functions as a link between primary antibody and the streptavidin-horse radish peroxidase-conjugate (Streptavidin-HRP-Conjugate). A second washing is followed by the application of this conjugate. It binds to the biotin at the secondary antibody. Any excess of unbound streptavidin-HRP-conjugate is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the horse radish peroxidase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen AEC (included only in kit MON-APP114) leads to the formation of a red-brown product of reaction at the place of the target antigen. The chromogen DAB (included only in kit MON-APP115) forms a dark brown precipitate.
Reagents provided:
8 ml Peroxide Block (ready-to-use) 8 ml Blocking Solution Reagent 1 (ready-to-use) 8 ml Biotinylated Secondary Antibody, polyvalent Reagent 2 (ready-to-use) 8 ml Streptavidin-HRP-Conjugate Reagent 3 (ready-to-use) 7 x 5 ml AEC Substrate Buffer 3 ml AEC Concentrate (Chromogen) Substrate systems recommended (if not included in the kit): Permanent AEC kit, AEC single solution, AEC substrate kit, DAB substrate kit, DAB high contrast kit. Materials required but not supplied Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O 3% H2O2 solution Reagents for enzyme digestion or heat pre-treatment Wash buffer Pink PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solutions should be stored at 2-8°C without fur ther dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. They should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support.
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. The tissue sections have to be completely covered with the different reagents in order to avoid drying out. Preparation of the chromogenic substrate AEC working solution (with MON-APP114 only): Add 2 drops (100 µl) of AEC Concentrate to one bottle of AEC Substrate Buffer and mix thoroughly. Preparation of the chromogenic substrate DAB working solution (with MON-APP115 only): Add 4 drops (200 µl) of DAB Concentrate to one bottle of DAB Substrate Buffer and mix thoroughly.
Procedure:
1. Peroxide Block (3% H2O2 solution) 10 min. 2. Washing with wash buffer 1 x 2 min. 3. Blocking Solution (protein block, Reagent 1) (This step is optional.) 5 min. 4. Washing with wash buffer 1 x 2 min. 5. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 6. Washing with wash buffer 3 x 2 min. 7. Biotinylated Secondary Antibody, polyvalent (Reagent 2, yellow) 10-15 min. 8. Washing with wash buffer 3 x 2 min. 9. Streptavidin-HRP-Conjugate (Reagent 3, red) 10-15 min. 10. Washing with wash buffer 3 x 2 min. 11. AEC or DAB (Controlling the colour intensity via light microscope is recommended.) 5-15 min. 12. Stopping the reaction with distilled H2O when the desired colour intensity is attained 13. Counterstaining and blueing 14. Mounting: aqueous with AEC, permanent with DAB or Permanent AEC
Expected results:
During the reaction of the substrate with horse radish peroxidase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support . No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pretreatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from mouse, rabbit, rat or guinea pig. 6. The antigen was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pretreatment or enzyme digestion. If you used a pre-treatment it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. Incubation times were too short or primary antibody concentration too low. 7. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme conjugate or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous horse radish peroxidase. Maybe the hydrogen peroxide solution used for blocking was inactivated. 9. Non-specific binding of the secondary antibody to endogenous biotin in the tissue section. Carry out an avidin-biotin block before incubation with the primary antibody.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous peroxidase or pseudoperoxidase activity or the endogenous biotin content may cause non-specific staining. The enzyme activity can be blocked by incubation with 3% H2O2 solution. Tissues containing Hepatitis B Surface Antigen (HBsAg) may give false positive results with HRP (horse radish peroxidase) detection systems (Omata et al, 1980). Background staining due to endogenous biotin can be blocked through an avidinbiotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the Blocking Solution instead of just draining it away as in other procedures. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear. ProClin 300 and sodium azide (NaN3) are used for stabilisation. Sodium azide deposits in drainage pipes made of lead or copper can result in the formation of highly explosive metallic azides. To avoid such deposits in drainage pipes, sodium azide should be discarded in a large volume of running water. Material safety data sheets (MSDS) are available upon request.
The Plus AP Kit, Broad Spectrum is based on the streptavidin-biotin system. It is designed for the qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. The kit is developed for use in combination with mono- and polyclonal primary antibodies and sera obtained from mouse, rabbit, rat, and guinea pig. The Plus AP Kit, Broad Spectrum can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus AP Kits, Broad Spectrum is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The method is based on the streptavidin-biotin system which means that a biotinylated secondary antibody binds to several molecules of a conjugate composed of streptavidin and alkaline phosphatase. Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The biotinylated secondary antibody in the Plus AP Kit, Broad Spectrum is polyvalent. With this kit it is therefore possible to detect mono- and polyclonal primary antibodies and sera obtained from mouse, rabbit, rat, and guinea pig.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Background staining caused by unspecific binding of the primary or secondary antibody is minimized via incubation with a protein blocking solution (Blocking Solution provided with the kit). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the biotinylated secondary antibody is applied and incubated. This secondary antibody functions as a link between primary antibody and streptavidin-alkaline phosphatase-conjugate (Streptavidin-AP-Conjugate). A second washing is followed by the application of this conjugate. It binds to biotin at the secondary antibody. Any excess of unbound streptavidin-AP-conjugate is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the alkaline phosphatase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen Permanent Red (included only in kit MON-APP110) leads to the formation of a magenta-red product of reaction at the place of the target antigen. Other suitable chromogens are Permanent AP Red (magenta-red) or NBT (blue-black) with its substrate BCIP.
Reagents provided:
4x 15 ml Blocking Solution Reagent 1 (ready-to-use) 4x 15 ml Biotinylated Secondary Antibody, polyvalent Reagent 2 (ready-to-use) 4x 15 ml Streptavidin-AP-Conjugate Reagent 3 (ready-to-use) 8 x 5 ml Permanent Red Buffer (Substrate Buffer) 2 ml Permanent Red Concentrate (Chromogen) Substrate systems recommended (if not included in the kit): Permanent AP Red Kit, BCIP/NBT Materials required but not supplied Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O Reagents for enzyme digestion or heat pre-treatment Wash buffer PBS or TBS PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solution should be stored at 2-8°C without further dilution. Please store the reagent in a dark place and do not freeze it. Under these conditions the solution is stable up to the expiry date. It should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support .
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion.Tissue sections have to be completely covered with the different reagents in order to avoid drying out. Preparation of the chromogenic substrate working solution (with MON-APP110 only): Add 2 drops (60 µl) of Permanent Red Concentrate to one bottle of Permanent Red Buffer (substrate buffer) and mix. This solution should be used directly after preparation.
Procedure:
1. Blocking Solution (protein block, Reagent 1) (This step is optional.) 5 min. 2. Washing with wash buffer 1 x 2 min. 3. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 4. Washing with wash buffer 3 x 2 min. 5. Biotinylated Secondary Antibody, polyvalent (Reagent 2, yellow) 10-15 min. 6. Washing with wash buffer 3 x 2 min. 7. Streptavidin-AP-Conjugate (Reagent 3, red) 10-15 min. 8. Washing with wash buffer 3 x 2 min. 9. Permanent Red substrate-chromogen solution (with MON-APP110) 5 min. 10. Wash with distilled H2O 1 min. 11. Permanent Red substrate-chromogen solution (with MON-APP110) 5 min. 12. Wash with distilled H2O 3 x 1 min. 13. Counterstaining and blueing 14. Mounting: aqueous or permanent after dehydration * The incubation times should be adjusted, when using other substrate-chromogen systems.
