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.
HIER Citrate Buffer pH 6.0 is a solution developed for heat induced epitope retrieval (HIER) in formalin-fixed paraffin-embedded tissue sections on slides. This procedure is primarily used in immunohistochemistry.
Immunohistochemical staining procedures consist of sequential incubation steps with blocking solutions, antibodies and secondary reagents, enzymes and chromogenic substrates carried out on tissue sections. These tissue sections are mostly prepared out of formalin-fixed paraffin-embedded tissue blocks. Cellular structures are very effectively stabilised by formalin fixation which results in optimal morphological preservation of the sample. On the other hand the formalin fixation leads to strong cross-links between proteins. This means that epitopes of antigens are being masked and often are no longer accessible for primary antibodies. In order to enable primary antibodies to bind to the antigens the epitopes have to be recovered. Heat induced epitope retrieval (HIER) in buffer solutions of different compositions and pH-values restore structures of the epitopes making them more accessible to specific antibodies. Enzymatic digestion with proteolytic enzymes is another way of recovering epitopes. The primary antibody used determines the appropriate method.
Principle of method:
HIER Citrate Buffer pH 6.0 is a 10fold concentrated citrate buffer solution with additives of stabilising substances. For preparation of the working strength solution the buffer concentrate is diluted 1:10 with deionised or distilled water. The resulting solution has a pH of 6.0 (5.8 to 6.2). HIER Citrate Buffer pH 6.0 is a very efficient epitope retrieval solution in immunohistochemical staining procedures to be used with primary antibodies of many different specificities.
Reagents provided:
500 ml HIER Citrate Buffer pH 6.0 (10fold concentrated, adequate for 5 litres ready-to-use citrate buffer)
Storage and handling:
The solution should be stored at 2-8°C. Do not free ze it. Under these conditions the solution is stable up to the expiry date indicated on the label. Do not use product after the expiry date. If stored at room temperature the solution is stable for at least 10 month from the date of delivery. The prepared working strength solution is stable for 1 month, if stored at 2-8°C. A positive and a negat ive control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by this reagent, please contact our technical support.
Reagent preparation:
Preparation of the citrate buffer working strength solution: Dilute HIER Citrate Buffer concentrate 1:10 with deionised or distilled water and mix thoroughly. The pH-value should be at 6.0 (5.8 to 6.2). If necessary adjust pH-value with diluted NaOH or HCl solution.
Procedure:
HIER Citrate Buffer is suitable for various HIER-methods such as steamer, pressure cooker, autoclave, water bath, and microwave oven. Tissue sections used in heat induced epitope retrieval should always be placed on adhesive slides. Epitope retrieval is carried out after dewaxing and rehydration of the sections. Exemplary protocol using steamer: 1. Prepare the working strength solution by diluting the buffer concentrate as described above and transfer to a Coplin jar. Please make sure that there is enough volume to cover the tissue sections on the slides completely. 2. Fill steamer with water according to instruction manual, close lid and start. 3. After 10 minutes place Coplin jar with citrate buffer in the steamer, close the lid and heat the solution for 20 minutes. 4. Place slides with tissue sections into the preheated solution and close the lid. Tissue sections have to be completely covered with citrate buffer solution. 5. Incubate slides 20 40 minutes. The optimal incubation time needs to be elaborated by the operator. 6. After the incubation take the Coplin jar with slides out of steamer and let cool down at room temperature for about 20 minutes. 7. Remove citrate buffer, rinse slides with wash buffer and proceed with immunohistological staining.
Expected results:
During the reaction of the substrate with horse radish peroxidase or alkaline phosphatase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results please read these instructions carefully, or contact our technical support. Also refer to the instructions of the detection systems for guidance on general troubleshooting.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific binding. Please refer to the instructions of the detection system for guidance on general quality control procedures.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex technique involving both histological and immunological detection methods. It requires a highly trained histotechnologist. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Inadequate counterstaining and mounting can influence the interpretation of the results. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid contact of reagent or specimen with eye, skin or mucous membrane. In case of the reagent or specimen coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagent must be avoided, since otherwise non-specific staining may occur. ProClin 300 is used for stabilisation. A material safety data sheet (MSDS) is available upon request.
