The TRIS/EDTA buffer, is designed for use during heat-induced epitope retrieval (HIER) on formalinfixed paraffin-embedded tissue sections prior to antibody application. In immunohistochemistry (IHC), protein cross-links are formed during formalin or other aldehyde fixation. These cross-links mask the antigenic sites in tissue specimens and results in weak or false negative staining in immunohistochemical detection of proteins. The use of this citrate buffer in combination with heat ensures that the antigenicity of proteins modified, thus enhancing staining intensity of antibodies. For the recommended pre-treatment technique, please refer to the individual antibody's instructions for use. This buffer is a 10x concentrate solution and must be diluted 1:10 with deionized water before using. The final readyto-use solution should have a pH of 9.0 ± 0.3. The buffer can be used for a maximum of three runs within five days after dilution. Also available in 250 ml, 500 ml en 1000 ml.
Principle of method:
Heat-Induced Epitope Retrieval
Reagents provided:
100 ml 10x concentrate solution. TRIS/EDTA buffer, Heat-Induced Epitope Retrieval (10x, Tween20), Blue
Storage and handling:
Store at room temperature
Reagent preparation:
Before use dilute 1:10 with deionized water. The final ready-to-use solution should have a pH of 9.0 ± 0,3,
Procedure:
1. Deparaffinize and rehydrate the tissue slides. 2. Fill the PT Module tank with 150 ml of TRIS?EDTA buffer and 1,350 ml of deionized water, to obtain 1,500 ml of ready-to-use solution (1:10 dilution). 3. Pre-heat the PT Module until temperature reaches 65°C and place the formalin-fixed slides in slide racks into the PT Module tank. 4. Heat the TRIS/EDTA buffer and slides to 98°C and incubate for 15 minutes. 5. Cool slides in the PT Module to 65°C. 6. Remove slides and cool to room temperature for at least 5 minutes in a PBS or TBS based buffer. 7. Continue with staining according to IHC protocol. Note: Alternative heating sources, such as a microwave or a pressure cooker, can be used to replace the PT Module tank. The optimal incubation time for these heating sources should be determined by authorised personnel / researchers.
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.
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.
CryoCompound freezing medium is an aqueous based frozen tissue embedding medium designed to support tissue blocks in cryostat sectioning. Formulated to promote rapid freezing, enhanced sectioning and consistent results at a working temperature of -20° C. The ready-to-use Cryocompound is available in different colors: MON-APP900C (Transparant/colorless), MON-APP900B (Blue), MON-APP900Y (Yellow), MON-APP900O (Orange), MON-APP900G (green), MON-APP900R (Red) or Multicolorkit MON-APP900BYGR (Blue, Yellow, Green, Red).
Principle of method:
Freezing medium
Reagents provided:
4x 100 ml Cryocompound, freezing medium (ready-to-use)
Storage and handling:
Store at room temperature
Procedure:
1. Place few drops of CryoCompound (depends on the size of the sample to be embedded) onto the center of the bottom of cryomold. Be careful to select the proper size embedding mold according to the size of the sample to be embedded. 2. Try to avoid the formation of air bubbles. Remove any bubbles inside the CryoCopound. This is important because the air bubbles will create problems when cutting sections. Air bubbles create freeze- thaw-freeze cycle and ice crystal will form inside of it and result in a very bad morphology due to the ice crystal artefact. 3. Let it settle for 15-30 seconds to allow the CryoCompound to completely wet the surface of the tissue. Hardening of the CryoCompound included the sample (it will happen in 0.5-1 minute) before preparing the CryoCompoundsample-block.
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.
Trypsin Pretreatment Kit consists of 2 reagents for the preparation of a trypsin solution used for enzymatic epitope retrieval on formalin-fixed tissue sections on slides. This procedure (sometimes called PIER, Protease Induced Epitope Retrieval) is primarily used in immunohistochemical staining procedures. Trypsin Pretreatment Kit is for research use only.