Expected results:
During the reaction of the substrate with alkaline phosphatase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support. No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from mouse, rabbit, rat or guinea pig. 6. The antigen was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. If you are using PBS-based wash buffer: the activity of alkaline phosphatase in the reagents is blocked if too much wash buffer remains on the slides. 7. Incubation times were too short or primary antibody concentration too low. 8. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme conjugate or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolized by endogenous alkaline phosphatase in the tissue. This undesired activity can often be suppressed using levamisole (see also Limitations of the procedure). 9. Non-specific binding of the secondary antibody to endogenous biotin in the tissue section. Carry out an avidin-biotin block before incubation with the primary antibody.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous alkaline phosphatase activity or the endogenous biotin content may cause non-specific staining. The enzyme activity can be blocked by incubation with levamisole. However, neither intestinal nor placental alkaline phosphatase can be blocked with levamisole. Background staining due to endogenous biotin can be blocked through an avidin-biotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the Blocking Solution instead of just draining it away as in other procedures. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. ProClin 300 and sodium azide (NaN3), used for stabilisation. Sodium azide deposits in drainage pipes made of lead or copper can result in the formation of highly explosive metallic azides. To avoid such deposits in drainage pipes, sodium azide should be discarded in a large volume of running water. Material safety data sheets (MSDS) for the pure substances are available upon request. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear.
The Plus AP Kit, Broad Spectrum is based on the streptavidin-biotin system. It is designed for the qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. The kit is developed for use in combination with mono- and polyclonal primary antibodies and sera obtained from mouse, rabbit, rat, and guinea pig. The Plus AP Kit, Broad Spectrum can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus AP Kits, Broad Spectrum is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The method is based on the streptavidin-biotin system which means that a biotinylated secondary antibody binds to several molecules of a conjugate composed of streptavidin and alkaline phosphatase. Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The biotinylated secondary antibody in the Plus AP Kit, Broad Spectrum is polyvalent. With this kit it is therefore possible to detect mono- and polyclonal primary antibodies and sera obtained from mouse, rabbit, rat, and guinea pig.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Background staining caused by unspecific binding of the primary or secondary antibody is minimized via incubation with a protein blocking solution (Blocking Solution provided with the kit). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the biotinylated secondary antibody is applied and incubated. This secondary antibody functions as a link between primary antibody and streptavidin-alkaline phosphatase-conjugate (Streptavidin-AP-Conjugate). A second washing is followed by the application of this conjugate. It binds to biotin at the secondary antibody. Any excess of unbound streptavidin-AP-conjugate is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the alkaline phosphatase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen Permanent Red (included only in kit MON-APP110) leads to the formation of a magenta-red product of reaction at the place of the target antigen. Other suitable chromogens are Permanent AP Red (magenta-red) or NBT (blue-black) with its substrate BCIP.
Reagents provided:
500 ml Blocking Solution Reagent 1 (ready-to-use) 500 ml Biotinylated Secondary Antibody, polyvalent Reagent 2 (ready-to-use) 500 ml Streptavidin-AP-Conjugate Reagent 3 (ready-to-use) Substrate systems recommended (if not included in the kit): Permanent AP Red Kit, BCIP/NBT Materials required but not supplied Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O Reagents for enzyme digestion or heat pre-treatment Wash buffer PBS or TBS PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solution should be stored at 2-8°C without further dilution. Please store the reagent in a dark place and do not freeze it. Under these conditions the solution is stable up to the expiry date. It should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support .
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion.Tissue sections have to be completely covered with the different reagents in order to avoid drying out. Preparation of the chromogenic substrate working solution (with MON-APP110 only): Add 2 drops (60 µl) of Permanent Red Concentrate to one bottle of Permanent Red Buffer (substrate buffer) and mix. This solution should be used directly after preparation.
Procedure:
1. Blocking Solution (protein block, Reagent 1) (This step is optional.) 5 min. 2. Washing with wash buffer 1 x 2 min. 3. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 4. Washing with wash buffer 3 x 2 min. 5. Biotinylated Secondary Antibody, polyvalent (Reagent 2, yellow) 10-15 min. 6. Washing with wash buffer 3 x 2 min. 7. Streptavidin-AP-Conjugate (Reagent 3, red) 10-15 min. 8. Washing with wash buffer 3 x 2 min. 9. Permanent Red substrate-chromogen solution (with MON-APP110) 5 min. 10. Wash with distilled H2O 1 min. 11. Permanent Red substrate-chromogen solution (with MON-APP110) 5 min. 12. Wash with distilled H2O 3 x 1 min. 13. Counterstaining and blueing 14. Mounting: aqueous or permanent after dehydration * The incubation times should be adjusted, when using other substrate-chromogen systems.
Expected results:
During the reaction of the substrate with alkaline phosphatase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support. No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from mouse, rabbit, rat or guinea pig. 6. The antigen was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. If you are using PBS-based wash buffer: the activity of alkaline phosphatase in the reagents is blocked if too much wash buffer remains on the slides. 7. Incubation times were too short or primary antibody concentration too low. 8. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme conjugate or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolized by endogenous alkaline phosphatase in the tissue. This undesired activity can often be suppressed using levamisole (see also Limitations of the procedure). 9. Non-specific binding of the secondary antibody to endogenous biotin in the tissue section. Carry out an avidin-biotin block before incubation with the primary antibody.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous alkaline phosphatase activity or the endogenous biotin content may cause non-specific staining. The enzyme activity can be blocked by incubation with levamisole. However, neither intestinal nor placental alkaline phosphatase can be blocked with levamisole. Background staining due to endogenous biotin can be blocked through an avidin-biotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the Blocking Solution instead of just draining it away as in other procedures. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. ProClin 300 and sodium azide (NaN3), used for stabilisation. Sodium azide deposits in drainage pipes made of lead or copper can result in the formation of highly explosive metallic azides. To avoid such deposits in drainage pipes, sodium azide should be discarded in a large volume of running water. Material safety data sheets (MSDS) for the pure substances are available upon request. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear.
The Plus AP Kit, Broad Spectrum is based on the streptavidin-biotin system. It is designed for the qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. The kit is developed for use in combination with mono- and polyclonal primary antibodies and sera obtained from mouse, rabbit, rat, and guinea pig. The Plus AP Kit, Broad Spectrum can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus AP Kits, Broad Spectrum is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The method is based on the streptavidin-biotin system which means that a biotinylated secondary antibody binds to several molecules of a conjugate composed of streptavidin and alkaline phosphatase. Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The biotinylated secondary antibody in the Plus AP Kit, Broad Spectrum is polyvalent. With this kit it is therefore possible to detect mono- and polyclonal primary antibodies and sera obtained from mouse, rabbit, rat, and guinea pig.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Background staining caused by unspecific binding of the primary or secondary antibody is minimized via incubation with a protein blocking solution (Blocking Solution provided with the kit). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the biotinylated secondary antibody is applied and incubated. This secondary antibody functions as a link between primary antibody and streptavidin-alkaline phosphatase-conjugate (Streptavidin-AP-Conjugate). A second washing is followed by the application of this conjugate. It binds to biotin at the secondary antibody. Any excess of unbound streptavidin-AP-conjugate is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the alkaline phosphatase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen Permanent Red (included only in kit MON-APP110) leads to the formation of a magenta-red product of reaction at the place of the target antigen. Other suitable chromogens are Permanent AP Red (magenta-red) or NBT (blue-black) with its substrate BCIP.
Reagents provided:
125 ml Blocking Solution Reagent 1 (ready-to-use) 125 ml Biotinylated Secondary Antibody, polyvalent Reagent 2 (ready-to-use) 125 ml Streptavidin-AP-Conjugate Reagent 3 (ready-to-use) Substrate systems recommended (if not included in the kit): Permanent AP Red Kit, BCIP/NBT Materials required but not supplied Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O Reagents for enzyme digestion or heat pre-treatment Wash buffer PBS or TBS PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solution should be stored at 2-8°C without further dilution. Please store the reagent in a dark place and do not freeze it. Under these conditions the solution is stable up to the expiry date. It should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support .