HIER EDTA Buffer pH 8.0 is a solution developed for heat induced epitope retrieval (HIER) in formalin-fixed paraffinembedded tissue sections on slides. This procedure is primarily used in immunohistochemistry.
Immunohistochemical staining procedures consist of sequential incubation steps with blocking solutions, antibodies and secondary reagents, enzymes and chromogenic substrates carried out on tissue sections. These tissue sections are mostly prepared out of formalin-fixed paraffin-embedded tissue blocks. Cellular structures are very effectively stabilised by formalin fixation which results in optimal morphological preservation of the sample. On the other hand the formalin fixation leads to strong cross-links between proteins. This means that epitopes of antigens are being masked and often are no longer accessible for primary antibodies. In order to enable primary antibodies to bind to the antigens the epitopes have to be recovered. Heat induced epitope retrieval (HIER) in buffer solutions of different compositions and pH-values restore structures of the epitopes making them more accessible to specific antibodies. Enzymatic digestion with proteolytic enzymes is another way of recovering epitopes. The primary antibody used determines the appropriate method.
Principle of method:
HIER EDTA Buffer pH 8.0 is a 10fold concentrated, buffered EDTA solution with additives of detergent and stabilising substances. For preparation of the working strength solution the buffer concentrate is diluted 1:10 with deionised or distilled water. The resulting solution has a pH of 8.0 (7.8 to 8.2). HIER EDTA Buffer pH 8.0 is a very efficient epitope retrieval solution in immunohistochemical staining procedures to be used with primary antibodies of different specificity. HIER EDTA Buffer pH 8.0 leads to considerably stronger signals compared with usually used citrate buffer.
Reagents provided:
500 ml HIER EDTA Buffer pH 8.0 (10fold concentrated, adequate for 5 litres ready-to-use EDTA Buffer)
Storage and handling:
The solution should be stored at 2-8°C. Do not free ze it. Under these conditions the solution is stable up to the expiry date indicated on the label. Do not use product after the expiry date. If stored at room temperature the solution is stable for at least 10 month from the date of delivery. The prepared working strength solution is stable for 1 month, if stored at 2-8°C. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by this reagent, please contact our technical support.
Reagent preparation:
Preparation of the EDTA buffer working strength solution: Dilute HIER EDTA Buffer concentrate 1:10 with deionised or distilled water and mix thoroughly. The pH-value should be at 8.0 (7.8 to 8.2). If necessary adjust pH-value with diluted NaOH or HCl solution.
Procedure:
HIER EDTA Buffer is suitable for various HIER-methods such as steamer, pressure cooker, autoclave, water bath, and microwave oven. Tissue sections used in heat induced epitope retrieval should always be placed on adhesive slides. Epitope retrieval is carried out after dewaxing and rehydration of the sections. Exemplary protocol using steamer: 1. Prepare the working strength solution by diluting the buffer concentrate as described above and transfer to a Coplin jar. Please make sure that there is enough volume to cover the tissue sections on the slides completely. 2. Fill steamer with water according to instruction manual, close lid and start. 3. After 10 minutes place Coplin jar with EDTA buffer in the steamer, close lid and heat the solution for 20 minutes. 4. Place slides with tissue sections into the preheated solution and close the lid. Tissue sections have to be completely covered with EDTA buffer solution. 5. Incubate slides 20 40 minutes. The optimal incubation time needs to be elaborated by the operator. 6. After the incubation take the Coplin jar with slides out of steamer and let cool down at room temperature for about 20 minutes. 7. Remove EDTA buffer, rinse slides with wash buffer and proceed with immunohistological staining.
Expected results:
During the reaction of the substrate with horse radish peroxidase or alkaline phosphatase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results please read these instructions carefully, or contact our technical support. Also refer to the instructions of the detection systems for guidance on general troubleshooting.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific binding. Please refer to the instructions of the detection system for guidance on general quality control procedures.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex technique involving both histological and immunological detection methods. It requires a highly trained histotechnologist. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Inadequate counterstaining and mounting can influence the interpretation of the results. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid contact of reagent and specimen with eye, skin or mucous membranes. In case of reagent or specimen coming into contact with a sensitive area, wash area with large amounts of water. Microbial contamination of the reagent must be avoided, since otherwise non-specific staining may occur. ProClin 300 is used for stabilisation. A material safety data sheet (MSDS) is available upon request.