Immunohistochemical staining procedures consist of sequential incubation steps with blocking solutions, antibodies and secondary reagents, enzymes and chromogenic substrates carried out on tissue sections. These tissue sections are mostly prepared out of formalin-fixed paraffin-embedded tissue blocks. Cellular structures are very effectively stabilised by formalin fixation which results in optimal morphological preservation of the sample. On the other hand the formalin fixation leads to strong cross-links between proteins. This means that epitopes of antigens are being masked and often are no longer accessible for primary antibodies. In order to enable primary antibodies to bind to antigens the epitopes have to be recovered. Enzymatic digestion with proteolytic enzymes (PIER) restores structures of the epitopes making them more accessible to specific antibodies. Heat induced epitope retrieval (HIER) in buffer solutions of different compositions and pH-values is another way of recovering epitopes. The primary antibody used determines the appropriate method.
Principle of method:
The components of this Trypsin Pretreatment Kit allow for the preparation of a buffered trypsin solution for enzymatic epitope retrieval.
Reagents provided:
30 ml Trypsin Solution 125 ml Trypsin Buffer
Storage and handling:
The solutions should be stored at 2-8°C. Please sto re 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. 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 this reagent, please contact our technical support.
Reagent preparation:
Mix 1 part Trypsin Solution with 3 parts Trypsin Buffer. The activity of the resulting trypsin solution can be adjusted by variation of the mixing ratio. Mix the two components in the ratio 1:1 when strong epitope retrieval is desired. The working solution is stable for at least one week if stored at 2-8°C.
Procedure:
Trypsin Pretreatment Kit is suitable for enzymatic epitope retrieval carried out after the dewaxing and rehydration of the sections. 1. Cover deparaffinised and rehydrated tissue sections with trypsin working solution. 2. Incubate for 10 - 20 minutes at 37°C. 3. Rinse carefully (3 x) with wash buffer. 4. Proceed with immunohistological staining as usual.
Expected results:
During the reaction of the substrate with horse radish peroxidase or 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 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 technique involving both histological and immunological detection methods. It requires a highly trained histotechnologist. 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. 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 reagent and specimen with eye, skin or mucous membranes. In case of reagent or specimen coming into contact with a sensitive area, wash area with large amounts of water. A material safety data sheet (MSDS) is available upon request.
The blocking solution Peroxide Block is intended for inhibition of endogenous peroxidase activity in tissue sections. It is primarily intended to be used in immunohistochemistry on formalin-fixed paraffin-embedded samples if using a detection system with horse radish peroxidase.
Endogenous peroxidase activity in the tissue can result in unspecific background staining in immunohistochemical staining procedures using horse radish peroxidase (HRP) as detection enzyme. This effect can be eliminated when tissue sections are incubated with Peroxide Block prior to immunohistochemical staining. Hydrogen peroxide in the solution blocks the activity of endogenous peroxidase.
Principle of method:
Peroxide Block is applied onto tissue sections to reduce non-specific staining due to endogenous peroxidase activity in immunohistochemistry. The step is carried out before incubation with primary antibody but after dewaxing and rehydration. If a heat induced epitope retrieval (HIER) or enzymatic digestion is necessary for immunohistochemical detection it is of no importance if the Peroxide Block is used before or after this step. In some cases it has been shown, that blocking of endogenous peroxidase before the epitope retrieval leads to better results.
Reagents provided:
100 ml Peroxide Block (ready-to-use)
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. Avoid exposure to strong light. Under these conditions the solution is stable up to the expiry date indicated on the label. Do not use product 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 this reagent, please contact our technical support.
Procedure:
1. Apply Peroxide Block for 10 minutes at room temperature. The section should be covered completely. 2. Rinse with wash buffer. 3. Proceed with next steps for immunohistochemical staining as usual starting with protein blocking or the primary antibody.