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion.Tissue sections have to be completely covered with the different reagents in order to avoid drying out. Preparation of the chromogenic substrate working solution (with MON-APP110 only): Add 2 drops (60 µl) of Permanent Red Concentrate to one bottle of Permanent Red Buffer (substrate buffer) and mix. This solution should be used directly after preparation.
Procedure:
1. Blocking Solution (protein block, Reagent 1) (This step is optional.) 5 min. 2. Washing with wash buffer 1 x 2 min. 3. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 4. Washing with wash buffer 3 x 2 min. 5. Biotinylated Secondary Antibody, polyvalent (Reagent 2, yellow) 10-15 min. 6. Washing with wash buffer 3 x 2 min. 7. Streptavidin-AP-Conjugate (Reagent 3, red) 10-15 min. 8. Washing with wash buffer 3 x 2 min. 9. Permanent Red substrate-chromogen solution (with MON-APP110) 5 min. 10. Wash with distilled H2O 1 min. 11. Permanent Red substrate-chromogen solution (with MON-APP110) 5 min. 12. Wash with distilled H2O 3 x 1 min. 13. Counterstaining and blueing 14. Mounting: aqueous or permanent after dehydration * The incubation times should be adjusted, when using other substrate-chromogen systems.
Expected results:
During the reaction of the substrate with alkaline phosphatase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support. No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from mouse, rabbit, rat or guinea pig. 6. The antigen was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. If you are using PBS-based wash buffer: the activity of alkaline phosphatase in the reagents is blocked if too much wash buffer remains on the slides. 7. Incubation times were too short or primary antibody concentration too low. 8. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme conjugate or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolized by endogenous alkaline phosphatase in the tissue. This undesired activity can often be suppressed using levamisole (see also Limitations of the procedure). 9. Non-specific binding of the secondary antibody to endogenous biotin in the tissue section. Carry out an avidin-biotin block before incubation with the primary antibody.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous alkaline phosphatase activity or the endogenous biotin content may cause non-specific staining. The enzyme activity can be blocked by incubation with levamisole. However, neither intestinal nor placental alkaline phosphatase can be blocked with levamisole. Background staining due to endogenous biotin can be blocked through an avidin-biotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the Blocking Solution instead of just draining it away as in other procedures. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. ProClin 300 and sodium azide (NaN3), used for stabilisation. Sodium azide deposits in drainage pipes made of lead or copper can result in the formation of highly explosive metallic azides. To avoid such deposits in drainage pipes, sodium azide should be discarded in a large volume of running water. Material safety data sheets (MSDS) for the pure substances are available upon request. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear.
The Plus AP Kit, Broad Spectrum is based on the streptavidin-biotin system. It is designed for the qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. The kit is developed for use in combination with mono- and polyclonal primary antibodies and sera obtained from mouse, rabbit, rat, and guinea pig. The Plus AP Kit, Broad Spectrum can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus AP Kits, Broad Spectrum is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The method is based on the streptavidin-biotin system which means that a biotinylated secondary antibody binds to several molecules of a conjugate composed of streptavidin and alkaline phosphatase. Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The biotinylated secondary antibody in the Plus AP Kit, Broad Spectrum is polyvalent. With this kit it is therefore possible to detect mono- and polyclonal primary antibodies and sera obtained from mouse, rabbit, rat, and guinea pig.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Background staining caused by unspecific binding of the primary or secondary antibody is minimized via incubation with a protein blocking solution (Blocking Solution provided with the kit). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the biotinylated secondary antibody is applied and incubated. This secondary antibody functions as a link between primary antibody and streptavidin-alkaline phosphatase-conjugate (Streptavidin-AP-Conjugate). A second washing is followed by the application of this conjugate. It binds to biotin at the secondary antibody. Any excess of unbound streptavidin-AP-conjugate is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the alkaline phosphatase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen Permanent Red (included only in kit MON-APP110) leads to the formation of a magenta-red product of reaction at the place of the target antigen. Other suitable chromogens are Permanent AP Red (magenta-red) or NBT (blue-black) with its substrate BCIP.
Reagents provided:
8 ml Blocking Solution Reagent 1 (ready-to-use) 8 ml Biotinylated Secondary Antibody, polyvalent Reagent 2 (ready-to-use) 8 ml Streptavidin-AP-Conjugate Reagent 3 (ready-to-use) 8 x 5 ml Permanent Red Buffer (Substrate Buffer) 2 ml Permanent Red Concentrate (Chromogen) Substrate systems recommended (if not included in the kit): Permanent AP Red Kit, BCIP/NBT Materials required but not supplied Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O Reagents for enzyme digestion or heat pre-treatment Wash buffer PBS or TBS PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solution should be stored at 2-8°C without further dilution. Please store the reagent in a dark place and do not freeze it. Under these conditions the solution is stable up to the expiry date. It should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support .
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion.Tissue sections have to be completely covered with the different reagents in order to avoid drying out. Preparation of the chromogenic substrate working solution (with MON-APP110 only): Add 2 drops (60 µl) of Permanent Red Concentrate to one bottle of Permanent Red Buffer (substrate buffer) and mix. This solution should be used directly after preparation.
Procedure:
1. Blocking Solution (protein block, Reagent 1) (This step is optional.) 5 min. 2. Washing with wash buffer 1 x 2 min. 3. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 4. Washing with wash buffer 3 x 2 min. 5. Biotinylated Secondary Antibody, polyvalent (Reagent 2, yellow) 10-15 min. 6. Washing with wash buffer 3 x 2 min. 7. Streptavidin-AP-Conjugate (Reagent 3, red) 10-15 min. 8. Washing with wash buffer 3 x 2 min. 9. Permanent Red substrate-chromogen solution (with MON-APP110) 5 min. 10. Wash with distilled H2O 1 min. 11. Permanent Red substrate-chromogen solution (with MON-APP110) 5 min. 12. Wash with distilled H2O 3 x 1 min. 13. Counterstaining and blueing 14. Mounting: aqueous or permanent after dehydration * The incubation times should be adjusted, when using other substrate-chromogen systems.
Expected results:
During the reaction of the substrate with alkaline phosphatase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support. No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from mouse, rabbit, rat or guinea pig. 6. The antigen was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. If you are using PBS-based wash buffer: the activity of alkaline phosphatase in the reagents is blocked if too much wash buffer remains on the slides. 7. Incubation times were too short or primary antibody concentration too low. 8. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme conjugate or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolized by endogenous alkaline phosphatase in the tissue. This undesired activity can often be suppressed using levamisole (see also Limitations of the procedure). 9. Non-specific binding of the secondary antibody to endogenous biotin in the tissue section. Carry out an avidin-biotin block before incubation with the primary antibody.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous alkaline phosphatase activity or the endogenous biotin content may cause non-specific staining. The enzyme activity can be blocked by incubation with levamisole. However, neither intestinal nor placental alkaline phosphatase can be blocked with levamisole. Background staining due to endogenous biotin can be blocked through an avidin-biotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the Blocking Solution instead of just draining it away as in other procedures. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. ProClin 300 and sodium azide (NaN3), used for stabilisation. Sodium azide deposits in drainage pipes made of lead or copper can result in the formation of highly explosive metallic azides. To avoid such deposits in drainage pipes, sodium azide should be discarded in a large volume of running water. Material safety data sheets (MSDS) for the pure substances are available upon request. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear.