HIER T-EDTA Buffer pH 9.0 is a solution developed for heat induced epitope retrieval (HIER) in formalin-fixed paraffinembedded tissue sections on slides. This procedure is primarily used in immunohistochemistry.
Immunohistochemical staining procedures consists of sequential incubation steps with blocking solutions, antibodies and secondary reagents, enzymes and chromogenic substrates carried out on tissue sections. These tissue sections are mostly prepared out of formalin-fixed paraffin-embedded tissue blocks. Cellular structures are very effectively stabilised by formalin fixation which results in optimal morphological preservation of the sample. On the other hand the formalin fixation leads to strong cross-links between proteins. This means that epitopes of antigens are being masked and often are no longer accessible for primary antibodies. In order to enable primary antibodies to bind to antigens the epitopes have to be recovered. Heat induced epitope retrieval (HIER) in buffer solutions of different compositions and pH-values restore structures of the epitopes making them more accessible to specific antibodies. Enzymatic digestion with proteolytic enzymes is another way of recovering epitopes. The primary antibody used determines the appropriate method.
Principle of method:
HIER T-EDTA Buffer pH 9.0 is a 10fold concentrated EDTA solution in Tris buffer with additives of detergent and stabilising substances. For preparation of the working strength solution the buffer concentrate is diluted 1:10 with deionised or distilled water. The resulting solution has a pH of 9.0 (8.8 to 9.2). HIER T-EDTA Buffer pH 9.0 is a very efficient epitope retrieval solution in immunohistochemical staining procedures to be used with primary antibodies of many different specificities. It leads to considerably stronger signals compared with usually used citrate buffer.
Reagents provided:
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 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.
Pepsin Solution is a ready-to-use solution developed for enzymatic epitope retrieval on formalin-fixed tissue sections on slides. This procedure (sometimes called PIER, Protease Induced Epitope Retrieval) is primarily used in immunohistochemical staining procedures.
Immunohistochemical staining procedures consist of sequential incubation steps with blocking solutions, antibodies and secondary reagents, enzymes and chromogenic substrates carried out on tissue sections. These tissue sections are mostly prepared out of formalin-fixed paraffin-embedded tissue blocks. Cellular structures are very effectively stabilised by formalin fixation which results in optimal morphological preservation of the sample. On the other hand the formalin fixation leads to strong cross-links between proteins. This means that epitopes of antigens are being masked and often are no longer accessible for primary antibodies. In order to enable primary antibodies to bind to antigens the epitopes have to be recovered. Enzymatic digestion with proteolytic enzymes (PIER) restores structures of the epitopes making them more accessible to specific antibodies. Heat induced epitope retrieval (HIER) in buffer solutions of different compositions and pH-values is another way of recovering epitopes. The primary antibody used determines the appropriate method.
Principle of method:
Pepsin Solution is a ready-to-use stabilised pepsin solution for enzymatic epitope retrieval.
Reagents provided:
60 ml Pepsin Solution (Ready-To-Use)
Storage and handling:
The solution should be stored at 2-8°C without furt her dilution. Do not freeze it. Under these conditions the solution is stable up to the expiry date indicated on the label. Do not use product after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by this reagent, please contact our technical support
Reagent preparation:
The solution is ready-to-use.
Procedure:
Pepsin Solution is suitable for enzymatic epitope retrieval carried out after the dewaxing and rehydration of the tissue sections. 1. Cover deparaffinised and rehydrated tissue sections with ready-to-use Pepsin Solution. 2. Incubate for 10 - 15 minutes at 37°C or 20 30 minutes at room temperature. The optimal incubation time needs to be elaborated by the operator. It was shown that in individual cases a stronger signal can be obtained when the incubation time is elongated up to 120 minutes (e. g. for detection of Collagen IV with different antibodies). 3. Rinse carefully (3 x) with wash buffer. 4. Proceed with immunohistological staining as usual.