Expected results:
During the reaction of the substrate with horse radish peroxidase or 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 please read these instructions carefully, contact our technical support. Also refer to the instructions of the detection systems containing Peroxide Block 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 binding. 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 technique involving both histological and immunological detection methods. It requires a highly trained histotechnologist. 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. 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 reagent or specimen with eye, skin or mucous membrane. In case of the reagent or specimen 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 may occur. 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.
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 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 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.
Pepsin Solution is a ready-to-use solution developed for enzymatic epitope retrieval on formalin-fixed tissue sections on slides. This procedure (sometimes called PIER, Protease Induced Epitope Retrieval) is primarily used in immunohistochemical staining procedures.
Immunohistochemical staining procedures consist of sequential incubation steps with blocking solutions, antibodies and secondary reagents, enzymes and chromogenic substrates carried out on tissue sections. These tissue sections are mostly prepared out of formalin-fixed paraffin-embedded tissue blocks. Cellular structures are very effectively stabilised by formalin fixation which results in optimal morphological preservation of the sample. On the other hand the formalin fixation leads to strong cross-links between proteins. This means that epitopes of antigens are being masked and often are no longer accessible for primary antibodies. In order to enable primary antibodies to bind to antigens the epitopes have to be recovered. Enzymatic digestion with proteolytic enzymes (PIER) restores structures of the epitopes making them more accessible to specific antibodies. Heat induced epitope retrieval (HIER) in buffer solutions of different compositions and pH-values is another way of recovering epitopes. The primary antibody used determines the appropriate method.
Principle of method:
Pepsin Solution is a ready-to-use stabilised pepsin solution for enzymatic epitope retrieval.
Reagents provided:
60 ml Pepsin Solution (Ready-To-Use)
Storage and handling:
The solution should be stored at 2-8°C without furt her dilution. 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. 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 this reagent, please contact our technical support
Reagent preparation:
The solution is ready-to-use.
Procedure:
Pepsin Solution is suitable for enzymatic epitope retrieval carried out after the dewaxing and rehydration of the tissue sections. 1. Cover deparaffinised and rehydrated tissue sections with ready-to-use Pepsin Solution. 2. Incubate for 10 - 15 minutes at 37°C or 20 30 minutes at room temperature. The optimal incubation time needs to be elaborated by the operator. It was shown that in individual cases a stronger signal can be obtained when the incubation time is elongated up to 120 minutes (e. g. for detection of Collagen IV with different antibodies). 3. Rinse carefully (3 x) with wash buffer. 4. Proceed with immunohistological staining as usual.
Expected results:
During the reaction of the substrate with horse radish peroxidase or 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 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 technique involving both histological and immunological detection methods. It requires a highly trained histotechnologist. 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. 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 reagent and specimen with eye, skin or mucous membranes. In case of reagent or specimen coming into contact with a sensitive area, wash area with large amounts of water. 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 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.
HIER Citrate Buffer pH 6.0 is a solution developed for heat induced epitope retrieval (HIER) in formalin-fixed paraffin-embedded tissue sections on slides. This procedure is primarily used in immunohistochemistry.
Immunohistochemical staining procedures consist of sequential incubation steps with blocking solutions, antibodies and secondary reagents, enzymes and chromogenic substrates carried out on tissue sections. These tissue sections are mostly prepared out of formalin-fixed paraffin-embedded tissue blocks. Cellular structures are very effectively stabilised by formalin fixation which results in optimal morphological preservation of the sample. On the other hand the formalin fixation leads to strong cross-links between proteins. This means that epitopes of antigens are being masked and often are no longer accessible for primary antibodies. In order to enable primary antibodies to bind to the antigens the epitopes have to be recovered. Heat induced epitope retrieval (HIER) in buffer solutions of different compositions and pH-values restore structures of the epitopes making them more accessible to specific antibodies. Enzymatic digestion with proteolytic enzymes is another way of recovering epitopes. The primary antibody used determines the appropriate method.