The Plus HRP One-Step Polymer anti-Mouse/Rabbit is designed for the qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. It was developed for use in combination with mono- and polyclonal primary antibodies and sera obtained from mice or rabbit. The kit can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus HRP One-Step Polymer anti-Mouse/Rabbit is a highly sensitive detection reagent intended for use in immunohistochemistry and immunocytochemistry. The enzyme polymer consists of several molecules of secondary antibodies covalently bound to several molecules of horseradish peroxidase (HRP). Visualisation occurs via an enzyme-substrate reaction in the presence of a colorising reagent which permits microscopical analysis. The test system is suitable for the detection of mono- and polyclonal primary antibodies and sera obtained from mice or rabbit. In contrast to other detection techniques, which often use the streptavidin-biotin system the Plus HRP One-Step Polymer kits avoid the problem of background staining caused by endogenous biotin in the tissue.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Endogenous peroxidase activity in the tissue may cause non-specific staining. This enzyme activity can be blocked by incubation with 3% H2O2-solution (peroxide block). Background staining caused by unspecific binding of the primary antibody or the secondary antibody in the HRP One-Step Polymer is minimized by incubation with a protein blocking solution. This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the HRP One-Step Polymer is applied and incubated. Any excess of unbound polymer is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the peroxidase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen AEC leads to the formation of a red-brown product of reaction at the place of the target antigen. The chromogen DAB forms a dark brown precipitate.
Reagents provided:
100 mL HRP One-Step-Polymer anti-Mouse/Rabbit (ready-to-use) Substrate systems recommended: Permanent AEC kit, AEC single solution, AEC substrate kit, DAB substrate kit, DAB High contrast kit. Materials required but not supplied: Positive and negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O 3% H2O2 solution Reagents for enzyme digestion or heat pre-treatment Wash buffer PBS or TBS Pink PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solution should be stored at 2-8°C without further dilution. Please store the reagent in a dark place and do not freeze it. Under these conditions the solution is stable up to the expiry date. It should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support .
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. Tissue sections have to be completely covered with the different reagents in order to avoid drying out.
Procedure:
1. Peroxide blocking (3 % H2O2 solution) 10 min. 2. Washing with wash buffer 1 x 2 min. 3. Blocking Solution (This step is optional.) 5 min. 4. Washing with wash buffer 1 x 2 min. 5. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 6. Washing with wash buffer 3 x 5 min. 7. HRP One-Step Polymer anti-Mouse/Rabbit 30 min. 8. Washing with wash buffer 3 x 2 min. 9. AEC or DAB (Controlling the colour intensity via light microscope is recommended.) 5-15 min. 10. Stopping the reaction with distilled H2O when the desired colour intensity is attained 11. Counterstaining and blueing 12. Mounting: aqueous with AEC, permanent with DAB
Expected results:
During the reaction of the substrate with horseradish peroxidase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support. No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from mouse or rabbit but from a different species. 6. The antigen/epitope was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. If you used heat pre-treatment or enzyme digestion it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. Incubation times were too short or primary antibody concentration too low. 7. Chromogenic substrate solution was too old. Nonspecific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme polymer or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use Blocking Solution or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous horse radish peroxidase in the tissue. Maybe the hydrogen peroxide solution used for blocking was inactivated
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous peroxidase or pseudoperoxidase activity may cause non-specific staining. The enzyme activity is blocked by incubation with hydrogen peroxide solution. Tissues containing Hepatitis B Surface Antigen (HBsAg) may give false positive results with HRP (horseradish peroxidase) detection systems (Omata et al, 1980). Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution can result in decreasing signal intensity. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear. ProClin 950 is used for stabilisation. A Material safety data sheet (MSDS) is available upon request.
The Plus HRP One-Step Polymer anti-Mouse/Rabbit is designed for the qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. It was developed for use in combination with mono- and polyclonal primary antibodies and sera obtained from mice or rabbit. The kit can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus HRP One-Step Polymer anti-Mouse/Rabbit is a highly sensitive detection reagent intended for use in immunohistochemistry and immunocytochemistry. The enzyme polymer consists of several molecules of secondary antibodies covalently bound to several molecules of horseradish peroxidase (HRP). Visualisation occurs via an enzyme-substrate reaction in the presence of a colorising reagent which permits microscopical analysis. The test system is suitable for the detection of mono- and polyclonal primary antibodies and sera obtained from mice or rabbit. In contrast to other detection techniques, which often use the streptavidin-biotin system the Plus HRP One-Step Polymer kits avoid the problem of background staining caused by endogenous biotin in the tissue.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Endogenous peroxidase activity in the tissue may cause non-specific staining. This enzyme activity can be blocked by incubation with 3% H2O2-solution (peroxide block). Background staining caused by unspecific binding of the primary antibody or the secondary antibody in the HRP One-Step Polymer is minimized by incubation with a protein blocking solution. This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the HRP One-Step Polymer is applied and incubated. Any excess of unbound polymer is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the peroxidase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen AEC leads to the formation of a red-brown product of reaction at the place of the target antigen. The chromogen DAB forms a dark brown precipitate.
Reagents provided:
6 mL HRP One-Step-Polymer anti-Mouse/Rabbit (ready-to-use) Substrate systems recommended: Permanent AEC kit, AEC single solution, AEC substrate kit, DAB substrate kit, DAB High contrast kit. Materials required but not supplied: Positive and negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O 3% H2O2 solution Reagents for enzyme digestion or heat pre-treatment Wash buffer PBS or TBS Pink PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solution should be stored at 2-8°C without further dilution. Please store the reagent in a dark place and do not freeze it. Under these conditions the solution is stable up to the expiry date. It should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support .
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. Tissue sections have to be completely covered with the different reagents in order to avoid drying out.
Procedure:
1. Peroxide blocking (3 % H2O2 solution) 10 min. 2. Washing with wash buffer 1 x 2 min. 3. Blocking Solution (This step is optional.) 5 min. 4. Washing with wash buffer 1 x 2 min. 5. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 6. Washing with wash buffer 3 x 5 min. 7. HRP One-Step Polymer anti-Mouse/Rabbit 30 min. 8. Washing with wash buffer 3 x 2 min. 9. AEC or DAB (Controlling the colour intensity via light microscope is recommended.) 5-15 min. 10. Stopping the reaction with distilled H2O when the desired colour intensity is attained 11. Counterstaining and blueing 12. Mounting: aqueous with AEC, permanent with DAB
Expected results:
During the reaction of the substrate with horseradish peroxidase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support. No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from mouse or rabbit but from a different species. 6. The antigen/epitope was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. If you used heat pre-treatment or enzyme digestion it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. Incubation times were too short or primary antibody concentration too low. 7. Chromogenic substrate solution was too old. Nonspecific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme polymer or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use Blocking Solution or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous horse radish peroxidase in the tissue. Maybe the hydrogen peroxide solution used for blocking was inactivated
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous peroxidase or pseudoperoxidase activity may cause non-specific staining. The enzyme activity is blocked by incubation with hydrogen peroxide solution. Tissues containing Hepatitis B Surface Antigen (HBsAg) may give false positive results with HRP (horseradish peroxidase) detection systems (Omata et al, 1980). Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution can result in decreasing signal intensity. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear. ProClin 950 is used for stabilisation. A Material safety data sheet (MSDS) is available upon request.