Expected results:
During the reaction of the substrate with horse radish peroxidase or alkaline phosphatase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results please read these instructions carefully, contact our technical support. Also refer to the instructions of the detection systems for guidance on general troubleshooting.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining. Please refer to the instructions of the detection system for guidance on general quality control procedures.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex technique involving both histological and immunological detection methods. It requires a highly trained histotechnologist. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Inadequate counterstaining and mounting can influence the interpretation of the results. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid contact of reagent and specimen with eye, skin or mucous membranes. In case of reagent or specimen coming into contact with a sensitive area, wash area with large amounts of water. A material safety data sheet (MSDS) is available upon request.
Permanent AEC Kit is intended for immunohistochemical and in situ-hybridisation staining procedures with horse radish peroxidase (HRP). AEC (3-Amino-9-ethylcarbazol) leads to the formation of a red-brown precipitate at the location of the target antigen or target nucleic acid. The precipitate is insoluble in aqueous and organic solvents and can be observed by light microscopy. Permanent AEC Kit is intended for research use only.
5.5 ml Reagent 1 3 ml Reagent 2 3 ml Reagent 3 (Chromogen) 4.5 ml Reagent 4 (H2O2) 1 Dilution Vial
Storage and handling:
The solutions should be stored at 2-8°C without fur ther dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. Do not use product after the expiry date. The working solution should be prepared freshly at the day of use. Excess working solution should be disposed. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents please contact our technical support.
Reagent preparation:
1) Pipette 5 ml distilled or deionised water into the provided dilution vial. 2) Add 3 drops buffer concentrate (Reagent 1). Mix thoroughly. 3) Add 2 drops Reagent 2. Mix thoroughly. 4) Add 2 drops AEC chromogen (Reagent 3). Mix thoroughly. 5) Add 2 drops H2O2 substrate (Reagent 4). Mix thoroughly. This working solution is stable for at least 16 hours if stored at 2-8°C in a dark place.
Procedure:
1) Apply the Permanent AEC working solution onto the slide. Incubate for 5-15 minutes. (Incubation time can be extended, if desired.) 2) Rinse with distilled or deionised H2O. 3) Counterstain with haematoxylin for about 30 seconds up to 5 minutes (depending on the desired staining intensity). 4) Rinse with distilled or deionised H2O. 5) Blueing in tap water for at least 5 minutes. 6) Dehydrate through a graded series of ethanol and clear in xylene. Mount with a permanent mounting medium.
Expected results:
During the reaction of the substrate with horse radish peroxidase in presence of the chromogen AEC, a red-brown precipitate is formed at the location of the target antigen or nucleic acid. The precipitate is insoluble in aqueous and organic solvents and can be observed by light microscopy.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results please read these instructions carefully, or contact our technical support. Also refer to the instructions of the detection systems for guidance on general troubleshooting.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining. Please refer to the instructions of the detection system for guidance on general quality control procedures.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). In some tissues endogenous peroxidase activity may cause non-specific staining. The enzyme activity should be blocked by incubation with hydrogen peroxide solution (H2O2 solution). The step is carried out before incubation with primary antibody but after dewaxing and rehydration. Background staining due to endogenous biotin can be blocked through an avidin-biotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. A longer exposure to absolute ethanol can result in decreasing staining intensity. Use of recycled alcohol to dehydrate tissue slides after staining is not recommended. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid contact of reagents or specimen with eye, skin or mucous membrane. In case of a reagent or specimen coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining may occur. A material safety data sheet (MSDS) is available upon request.
Permanent AP Red Kit is developed for immunohistochemical and in situ-hybridisation staining procedures with alkaline phosphatase. Permanent AP Red leads to the formation of a magenta-red precipitate at the location of the target antigen or target nucleic acid. The precipitate is insoluble in aqueous and organic solvents and can be observed by light or fluorescence microscopy.
125 ml Permanent AP Red Buffer 2 ml Permanent AP Red Chromogen 1 Dilution Vial
Storage and handling:
The solutions should be stored at 2-8°C without further dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. Do not use product after the expiry date. The working solution prepared is stable for about 60 minutes and should therefore be used directly after preparation. Excess working solution should be discarded. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents please contact our technical support.