Principle of method:
HIER Citrate Buffer pH 6.0 is a 10fold concentrated citrate buffer solution with additives of stabilising substances. For preparation of the working strength solution the buffer concentrate is diluted 1:10 with deionised or distilled water. The resulting solution has a pH of 6.0 (5.8 to 6.2). HIER Citrate Buffer pH 6.0 is a very efficient epitope retrieval solution in immunohistochemical staining procedures to be used with primary antibodies of many different specificities.
Reagents provided:
500 ml HIER Citrate Buffer pH 6.0 (10fold concentrated, adequate for 5 litres ready-to-use citrate buffer)
Storage and handling:
The solution should be stored at 2-8°C. Do not free ze 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 stored at room temperature the solution is stable for at least 10 month from the date of delivery. The prepared working strength solution is stable for 1 month, if stored at 2-8°C. A positive and a negat ive 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 this reagent, please contact our technical support.
Reagent preparation:
Preparation of the citrate buffer working strength solution: Dilute HIER Citrate Buffer concentrate 1:10 with deionised or distilled water and mix thoroughly. The pH-value should be at 6.0 (5.8 to 6.2). If necessary adjust pH-value with diluted NaOH or HCl solution.
Procedure:
HIER Citrate Buffer is suitable for various HIER-methods such as steamer, pressure cooker, autoclave, water bath, and microwave oven. Tissue sections used in heat induced epitope retrieval should always be placed on adhesive slides. Epitope retrieval is carried out after dewaxing and rehydration of the sections. Exemplary protocol using steamer: 1. Prepare the working strength solution by diluting the buffer concentrate as described above and transfer to a Coplin jar. Please make sure that there is enough volume to cover the tissue sections on the slides completely. 2. Fill steamer with water according to instruction manual, close lid and start. 3. After 10 minutes place Coplin jar with citrate buffer in the steamer, close the lid and heat the solution for 20 minutes. 4. Place slides with tissue sections into the preheated solution and close the lid. Tissue sections have to be completely covered with citrate buffer solution. 5. Incubate slides 20 40 minutes. The optimal incubation time needs to be elaborated by the operator. 6. After the incubation take the Coplin jar with slides out of steamer and let cool down at room temperature for about 20 minutes. 7. Remove citrate buffer, rinse slides with wash buffer and proceed with immunohistological staining.
Expected results:
During the reaction of the substrate with horse radish peroxidase or 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 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 binding. 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 technique involving both histological and immunological detection methods. It requires a highly trained histotechnologist. 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. 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 reagent or specimen with eye, skin or mucous membrane. In case of the reagent or specimen 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 may occur. ProClin 300 is used for stabilisation. A material safety data sheet (MSDS) is available upon request.
HIER EDTA Buffer pH 8.0 is a solution developed for heat induced epitope retrieval (HIER) in formalin-fixed paraffinembedded tissue sections on slides. This procedure is primarily used in immunohistochemistry.
Immunohistochemical staining procedures consist of sequential incubation steps with blocking solutions, antibodies and secondary reagents, enzymes and chromogenic substrates carried out on tissue sections. These tissue sections are mostly prepared out of formalin-fixed paraffin-embedded tissue blocks. Cellular structures are very effectively stabilised by formalin fixation which results in optimal morphological preservation of the sample. On the other hand the formalin fixation leads to strong cross-links between proteins. This means that epitopes of antigens are being masked and often are no longer accessible for primary antibodies. In order to enable primary antibodies to bind to the antigens the epitopes have to be recovered. Heat induced epitope retrieval (HIER) in buffer solutions of different compositions and pH-values restore structures of the epitopes making them more accessible to specific antibodies. Enzymatic digestion with proteolytic enzymes is another way of recovering epitopes. The primary antibody used determines the appropriate method.