The Plus HRP Polymer anti-Mouse kit is designed for the qualitative detection of antigens in fixed paraffinembedded tissue sections, in frozen tissue sections, and in cytological samples. It was developed for use in combination with monoclonal primary antibodies and sera obtained from mice. The reagent can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus HRP Polymer anti-Mouse kit is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The enzyme polymer consists of several molecules of secondary antibodies covalently bound to several molecules of horse radish peroxidase (HRP). Visualisation occurs via an enzyme-substrate reaction in the presence of a colorising reagent which permits microscopical analysis. The test system is suitable for the detection of monoclonal primary antibodies and sera obtained from mice. In contrast to other detection techniques, which often use the streptavidin-biotin system the Plus HRP Polymer antiMouse kit avoids the problem of background staining caused by endogenous biotin in the tissue
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Endogenous peroxidase activity in the tissue may cause non-specific staining. This enzyme activity can be blocked by incubation with 3% H2O2-solution (peroxide block). Background staining caused by unspecific binding of the primary antibody or the secondary antibody in the HRPpolymer is minimized by incubation with a protein blocking solution. This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the HRP-polymer is applied and incubated. Any excess of unbound HRP-polymer is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the peroxidase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen AEC leads to the formation of a red-brown product of reaction at the place of the target antigen. The chromogen DAB forms a dark brown precipitate.
Reagents provided:
100 mL HRP-Polymer anti-Mouse (ready-to-use) Substrate systems recommended: Permanent AEC kit, AEC single solution, AEC substrate kit, DAB substrate kit, DAB High contrast kit. Materials required but not supplied: Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O 3% H2O2 solution Reagents for enzyme digestion or heat pre-treatment Wash buffer PBS or TBS PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solution should be stored at 2-8°C without further dilution. Please store the reagent in a dark place and do not freeze it. Under these conditions the solution is stable up to the expiry date. It should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support .
Reagent preparation:
Reagent should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. Tissue sections have to be completely covered with the different reagents in order to avoid drying out.
Procedure:
1. Peroxide blocking (3 % H2O2 solution) 10 min. 2. Washing with wash buffer 1 x 2 min. 3. Blocking Solution (This step is optional.) 5 min. 4. Washing with wash buffer 1 x 2 min. 5. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 6. Washing with wash buffer 3 x 5 min. 7. HRP-polymer anti-Mouse 30 min. 8. Washing with wash buffer 3 x 2 min. 9. AEC or DAB (Controlling the colour intensity via light microscope is recommended.) 5-15 min. 10. Stopping the reaction with distilled H2O when the desired colour intensity is attained 11. Counterstaining and blueing 12. Mounting: aqueous with AEC, permanent with DAB or Permanent AEC
Expected results:
During the reaction of the substrate with horse radish peroxidase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support . No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from mouse but from a different species. 6. The antigen/epitope was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. If you used heat pre-treatment or enzyme digestion it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. Incubation times were too short or primary antibody concentration too low. 7. Chromogenic substrate solution was too old. Nonspecific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme polymer or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous horse radish peroxidase in the tissue. Maybe the hydrogen peroxide solution used for blocking was inactivated
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous peroxidase or pseudoperoxidase activity may cause non-specific staining. The enzyme activity is blocked by incubation with hydrogen peroxide solution. Tissues containing Hepatitis B Surface Antigen (HBsAg) may give false positive results with HRP (horse radish peroxidase) detection systems (Omata et al, 1980). Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution can result in decreasing signal intensity. Sanbio guarantee that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of the reagent coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagent must be avoided, since otherwise non-specific staining might appear. ProClin 950 is used for stabilisation. A Material safety data sheets (MSDS) is available upon request.
The Plus HRP Polymer anti-Mouse kit is designed for the qualitative detection of antigens in fixed paraffinembedded tissue sections, in frozen tissue sections, and in cytological samples. It was developed for use in combination with monoclonal primary antibodies and sera obtained from mice. The reagent can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus HRP Polymer anti-Mouse kit is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The enzyme polymer consists of several molecules of secondary antibodies covalently bound to several molecules of horse radish peroxidase (HRP). Visualisation occurs via an enzyme-substrate reaction in the presence of a colorising reagent which permits microscopical analysis. The test system is suitable for the detection of monoclonal primary antibodies and sera obtained from mice. In contrast to other detection techniques, which often use the streptavidin-biotin system the Plus HRP Polymer antiMouse kit avoids the problem of background staining caused by endogenous biotin in the tissue
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Endogenous peroxidase activity in the tissue may cause non-specific staining. This enzyme activity can be blocked by incubation with 3% H2O2-solution (peroxide block). Background staining caused by unspecific binding of the primary antibody or the secondary antibody in the HRPpolymer is minimized by incubation with a protein blocking solution. This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the HRP-polymer is applied and incubated. Any excess of unbound HRP-polymer is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the peroxidase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen AEC leads to the formation of a red-brown product of reaction at the place of the target antigen. The chromogen DAB forms a dark brown precipitate.
Reagents provided:
6 mL HRP-Polymer anti-Mouse (ready-to-use) Substrate systems recommended: Permanent AEC kit, AEC single solution, AEC substrate kit, DAB substrate kit, DAB High contrast kit. Materials required but not supplied: Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O 3% H2O2 solution Reagents for enzyme digestion or heat pre-treatment Wash buffer PBS or TBS PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solution should be stored at 2-8°C without further dilution. Please store the reagent in a dark place and do not freeze it. Under these conditions the solution is stable up to the expiry date. It should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support .
Reagent preparation:
Reagent should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. Tissue sections have to be completely covered with the different reagents in order to avoid drying out.
Procedure:
1. Peroxide blocking (3 % H2O2 solution) 10 min. 2. Washing with wash buffer 1 x 2 min. 3. Blocking Solution (This step is optional.) 5 min. 4. Washing with wash buffer 1 x 2 min. 5. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 6. Washing with wash buffer 3 x 5 min. 7. HRP-polymer anti-Mouse 30 min. 8. Washing with wash buffer 3 x 2 min. 9. AEC or DAB (Controlling the colour intensity via light microscope is recommended.) 5-15 min. 10. Stopping the reaction with distilled H2O when the desired colour intensity is attained 11. Counterstaining and blueing 12. Mounting: aqueous with AEC, permanent with DAB or Permanent AEC
Expected results:
During the reaction of the substrate with horse radish peroxidase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support . No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from mouse but from a different species. 6. The antigen/epitope was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. If you used heat pre-treatment or enzyme digestion it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. Incubation times were too short or primary antibody concentration too low. 7. Chromogenic substrate solution was too old. Nonspecific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme polymer or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous horse radish peroxidase in the tissue. Maybe the hydrogen peroxide solution used for blocking was inactivated
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous peroxidase or pseudoperoxidase activity may cause non-specific staining. The enzyme activity is blocked by incubation with hydrogen peroxide solution. Tissues containing Hepatitis B Surface Antigen (HBsAg) may give false positive results with HRP (horse radish peroxidase) detection systems (Omata et al, 1980). Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution can result in decreasing signal intensity. Sanbio guarantee that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of the reagent coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagent must be avoided, since otherwise non-specific staining might appear. ProClin 950 is used for stabilisation. A Material safety data sheets (MSDS) is available upon request.
