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.
Pronase 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. Proteolytic pre-treatments are also used in protocols for in situhybridization. Pronase solution is intended 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:
Pronase Solution is a ready-to-use solution for enzymatic epitope retrieval.
Reagents provided:
250 ml Pronase Solution (Ready-To-Use)
Storage and handling:
The solution should be stored in aliquots at -20°C without further dilution. The solution should be aliquoted in order to avoid repeated freeze and thawing. Under these conditions the solution is stable up to the expiry date indicated on the label. Do not use product after the expiry date.
Reagent preparation:
Pronase solution is ready-to-use and should be at room temperature prior to use.
Procedure:
Pronase 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 Pronase Solution. 2. Incubate for 15 - 20 minutes at room temperature. The optimal incubation time needs to be elaborated by the operator. 3. Rinse carefully (3 x), first with distilled water followed by 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
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 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 the reagent with eye, skin or mucous membrane. In case of reagent coming into contact with a sensitive area, wash the area with large amounts of water. Material safety data sheets (MSDS) are 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 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.
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:
500 ml Wash Buffer (20fold concentrated, adequate for 10 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.
The aqua mounting medium is a widely used for coverslipping from aqueous solutions and is non-fluorescing and has an antifade component to increase the viewing time of the specimen. Use Aqua-Poly/Mount with most fluorescent dyes and stains including DAB, Alkaline Phosphatase, Fast Red, AEC (aminoethylcarbazole) and a variety of other chromogens to enhance and retain fluorescent intensity. The mounting medium (water based) can be used for frozen sections, fat stains, chromogens for immunohistochemistry and in situ hybridization as well as other applications requiring a water-soluble mounting medium.
Principle of method:
Coverslipping from aqueous solutions
Reagents provided:
50 ml Mounting medium (water based), ready-to-use.
Storage and handling:
Store at room temperature
Procedure:
1. Prepare slides as required. 2. Prior to coverslipping rinse the slides in distilled or deionized water. 3. Place the bottle upside down with the end cap attached in a container before use. This will help clear of tiny bubbles. 4. Blot excess water from the slide without drying the tissue specimen. The tissue must be moist prior to mounting. 5. Apply enough drops of aqua mounting medium on the tissue section so that the tissue is completely covered. 6. Carefully lower the coverslip at an angle while gently applying pressure to force any excess medium and air bubbles away from the tissue and out from under the coverslip. 7. Slides can be dried at room temperature or at 4°C or in an oven but the temperature hight is depending what type of staining is used. 8. Allow the aqua mounting medium to dry before examine under the microscope.
The BrightDiluents universal IHC diluent is specially formulated to stabilize antibodies and eliminate backgrounds encountered in immunohistochemistry staining procedures. The diluent contains complex proteins and non-protein mixtures which eliminate histostaining background by blocking Fc receptors and prevents non-specific protein-protein interactions. It can also be used as blocking applied before primary antibodies. If primary antibodies are diluted in this diluent, a blocking step before primary antibodies can generally be omitted. The blocking /diluent solution is supplied in Ready-to-Use format. It should not be diluted to be effective. The diluent contains no azide, therefore, it can be used to dilute and stabilize HRP-conjugates as well as any other solution that need and stabilizing carrier protein, such as Blocking and Poly-AP conjugates. This product should be interpreted by a qualified pathologist with relevant clinical information, morphological and histological studies and with proper controls. The ready-to-use antibody diluent is available in different colors: MON-APP917 (Transparant/colorless), MON-APP917B (Blue), MON-APP917Y (Yellow), MON-APP917G (green), MON-APP917R (Red)
Principle of method:
Antibody diluent
Reagents provided:
125 ml BrightDiluent, normal antibody diluent (ready-to-use)
Permanent HRP Green Kit is intended for immunohistochemical and in situ-hybridisation staining procedures with horse radish peroxidase (HRP). It results in the formation of a green precipitate at the location of the target antigen or target nucleic acid. The precipitate is insoluble in organic solvents and can be observed by light microscopy. The HRP Green precipitate shows a very good contrast to red chromogenic substrates used with alkaline phosphatase detection systems and is therefore especially recommended for double stains. Permanent HRP Green Kit is intended for research use only.
100 ml HRP Green Substrate Buffer 3 ml HRP Green Chromogen 1 Dilution Vial
Storage and handling:
The solutions should be stored at 2-8°C without fur ther dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. Do not use product after the expiry date. The working solution should be prepared freshly at the day of use. It is stable for at least 4 hours. Excess working solution should be disposed. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents please contact our technical support.
Reagent preparation:
1) Pipette 1 ml HRP Green Substrate Buffer into the provided dilution vial. 2) Add 1 drop (30 µl) of HRP Green Chromogen. Mix thoroughly. 3) The resulting working solution is stable for at least 4 hours. If you want to prepare other quantities of the working solution, please use the same ratio HRP Green Substrate and HRP Chromogen.
Procedure:
1) Rinse the slide with wash buffer after the previous incubation step. 2) Apply the Permanent HRP Green working solution onto the slide. Incubate for 2-10 minutes. 3) Rinse with distilled H2O. 4) Counterstain with haematoxylin for about 30 seconds up to 5 minutes (depending on the desired staining intensity). 5) Rinse with distilled H2O. 6) Blueing in tap water for 5 minutes. 7) Dehydrate through a graded series of ethanol and clear in xylene. Do not exceed incubation times of 30 sec per dehydration step. Use only high purity xylene. Mount with a permanent mounting medium. Note: The colour intensity can be intensified by increasing the chromogen concentration (up to 3 drops or 90 µl chromogen) in the working solution. Lithium carbonate may have a negative effect on the staining result. We recommend to only bluing in tap water. Occasionally precipitates may appear in the HRP Green Chromogen solution. This doesnt affect the staining result.
Expected results:
During the reaction of the substrate with horse radish peroxidase in presence of the Permanent HRP Green chromogen, a green precipitate is formed at the location of the target antigen or nucleic acid. The precipitate is insoluble in organic solvents and can be observed by light microscopy.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results please read these instructions carefully, contact our technical support. Also refer to the instructions of the detection systems for guidance on general troubleshooting.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining. Please refer to the instructions of the detection system for guidance on general quality control procedures.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Too long incubation steps at the final dehydration serious can diminish the staining intensity. Also low grade xylene and some forms of recycled alcohol can have a negative effect on the staining result. In double stain procedures we recommend to use Permanent HRP Green as the last chromogen. Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid contact of reagents or specimen with eye, skin or mucous membrane. In case of a reagent or specimen coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining may occur. Material safety data sheets (MSDS) are available upon request.
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.
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.
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.
Expected results:
Epitope retrieval
Precautions:
Refer to SDS
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