Principle of method:
HIER EDTA Buffer pH 8.0 is a 10fold concentrated, buffered EDTA solution with additives of detergent and stabilising substances. For preparation of the working strength solution the buffer concentrate is diluted 1:10 with deionised or distilled water. The resulting solution has a pH of 8.0 (7.8 to 8.2). HIER EDTA Buffer pH 8.0 is a very efficient epitope retrieval solution in immunohistochemical staining procedures to be used with primary antibodies of different specificity. HIER EDTA Buffer pH 8.0 leads to considerably stronger signals compared with usually used citrate buffer.
Reagents provided:
500 ml HIER EDTA Buffer pH 8.0 (10fold concentrated, adequate for 5 litres ready-to-use EDTA Buffer)
Storage and handling:
The solution should be stored at 2-8°C. Do not free ze 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 stored at room temperature the solution is stable for at least 10 month from the date of delivery. The prepared working strength solution is stable for 1 month, if stored at 2-8°C. 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 this reagent, please contact our technical support.
Reagent preparation:
Preparation of the EDTA buffer working strength solution: Dilute HIER EDTA Buffer concentrate 1:10 with deionised or distilled water and mix thoroughly. The pH-value should be at 8.0 (7.8 to 8.2). If necessary adjust pH-value with diluted NaOH or HCl solution.
Procedure:
HIER EDTA Buffer is suitable for various HIER-methods such as steamer, pressure cooker, autoclave, water bath, and microwave oven. Tissue sections used in heat induced epitope retrieval should always be placed on adhesive slides. Epitope retrieval is carried out after dewaxing and rehydration of the sections. Exemplary protocol using steamer: 1. Prepare the working strength solution by diluting the buffer concentrate as described above and transfer to a Coplin jar. Please make sure that there is enough volume to cover the tissue sections on the slides completely. 2. Fill steamer with water according to instruction manual, close lid and start. 3. After 10 minutes place Coplin jar with EDTA buffer in the steamer, close lid and heat the solution for 20 minutes. 4. Place slides with tissue sections into the preheated solution and close the lid. Tissue sections have to be completely covered with EDTA buffer solution. 5. Incubate slides 20 40 minutes. The optimal incubation time needs to be elaborated by the operator. 6. After the incubation take the Coplin jar with slides out of steamer and let cool down at room temperature for about 20 minutes. 7. Remove EDTA buffer, rinse slides with wash buffer and proceed with immunohistological staining.
Expected results:
During the reaction of the substrate with horse radish peroxidase or 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 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 binding. 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 technique involving both histological and immunological detection methods. It requires a highly trained histotechnologist. 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. 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 reagent and specimen with eye, skin or mucous membranes. In case of reagent or specimen coming into contact with a sensitive area, wash area with large amounts of water. Microbial contamination of the reagent must be avoided, since otherwise non-specific staining may occur. ProClin 300 is used for stabilisation. A material safety data sheet (MSDS) is available upon request.
HIER T-EDTA Buffer pH 9.0 is a solution developed for heat induced epitope retrieval (HIER) in formalin-fixed paraffinembedded tissue sections on slides. This procedure is primarily used in immunohistochemistry.
Immunohistochemical staining procedures consists of sequential incubation steps with blocking solutions, antibodies and secondary reagents, enzymes and chromogenic substrates carried out on tissue sections. These tissue sections are mostly prepared out of formalin-fixed paraffin-embedded tissue blocks. Cellular structures are very effectively stabilised by formalin fixation which results in optimal morphological preservation of the sample. On the other hand the formalin fixation leads to strong cross-links between proteins. This means that epitopes of antigens are being masked and often are no longer accessible for primary antibodies. In order to enable primary antibodies to bind to antigens the epitopes have to be recovered. Heat induced epitope retrieval (HIER) in buffer solutions of different compositions and pH-values restore structures of the epitopes making them more accessible to specific antibodies. Enzymatic digestion with proteolytic enzymes is another way of recovering epitopes. The primary antibody used determines the appropriate method.