The Plus HRP Polymer Kit is designed for the qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. It is developed for use in combination with mono- and polyclonal primary antibodies and sera obtained from mice and rabbits. The kit can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus HRP Polymer Kit is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The enzyme polymer in this kit consists of several molecules of secondary antibodies covalently bound to several molecules of horse radish peroxidase (HRP). Visualisation occurs via an enzyme-substrate reaction in the presence of a colorising reagent which permits microscopical analysis. The test system is suitable for the detection of mono- and polyclonal primary antibodies and sera obtained from mice and rabbits. In contrast to other detection techniques, which often use the streptavidin-biotin system the Plus HRP Polymer Kit avoids the problem of background staining caused by endogenous biotin in the tissue
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Endogenous peroxidase activity in the tissue may cause non-specific staining. This enzyme activity can be blocked by incubation with 3% H2O2-solution (peroxide block). Background staining caused by unspecific binding of the primary antibody or the secondary antibody in the HRPpolymer is minimized by incubation with a protein blocking solution (Blocking Solution, provided with the kit). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the enhancement reagent (PostBlock) is applied and incubated. A second washing is followed by the application of the HRP-polymer. Any excess of unbound HRP-polymer is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the peroxidase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen AEC leads to the formation of a red-brown product of reaction at the place of the target antigen. The chromogen DAB forms a dark brown precipitate.
Reagents provided:
100 ml Blocking Solution Reagent 1 (ready-to-use) 100 ml PostBlock Reagent 2 (ready-to-use) 100 ml HRP-Polymer (Mouse/Rabbit) Reagent 3 (ready-to-use) Substrate systems recommended: Permanent AEC kit, AEC single solution, AEC substrate kit, DAB substrate kit, DAB High contrast kit. Materials required but not supplied: Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O 3% H2O2 solution Reagents for enzyme digestion or heat pre-treatment Wash buffer PBS or TBS Pink PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solutions should be stored at 2-8°C without further dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. They should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support.
Reagent preparation:
Reagents should be at room temperature when used. ? Deparaffinise and rehydrate paraffin-embedded tissue sections. ? Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. ? Tissue sections have to be completely covered with the different reagents in order to avoid drying out.
Procedure:
1. Peroxide blocking (3 % H2O2 solution) 10 min. 2. Washing with wash buffer 1 x 2 min. 3. Blocking Solution (protein block, Reagent 1) (This step is optional.) 5 min. 4. Washing with wash buffer 1 x 2 min. 5. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 6. Washing with wash buffer 3 x 5 min. 7. PostBlock (Reagent 2, yellow) 20 min. 8. Washing with wash buffer 3 x 5 min. 9. HRP-polymer (Reagent 3, red) 30 min. 10. Washing with wash buffer 3 x 2 min. 11. AEC or DAB (Controlling the colour intensity via light microscope is recommended.) 5-15 min. 12. Stopping the reaction with distilled H2O when the desired colour intensity is attained 13. Counterstaining and blueing 14. Mounting: aqueous with AEC, permanent with DAB
Expected results:
During the reaction of the substrate with horse radish peroxidase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support . No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pretreatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from mouse or rabbit, but from a different species. 6. The antigen/epitope was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. If you used heat pre-treatment or enzyme digestion it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. Incubation times were too short or primary antibody concentration too low. 7. Chromogenic substrate solution was too old. Nonspecific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme polymer or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous horse radish peroxidase in the tissue. Maybe the hydrogen peroxide solution used for blocking was inactivated.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous peroxidase or pseudoperoxidase activity may cause non-specific staining. The enzyme activity is blocked by incubation with hydrogen peroxide solution. Tissues containing Hepatitis B Surface Antigen (HBsAg) may give false positive results with HRP (horse radish peroxidase) detection systems (Omata et al, 1980). Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the Blocking Solution instead of just draining it away as in other procedures. We guarantee that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear. ProClin is used for stabilisation. A material safety data sheet is available upon request.
The Plus HRP Polymer Kit is designed for the qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. It is developed for use in combination with mono- and polyclonal primary antibodies and sera obtained from mice and rabbits. The kit can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus HRP Polymer Kit is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The enzyme polymer in this kit consists of several molecules of secondary antibodies covalently bound to several molecules of horse radish peroxidase (HRP). Visualisation occurs via an enzyme-substrate reaction in the presence of a colorising reagent which permits microscopical analysis. The test system is suitable for the detection of mono- and polyclonal primary antibodies and sera obtained from mice and rabbits. In contrast to other detection techniques, which often use the streptavidin-biotin system the Plus HRP Polymer Kit avoids the problem of background staining caused by endogenous biotin in the tissue
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Endogenous peroxidase activity in the tissue may cause non-specific staining. This enzyme activity can be blocked by incubation with 3% H2O2-solution (peroxide block). Background staining caused by unspecific binding of the primary antibody or the secondary antibody in the HRPpolymer is minimized by incubation with a protein blocking solution (Blocking Solution, provided with the kit). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the enhancement reagent (PostBlock) is applied and incubated. A second washing is followed by the application of the HRP-polymer. Any excess of unbound HRP-polymer is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the peroxidase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen AEC leads to the formation of a red-brown product of reaction at the place of the target antigen. The chromogen DAB forms a dark brown precipitate.
Reagents provided:
6 ml Blocking Solution Reagent 1 (ready-to-use) 6 ml PostBlock Reagent 2 (ready-to-use) 6 ml HRP-Polymer (Mouse/Rabbit) Reagent 3 (ready-to-use) Substrate systems recommended: Permanent AEC kit, AEC single solution, AEC substrate kit, DAB substrate kit, DAB High contrast kit. Materials required but not supplied: Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O 3% H2O2 solution Reagents for enzyme digestion or heat pre-treatment Wash buffer PBS or TBS Pink PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solutions should be stored at 2-8°C without further dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. They should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support.
Reagent preparation:
Reagents should be at room temperature when used. ? Deparaffinise and rehydrate paraffin-embedded tissue sections. ? Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. ? Tissue sections have to be completely covered with the different reagents in order to avoid drying out.
Procedure:
1. Peroxide blocking (3 % H2O2 solution) 10 min. 2. Washing with wash buffer 1 x 2 min. 3. Blocking Solution (protein block, Reagent 1) (This step is optional.) 5 min. 4. Washing with wash buffer 1 x 2 min. 5. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 6. Washing with wash buffer 3 x 5 min. 7. PostBlock (Reagent 2, yellow) 20 min. 8. Washing with wash buffer 3 x 5 min. 9. HRP-polymer (Reagent 3, red) 30 min. 10. Washing with wash buffer 3 x 2 min. 11. AEC or DAB (Controlling the colour intensity via light microscope is recommended.) 5-15 min. 12. Stopping the reaction with distilled H2O when the desired colour intensity is attained 13. Counterstaining and blueing 14. Mounting: aqueous with AEC, permanent with DAB
Expected results:
During the reaction of the substrate with horse radish peroxidase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support . No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pretreatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from mouse or rabbit, but from a different species. 6. The antigen/epitope was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. If you used heat pre-treatment or enzyme digestion it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. Incubation times were too short or primary antibody concentration too low. 7. Chromogenic substrate solution was too old. Nonspecific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme polymer or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous horse radish peroxidase in the tissue. Maybe the hydrogen peroxide solution used for blocking was inactivated.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous peroxidase or pseudoperoxidase activity may cause non-specific staining. The enzyme activity is blocked by incubation with hydrogen peroxide solution. Tissues containing Hepatitis B Surface Antigen (HBsAg) may give false positive results with HRP (horse radish peroxidase) detection systems (Omata et al, 1980). Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the Blocking Solution instead of just draining it away as in other procedures. We guarantee that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear. ProClin is used for stabilisation. A material safety data sheet is available upon request.