Reagent preparation:
Reagent preparation (Preparation of the working solution) 1) Pipette 2.5 ml AP Red Buffer into the provided dilution vial and let it come to room temperature. The chromogen should still be kept cool. 2) Directly prior to use add 1 drop of Permanent AP Red Chromogen into the buffer. Mix thoroughly. 3) The solution is stable for about 60 minutes. Preparation should be done directly before use. Make sure to pipet the chromogen/substrate mix on the last slide of the staining run within 40 min after mixing. If you want to prepare other quantities of the working solution, please use same ratio AP Red Buffer and Chromogen
Procedure:
1) Rinse the slide with wash buffer after the previous incubation step. 2) Apply freshly prepared Permanent AP Red working solution onto the slide. Incubate for 10 minutes. 3) Rinse with distilled H2O. 4) Counterstain with haematoxylin for about 30 seconds up to 5 minutes (depending on the desired staining intensity). 5) Rinse with distilled H2O. 6) Blueing in tap water for at least 5 minutes. 7) Dehydrate through a graded series of ethanol and clear in xylene. Mount with a permanent mounting medium. Note: It is also possible to mount Permanent AP Red with aqueous mounting media.
Expected results:
During the reaction of the substrate with alkaline phosphatase in presence of the chromogen Permanent AP Red, a magenta-red precipitate is formed at the location of the target antigen or nucleic acid. The precipitate is insoluble in aqueous and organic solvents and can be observed by light or fluorescence microscopy (Texas Red filter).
Trouble shooting:
If you observe unusual staining or other deviations from the expected results please read these instructions carefully, or contact our technical support. Also refer to the instructions of the detection systems for guidance on general troubleshooting.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining. Please refer to the instructions of the detection system for guidance on general quality control procedures.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). In some tissues endogenous alkaline phosphatase activity may cause non-specific staining. However, neither intestinal nor placental alkaline phosphatase can be blocked with levamisole. Therefore, tissues of this origin should be stained with peroxidase detection systems. A higher sensitivity can be obtained when a second chromogenic substrate step is used (i. e. 2 x 10 min Permanent AP Red). Background staining due to endogenous biotin can be blocked through an avidin-biotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. A longer exposure to absolute ethanol can result in decreasing staining intensity. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid contact of reagents or specimen with eye, skin or mucous membrane. In case of a reagent or specimen coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear. A material safety data sheet (MSDS) is available upon request.
Permanent AP Red Kit is developed for immunohistochemical and in situ-hybridisation staining procedures with alkaline phosphatase. Permanent AP Red leads to the formation of a magenta-red precipitate at the location of the target antigen or target nucleic acid. The precipitate is insoluble in aqueous and organic solvents and can be observed by light or fluorescence microscopy.
500 ml Permanent AP Red Buffer 8 ml Permanent AP Red Chromogen 1 Dilution Vial
Storage and handling:
The solutions should be stored at 2-8°C without further dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. Do not use product after the expiry date. The working solution prepared is stable for about 60 minutes and should therefore be used directly after preparation. Excess working solution should be discarded. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents please contact our technical support.
Reagent preparation:
Reagent preparation (Preparation of the working solution) 1) Pipette 2.5 ml AP Red Buffer into the provided dilution vial and let it come to room temperature. The chromogen should still be kept cool. 2) Directly prior to use add 1 drop of Permanent AP Red Chromogen into the buffer. Mix thoroughly. 3) The solution is stable for about 60 minutes. Preparation should be done directly before use. Make sure to pipet the chromogen/substrate mix on the last slide of the staining run within 40 min after mixing. If you want to prepare other quantities of the working solution, please use same ratio AP Red Buffer and Chromogen
Procedure:
1) Rinse the slide with wash buffer after the previous incubation step. 2) Apply freshly prepared Permanent AP Red working solution onto the slide. Incubate for 10 minutes. 3) Rinse with distilled H2O. 4) Counterstain with haematoxylin for about 30 seconds up to 5 minutes (depending on the desired staining intensity). 5) Rinse with distilled H2O. 6) Blueing in tap water for at least 5 minutes. 7) Dehydrate through a graded series of ethanol and clear in xylene. Mount with a permanent mounting medium. Note: It is also possible to mount Permanent AP Red with aqueous mounting media.
Expected results:
During the reaction of the substrate with alkaline phosphatase in presence of the chromogen Permanent AP Red, a magenta-red precipitate is formed at the location of the target antigen or nucleic acid. The precipitate is insoluble in aqueous and organic solvents and can be observed by light or fluorescence microscopy (Texas Red filter).