Principle of method:
HIER T-EDTA Buffer pH 9.0 is a 10fold concentrated EDTA solution in Tris buffer with additives of detergent and stabilising substances. For preparation of the working strength solution the buffer concentrate is diluted 1:10 with deionised or distilled water. The resulting solution has a pH of 9.0 (8.8 to 9.2). HIER T-EDTA Buffer pH 9.0 is a very efficient epitope retrieval solution in immunohistochemical staining procedures to be used with primary antibodies of many different specificities. It leads to considerably stronger signals compared with usually used citrate buffer.
Reagents provided:
500 ml HIER T-EDTA Buffer pH 9.0 (10fold concentrated, adequate for 5 litres ready-to-use T-EDTA Buffer)
Storage and handling:
The solution should be stored at 2-8°C. Do not free ze 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 stored at room temperature the solution is stable for at least 10 month from the date of delivery. The prepared working strength solution is stable for 1 month, if stored at 2-8°C. 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 this reagent, please contact our technical support.
Reagent preparation:
Preparation of the T-EDTA buffer working strength solution: Dilute HIER T-EDTA Buffer concentrate 1:10 with deionised or distilled water and mix thoroughly. The pH-value should be at 9.0 (8.8 to 9.2). If necessary adjust pH-value with diluted NaOH or HCl solution.
Procedure:
HIER T-EDTA Buffer is suitable for various HIER-methods such as steamer, pressure cooker, autoclave, water bath, and microwave oven. Tissue sections used in heat induced epitope retrieval should always be placed on adhesive slides. Epitope retrieval is carried out after dewaxing and rehydration of the sections. Exemplary protocol using steamer: 1. Prepare the working strength solution by diluting the buffer concentrate as described above and transfer to a Coplin jar. Please make sure that there is enough volume to cover the tissue sections on the slides completely. 2. Fill steamer with water according to instruction manual, close lid and start. 3. After 10 minutes place Coplin jar with T-EDTA buffer in the steamer, close the lid and heat the solution for 20 minutes. 4. Place slides with tissue sections into the preheated solution and close the lid. Tissue sections have to be completely covered with T-EDTA buffer solution. 5. Incubate slides 20 40 minutes. The optimal incubation time needs to be elaborated by the operator. 6. After the incubation take the Coplin jar with slides out of steamer and let cool down at room temperature for about 20 minutes. 7. Remove T-EDTA buffer, rinse slides with wash buffer and proceed with immunohistological staining.
Expected results:
During the reaction of the substrate with horse radish peroxidase or 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 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 binding. 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 technique involving both histological and immunological detection methods. It requires a highly trained histotechnologist. 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. 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 reagent and specimen with eye, skin or mucous membranes. In case of reagent or specimen coming into contact with a sensitive area, wash area with large amounts of water. Microbial contamination of the reagent must be avoided, since otherwise non-specific staining may occur. 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 wash Buffer is designed as washing solution for immunohistochemical and immunocytological staining procedures on slides. Wash Buffer is primarily used with formalin-fixed paraffin-embedded tissue sections, but also with frozen, HOPE-fixed, and cytological samples as well as in immunoblot procedures. The Wash Buffer is suitable for manually operated and automated immunohistochemical staining.
Immunohistochemical staining procedures consist of sequential incubation steps with blocking solutions, antibodies and secondary reagents, enzymes and chromogenic substrates carried out on tissue sections. Washing away the applied reagents after each incubation step is critical to receive optimally stained samples. The Wash Buffer is especially designed for effective washing and therefore ensures brilliant staining results.