The Plus AP Polymer Kit is designed for the qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. It is developed for use in combination with mono- and polyclonal primary antibodies and sera obtained from mice and rabbits. The kit can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus AP Polymer Kit is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The enzyme polymer in this kit consists of several molecules of secondary antibodies covalently bound to several molecules of alkaline phosphatase (AP). Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The test system is suitable for the detection of mono- and polyclonal primary antibodies and sera obtained from mice and rabbits. In contrast to other detection techniques, which often use the streptavidin-biotin system the Plus AP Polymer Kit avoids the problem of background staining caused by endogenous biotin in the tissue.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Background staining caused by unspecific binding of the primary antibody or the secondary antibody in the AP polymer is minimized by incubation with a protein blocking solution (Blocking Solution, provided with this kit). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the enhancement reagent (PostBlock) is applied and incubated. A second washing is followed by the application of the AP-polymer. Any excess of unbound APpolymer is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the alkaline phosphatase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen Permanent AP Red leads to the formation of a magentared product of reaction at the place of the target antigen.
Reagents provided:
100 ml Blocking Solution Reagent 1 (ready-to-use) 100 ml PostBlock Reagent 2 (ready-to-use) 100 ml AP-Polymer (Mouse/Rabbit) Reagent 3 (ready-to-use) Materials required but not supplied Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O Reagents for enzyme digestion or heat pre-treatment Wash buffer PBS or TBS PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solutions should be stored at 2-8°C without further dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. They should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support.
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. Tissue sections have to be completely covered with the different reagents in order to avoid drying out
Procedure:
1. Blocking Solution (protein block, Reagent 1) (This step is optional.) 5 min. 2. Washing with wash buffer 1 x 2 min. 3. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 4. Washing with wash buffer 3 x 5 min. 5. PostBlock (Reagent 2, yellow) 20 min. 6. Washing with wash buffer 3 x 5 min. 7. AP-polymer (Reagent 3, red) 30 min. 8. Washing with wash buffer 3 x 2 min. 9. Permanent AP Red 10-20 min. (Controlling the colour intensity via light microscope is recommended.) 10. Stopping the reaction with distilled H2O when the desired colour intensity is attained 11. Counterstaining and blueing 12. Mounting: permanent or aqueous with Permanent AP Red
Expected results:
During the reaction of the substrate with alkaline phosphatase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please contact our technical support. No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from mouse or rabbit, but from a different species. 6. The antigen/epitope was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. If you used heat pre-treatment or enzyme digestion it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. If you are using PBS-based wash buffer: the activity of alkaline phosphatase in the reagents is blocked if too much wash buffer remains on the slides. 7. Incubation times were too short or primary antibody concentration too low. 8. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme polymer or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous alkaline phosphatase in the tissue. This undesired activity can often be suppressed using levamisole (see section Limitations of the Procedure).
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous alkaline phosphatase activity may cause non-specific staining. The enzyme activity can be blocked by incubation with levamisole. However, neither intestinal nor placental alkaline phosphatase can be blocked with levamisole. Therefore, tissues of this origin should be stained with peroxidase detection systems (i.e. POLHRP-125). Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the BlockingSolution instead of just draining it away as in other procedures. We will guarantee that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear. ProClin 300 and ProClin 950 used for stabilisation. Material safety data sheets (MSDS) are available upon request.
The Plus AP Polymer Kit is designed for the qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. It is developed for use in combination with mono- and polyclonal primary antibodies and sera obtained from mice and rabbits. The kit can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus AP Polymer Kit is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The enzyme polymer in this kit consists of several molecules of secondary antibodies covalently bound to several molecules of alkaline phosphatase (AP). Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The test system is suitable for the detection of mono- and polyclonal primary antibodies and sera obtained from mice and rabbits. In contrast to other detection techniques, which often use the streptavidin-biotin system the Plus AP Polymer Kit avoids the problem of background staining caused by endogenous biotin in the tissue.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Background staining caused by unspecific binding of the primary antibody or the secondary antibody in the AP polymer is minimized by incubation with a protein blocking solution (Blocking Solution, provided with this kit). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the enhancement reagent (PostBlock) is applied and incubated. A second washing is followed by the application of the AP-polymer. Any excess of unbound APpolymer is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the alkaline phosphatase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen Permanent AP Red leads to the formation of a magentared product of reaction at the place of the target antigen.
Reagents provided:
6 ml Blocking Solution Reagent 1 (ready-to-use) 6 ml PostBlock Reagent 2 (ready-to-use) 6 ml AP-Polymer (Mouse/Rabbit) Reagent 3 (ready-to-use) Materials required but not supplied Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O Reagents for enzyme digestion or heat pre-treatment Wash buffer PBS or TBS PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solutions should be stored at 2-8°C without further dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. They should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support.
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. Tissue sections have to be completely covered with the different reagents in order to avoid drying out
Procedure:
1. Blocking Solution (protein block, Reagent 1) (This step is optional.) 5 min. 2. Washing with wash buffer 1 x 2 min. 3. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 4. Washing with wash buffer 3 x 5 min. 5. PostBlock (Reagent 2, yellow) 20 min. 6. Washing with wash buffer 3 x 5 min. 7. AP-polymer (Reagent 3, red) 30 min. 8. Washing with wash buffer 3 x 2 min. 9. Permanent AP Red 10-20 min. (Controlling the colour intensity via light microscope is recommended.) 10. Stopping the reaction with distilled H2O when the desired colour intensity is attained 11. Counterstaining and blueing 12. Mounting: permanent or aqueous with Permanent AP Red
Expected results:
During the reaction of the substrate with alkaline phosphatase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please contact our technical support. No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from mouse or rabbit, but from a different species. 6. The antigen/epitope was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. If you used heat pre-treatment or enzyme digestion it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. If you are using PBS-based wash buffer: the activity of alkaline phosphatase in the reagents is blocked if too much wash buffer remains on the slides. 7. Incubation times were too short or primary antibody concentration too low. 8. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme polymer or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous alkaline phosphatase in the tissue. This undesired activity can often be suppressed using levamisole (see section Limitations of the Procedure).
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous alkaline phosphatase activity may cause non-specific staining. The enzyme activity can be blocked by incubation with levamisole. However, neither intestinal nor placental alkaline phosphatase can be blocked with levamisole. Therefore, tissues of this origin should be stained with peroxidase detection systems (i.e. POLHRP-125). Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the BlockingSolution instead of just draining it away as in other procedures. We will guarantee that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear. ProClin 300 and ProClin 950 used for stabilisation. Material safety data sheets (MSDS) are available upon request.
The causes and effects of neuronal degeneration are of major interest to a wide variety of neuroscientists. Paralleling this growing interest is an increasing number of methods applicable to the detection of neuronal degeneration. Fluoro-Jade C stains all degenerating neurons regardless of specific insult or mechanism of cell death. Fluoro-Jade C exhibits the greatest signal to background ratio, as well as the highest resolution. This translates to a stain of maximal contrast and affinity for degenerating neurons. This makes it ideal for localising not only degenerating nerve cell bodies but also distal dendrites, axons and terminals. The dye is highly resistant to fading and is compatible with virtually all histological processing and staining protocols. Note: This product is equivalent to discontinued product AG325 from Merck-Millipore.
Product Type:
Staining Reagent
Format:
Dry, Coffee brown to brick red powder; hygroscopic powder keep dessicated.
Species Reactivity:
Human,Mouse,Other Mammals (Predicted),Rat
Applications:
FC,ICC
Application Details:
Following our detailed protocol, Fluoro-Jade B labels degenerating neurons which are visualised with blue light excitation, while DAPI (not included) counter stains cell nuclei, visualised with ultra-violet illumination. The Fluoro-Jade C dye can be used on all kinds of preserved tissues, including fresh-frozen, paraformaldehyde or formalin fixed, and formalin fixed, paraffin-embedded tissues.