Trouble shooting:
If you observe unusual staining or other deviations from the expected results please read these instructions carefully, or contact our technical support. Also refer to the instructions of the detection systems for guidance on general troubleshooting.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining. Please refer to the instructions of the detection system for guidance on general quality control procedures.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). In some tissues endogenous alkaline phosphatase activity may cause non-specific staining. However, neither intestinal nor placental alkaline phosphatase can be blocked with levamisole. Therefore, tissues of this origin should be stained with peroxidase detection systems. A higher sensitivity can be obtained when a second chromogenic substrate step is used (i. e. 2 x 10 min Permanent AP Red). Background staining due to endogenous biotin can be blocked through an avidin-biotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. A longer exposure to absolute ethanol can result in decreasing staining intensity. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid contact of reagents or specimen with eye, skin or mucous membrane. In case of a reagent or specimen coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear. A material safety data sheet (MSDS) is available upon request.
The blocking solution Peroxide Block is intended for inhibition of endogenous peroxidase activity in tissue sections. It is primarily intended to be used in immunohistochemistry on formalin-fixed paraffin-embedded samples if using a detection system with horse radish peroxidase.
Endogenous peroxidase activity in the tissue can result in unspecific background staining in immunohistochemical staining procedures using horse radish peroxidase (HRP) as detection enzyme. This effect can be eliminated when tissue sections are incubated with Peroxide Block prior to immunohistochemical staining. Hydrogen peroxide in the solution blocks the activity of endogenous peroxidase.
Principle of method:
Peroxide Block is applied onto tissue sections to reduce non-specific staining due to endogenous peroxidase activity in immunohistochemistry. The step is carried out before incubation with primary antibody but after dewaxing and rehydration. If a heat induced epitope retrieval (HIER) or enzymatic digestion is necessary for immunohistochemical detection it is of no importance if the Peroxide Block is used before or after this step. In some cases it has been shown, that blocking of endogenous peroxidase before the epitope retrieval leads to better results.
Reagents provided:
8 ml Peroxide Block (ready-to-use)
Storage and handling:
The solution should be stored at 2-8°C without furt her dilution. Please store the reagent in a dark place and do not freeze it. Avoid exposure to strong light. Under these conditions the solution is stable up to the expiry date indicated on the label. Do not use product after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by this reagent, please contact our technical support.
Procedure:
1. Apply Peroxide Block for 10 minutes at room temperature. The section should be covered completely. 2. Rinse with wash buffer. 3. Proceed with next steps for immunohistochemical staining as usual starting with protein blocking or the primary antibody.
Expected results:
During the reaction of the substrate with horse radish peroxidase or alkaline phosphatase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope
Trouble shooting:
If you observe unusual staining or other deviations from the expected results please read these instructions carefully, contact our technical support. Also refer to the instructions of the detection systems containing Peroxide Block for guidance on general troubleshooting
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific binding. Please refer to the instructions of the detection system for guidance on general quality control procedures.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex technique involving both histological and immunological detection methods. It requires a highly trained histotechnologist. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Inadequate counterstaining and mounting can influence the interpretation of the results. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid contact of reagent or specimen with eye, skin or mucous membrane. In case of the reagent or specimen coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagent must be avoided, since otherwise non-specific staining may occur. A material safety data sheet (MSDS) is available upon request
The blocking solution Peroxide Block is intended for inhibition of endogenous peroxidase activity in tissue sections. It is primarily intended to be used in immunohistochemistry on formalin-fixed paraffin-embedded samples if using a detection system with horse radish peroxidase.
Endogenous peroxidase activity in the tissue can result in unspecific background staining in immunohistochemical staining procedures using horse radish peroxidase (HRP) as detection enzyme. This effect can be eliminated when tissue sections are incubated with Peroxide Block prior to immunohistochemical staining. Hydrogen peroxide in the solution blocks the activity of endogenous peroxidase.
Principle of method:
Peroxide Block is applied onto tissue sections to reduce non-specific staining due to endogenous peroxidase activity in immunohistochemistry. The step is carried out before incubation with primary antibody but after dewaxing and rehydration. If a heat induced epitope retrieval (HIER) or enzymatic digestion is necessary for immunohistochemical detection it is of no importance if the Peroxide Block is used before or after this step. In some cases it has been shown, that blocking of endogenous peroxidase before the epitope retrieval leads to better results.