Principle of method:
The Wash Buffer is a 20fold concentrated phosphate buffer with additives of sodium chloride, detergent, and stabilising substances. For preparation of the working strength solution the buffer concentrate is diluted 1:20 with deionised or distilled water. The resulting solution has a pH of 7.2 (7.0 to 7.4). The Wash Buffer is wetting the tissue sections with detergent and thus reduces surface tension and improves spreading the reagents on the tissue section, reduces unspecific binding of reagents on the tissue sample, and because of the exact tuned salt concentration effects an excellent preservation of cell morphology.
Reagents provided:
2500 ml Wash Buffer (20fold concentrated, adequate for 50 litres ready-to-use wash buffer)
Storage and handling:
The solution should be stored at room temperature. It is stable up to the expiry date indicated on the label if undiluted. Do not use product after the expiry date. The diluted working strength solution is stable for about 1 week depending on the ambient temperature. 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 this reagent, please contact our technical support.
Reagent preparation:
Preparation of the Wash Buffer working strength solution: Dilute Wash Buffer concentrate 1:20 with deionised or distilled water and mix thoroughly. The pH-value should be at 7.2 (7.0 to 7.4). If necessary adjust pH-value with diluted NaOH or HCl solution.
Expected results:
During the reaction of the substrate with horse radish peroxidase or 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 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 technique involving both histological and immunological detection methods. It requires a highly trained histotechnologist. 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 Wash Buffer is a 20fold concentrated solution with a mildly acidic pH-value. The correct pHvalue of about 7.0 (+/- 0.2) is achieved after diluting the solution 1:20. Sometimes deionised water has pH-values considerably different from the neutral point (pH 7.0) depending on the preparation method. Experiments have shown that the Wash Buffer can successfully be diluted with deionised or distilled with water in the pH-range of 5.5 up to 9.5. If a detection system with alkaline phosphatase is used please note: larger amounts of wash buffer remaining on the slides can lead to decreasing enzyme activity. 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 reagent or specimen with eye, skin or mucous membrane. In case of the reagent or specimen 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 may occur. ProClin 300 is used for stabilisation. A material safety data sheet (MSDS) is available upon request.
Blocking Solution is developed to eliminate unspecific binding of primary and secondary antibodies to tissue sections. It is primarily intended to be used in immunohistochemistry on formalin-fixed paraffin-embedded samples.
Unspecific binding of primary and secondary antibodies to tissue sections in immunohistochemical staining procedures can result in background staining. This effect can be eliminated when tissue sections are incubated with Blocking Solution prior to incubation with the primary antibody. The protein in Blocking Solution abolishes unspecific binding. Blocking Solution is a universal blocking reagent. Unlike other frequently used blocking solutions (e. g. serum blocks) the reagent can be used regardless of the origin species of the secondary antibody. Interferences with secondary antibodies or other components of detection systems are not observed. In contrast to other blocking reagents this Blocking Solution should not be incubated longer than 10 minutes and should be rinsed away with wash buffer. Otherwise the signal intensity of the immunohistochemical staining reaction could be decreased.
Principle of method:
Blocking Solution is applied on tissue sections to reduce background staining due to unspecific binding of primary and secondary antibodies in immunohistochemistry. The step is carried out prior to incubation with the primary antibody
Reagents provided:
100 ml Blocking Solution (ready-to-use)
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. Do not use product 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 this reagent, please contact our technical support.
Procedure:
1. Apply Blocking Solution for 5 minutes at room temperature. The section should be covered completely. 2. Rinse with wash buffer. 3. Proceed with next steps for immunohistochemical staining as usual starting with the primary antibody.
Expected results:
During the reaction of the substrate with horse radish peroxidase or 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 please read these instructions carefully, contact our technical support . Also refer to the instructions of the detection systems containing Blocking Solution 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 technique involving both histological and immunological detection methods. It requires a highly trained histotechnologist. 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). 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. 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. Wear protective clothing to avoid contact of reagent or specimen with eye, skin or mucous membrane. In case of the reagent or specimen 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 may occur. ProClin 950 is used for stabilisation. A Material safety data sheet (MSDS) is available upon request.
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