Alternative Names:
FJC, Fluoro-Jade
Biosensis Brand:
Biosensis®
Detection Method:
Fluorescence
Excitation/Emission:
FJC visualization is accomplished using blue light or a 488 nm Laser. <br>Excitation Peak: 495 nm<br>Emission Peak: 521 nm<br>Filter system for visualizing: Fluorescein/FITC
Shelf Life:
6 months after date of receipt (unopened vial).
Use:
For research use only.
Kit Components:
Materials Provided: 30 mg Fluoro-Jade C, dry powder Detailed protocol Equipment and Reagents Required: Distilled water ACS grade Ethanol (200 proof) for slide & solution preparation 1% sodium hydroxide in 80% ethanol (basic alcohol solution) 0.1% Acetic Acid solution (in water) 70% ethanol in distilled water 0.06% (KMnO4) potassium permanganate solution DAPI powder or 100X solution (working range is 0.5-5 µg/mL) Xylene liquid Staining dishes/Coplin jars Cover slips DPX mounting media or another permanent mounting medium. Non-polar media are preferred over aqueous mounting media such as glycerin/water to obtain high- contrast images (refer to Appendix B in the protocol for a comparative analysis). Traditional fluorescent mounting mediums are not recommended because of their high pH. Slide warmer Convection oven
Specificity:
Degenerating neurons, and neuronal degeneration. There is no specific staining in normal healthy brain. Note: Some researchers under some conditions report blood vessel staining with Fluoro Jade. This may be because Fluoro Jade is an analogue of eosin (which stains blood cells). In general, good perfusion and preparation of the tissue should help prevent blood vessel staining but it may not be possible to eliminate it entirely. In our experience it is generally possible to distinguish neuronal from blood vessels staining by eye.
Storage:
The powdered dye can be stored desiccated at room temperature in the dark. Storage in a desiccator is recommended as FJB is hydroscopic. The 0.01% stock solution will remain stable for 3 months when stored in a refrigerator, in the dark. The 0.0001-0.0004% working solution in 0.1% acetic acid should be used within 4 hours of preparation. Diluted FJB dye solutions are not stable and should not be stored. The other diluted solutions can be reused and stored for up to 48 hours if refrigerated and protected from light. Best results require freshly diluted solutions.
Purification:
Silica TLC (acetanitrile/water, 6/4) revealed the presence of 2 fluorescent spots, presumably corresponding to the mono and di sulphate homologues. The presence of precursors or free fluorescein was not detected.
The causes and effects of neuronal degeneration are of major interest to a wide variety of neuroscientists. Paralleling this growing interest is an increasing number of methods applicable to the detection of neuronal degeneration. The fluorescent dye Fluoro-Jade B (FJB), like its more purified brother Fluoro-Jade C (FJC), is an anionic fluorescein derivative useful for the histological staining of neurons undergoing degeneration. Fluoro-Jade B differs from FJC in that it is a slightly less refined chemical formulation and thus it does not quite provide the same level of signal to noise or high resolution as FJC. Nonetheless FJB is still widely used and works very well as a marker of degenerating neurons and even glia (see Damjanac M et al., Brain Res. 2007;1128(1):40-9). FJB operates nearly identically in protocol to that of FJC, and Fluoro-Jade B is compatible with several other labeling procedures including immunofluorescent and fluorescent Nissl techniques. Fluoro-Jade B stains all degenerating neurons regardless of specific insult or mechanism of cell death. Fluoro-Jade B exhibits the greatest signal to background ratio, as well as the highest resolution. This translates to a stain of maximal contrast and affinity for degenerating neurons. This makes it ideal for localising not only degenerating nerve cell bodies but also distal dendrites, axons and terminals. The dye is highly resistant to fading and is compatible with virtually all histological processing and staining protocols. Note: This product is equivalent to discontinued product AG310 from Merck-Millipore.
Product Type:
Staining Reagent
Format:
Dry, Coffee brown to brick red powder; hygroscopic powder keep dessicated.
Species Reactivity:
Human,Mouse,Other Mammals (Predicted),Rat
Applications:
FC,ICC
Application Details:
Following our detailed protocol, Fluoro-Jade C labels degenerating neurons which are visualised with blue light excitation, while DAPI (not included) counter stains cell nuclei, visualised with ultra-violet illumination. The Fluoro-Jade C dye can be used on all kinds of preserved tissues, including fresh-frozen, paraformaldehyde or formalin fixed, and formalin fixed, paraffin-embedded tissues.
Alternative Names:
FJB, Fluoro-Jade
Biosensis Brand:
Biosensis®
Detection Method:
Fluorescence
Excitation/Emission:
FJB visualization is accomplished using blue light or a 488 nm Laser. <br>Excitation Peak: 495 nm<br>Emission Peak: 521 nm<br>Filter system for visualizing: Fluorescein/FITC
Shelf Life:
6 months after date of receipt (unopened vial).
Use:
For research use only.
Kit Components:
Materials Provided: 30 mg Fluoro-Jade B, dry powder Detailed protocol Equipment and Reagents Required: Distilled water ACS grade Ethanol (200 proof) for slide & solution preparation 1% sodium hydroxide in 80% ethanol (basic alcohol solution) 0.1% Acetic Acid solution (in water) 70% ethanol in distilled water 0.06% (KMnO4) potassium permanganate solution DAPI powder or 100X solution (working range is 0.5-5 µg/mL) Xylene liquid Staining dishes/Coplin jars Cover slips DPX mounting media or another permanent mounting medium. Non-polar media are preferred over aqueous mounting media such as glycerin/water to obtain high- contrast images (refer to Appendix B in the protocol for a comparative analysis). Traditional fluorescent mounting mediums are not recommended because of their high pH. Slide warmer Convection oven
Product references:
Ikeda A et al. (2022) "Alteration of the neuronal and glial cell profiles in Neu1-deficient zebrafish" Glycoconj ; [Epub ahead of print]; Application: ICC/FC Species: Zebrafish
Choi I et al. (2022) "Interleukin-17A Mediates Hippocampal Damage and Aberrant Neurogenesis Contributing to Epilepsy-Associated Anxiety" Front. Mol. Neurosci. ; [Epub ahead of print]; Application: ICC/FC Species: Mouse
Cui Y et al. (2022) "Modified Citrus Pectin Alleviates Cerebral Ischemia/Reperfusion Injury by Inhibiting NLRP Inflammasome Activation via TLR4/NF-?B Signaling Pathway in Microglia" J Inflamm. Res. ; 15: 3369-3385; Application: ICC/FC Species: Mouse
Specificity:
Degenerating neurons, and neuronal degeneration. There is no specific staining in normal healthy brain. Note: Some researchers under some conditions report blood vessel staining with Fluoro Jade. This may be because Fluoro Jade is an analogue of eosin (which stains blood cells). In general, good perfusion and preparation of the tissue should help prevent blood vessel staining but it may not be possible to eliminate it entirely. In our experience it is generally possible to distinguish neuronal from blood vessels staining by eye.
Storage:
The powdered dye can be stored desiccated at room temperature in the dark. Storage in a desiccator is recommended as FJB is hydroscopic. The 0.01% stock solution will remain stable for 3 months when stored in a refrigerator, in the dark. The 0.0001-0.0004% working solution in 0.1% acetic acid should be used within 4 hours of preparation. Diluted FJB dye solutions are not stable and should not be stored. The other diluted solutions can be reused and stored for up to 48 hours if refrigerated and protected from light. Best results require freshly diluted solutions.
Purification:
Thin layer chromatograpy using cellulose plates and a solvent system of n-propinol, water, and ammonium hydroxide (6:5:2) revealed the presence of two fluorescent isomers and two trace non-fluorescent bands. No amount of fluorescein or Fluoro-Jade was present.
Target:
Degenerating neurons
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