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.
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 Citrate 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 6.0 ± 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. Citrate buffer, Heat-Induced Epitope Retrieval (10x, Tween20), Red
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 6.0 ± 0,3,
Procedure:
1. Deparaffinize and rehydrate the tissue slides. 2. Fill the PT Module tank with 150 ml of citrate 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 citrate buffer and slides to 98°C and incubate for 20 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 Citrate 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 6.0 ± 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 6.0 ± 0,3,
Procedure:
1. Deparaffinize and rehydrate the tissue slides. 2. Fill the PT Module tank with 150 ml of citrate 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 citrate buffer and slides to 98°C and incubate for 20 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 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.
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.
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:
100 ml HIER T-EDTA Buffer pH 9.0 (10fold concentrated, adequate for 1 litre 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.
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 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 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:
100 ml HIER Citrate Buffer pH 6.0 (10fold concentrated, adequate for 1 litre 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.
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.
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