Reagents provided:
500 ml Peroxide Block (ready-to-use)
Storage and handling:
The solution should be stored at 2-8°C without furt her dilution. Please store the reagent in a dark place and do not freeze it. Avoid exposure to strong light. Under these conditions the solution is stable up to the expiry date indicated on the label. Do not use product after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by this reagent, please contact our technical support.
Procedure:
1. Apply Peroxide Block for 10 minutes at room temperature. The section should be covered completely. 2. Rinse with wash buffer. 3. Proceed with next steps for immunohistochemical staining as usual starting with protein blocking or the primary antibody.
Expected results:
During the reaction of the substrate with horse radish peroxidase or alkaline phosphatase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope
Trouble shooting:
If you observe unusual staining or other deviations from the expected results please read these instructions carefully, contact our technical support. Also refer to the instructions of the detection systems containing Peroxide Block for guidance on general troubleshooting
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific binding. Please refer to the instructions of the detection system for guidance on general quality control procedures.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex technique involving both histological and immunological detection methods. It requires a highly trained histotechnologist. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Inadequate counterstaining and mounting can influence the interpretation of the results. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid contact of reagent or specimen with eye, skin or mucous membrane. In case of the reagent or specimen coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagent must be avoided, since otherwise non-specific staining may occur. A material safety data sheet (MSDS) is available upon request
The blocking solution Peroxide Block is intended for inhibition of endogenous peroxidase activity in tissue sections. It is primarily intended to be used in immunohistochemistry on formalin-fixed paraffin-embedded samples if using a detection system with horse radish peroxidase.
Endogenous peroxidase activity in the tissue can result in unspecific background staining in immunohistochemical staining procedures using horse radish peroxidase (HRP) as detection enzyme. This effect can be eliminated when tissue sections are incubated with Peroxide Block prior to immunohistochemical staining. Hydrogen peroxide in the solution blocks the activity of endogenous peroxidase.
Principle of method:
Peroxide Block is applied onto tissue sections to reduce non-specific staining due to endogenous peroxidase activity in immunohistochemistry. The step is carried out before incubation with primary antibody but after dewaxing and rehydration. If a heat induced epitope retrieval (HIER) or enzymatic digestion is necessary for immunohistochemical detection it is of no importance if the Peroxide Block is used before or after this step. In some cases it has been shown, that blocking of endogenous peroxidase before the epitope retrieval leads to better results.
Reagents provided:
100 ml Peroxide Block (ready-to-use)
Storage and handling:
The solution should be stored at 2-8°C without furt her dilution. Please store the reagent in a dark place and do not freeze it. Avoid exposure to strong light. Under these conditions the solution is stable up to the expiry date indicated on the label. Do not use product after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by this reagent, please contact our technical support.
Procedure:
1. Apply Peroxide Block for 10 minutes at room temperature. The section should be covered completely. 2. Rinse with wash buffer. 3. Proceed with next steps for immunohistochemical staining as usual starting with protein blocking or the primary antibody.
Expected results:
During the reaction of the substrate with horse radish peroxidase or alkaline phosphatase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope
Trouble shooting:
If you observe unusual staining or other deviations from the expected results please read these instructions carefully, contact our technical support. Also refer to the instructions of the detection systems containing Peroxide Block for guidance on general troubleshooting
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific binding. Please refer to the instructions of the detection system for guidance on general quality control procedures.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex technique involving both histological and immunological detection methods. It requires a highly trained histotechnologist. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Inadequate counterstaining and mounting can influence the interpretation of the results. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid contact of reagent or specimen with eye, skin or mucous membrane. In case of the reagent or specimen coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagent must be avoided, since otherwise non-specific staining may occur. A material safety data sheet (MSDS) is available upon request
The Plus AP Kit, Mouse is based on the streptavidin-biotin system. It is designed for the qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. The kit is developed for use in combination with monoclonal primary antibodies and sera obtained from mice. The Plus AP Kit, Mouse can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of immunohistochemical staining is to make tissue and cell antigens visible. The Plus AP Kit, Mouse is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The method is based on the streptavidin-biotin system which means that a biotinylated secondary antibody binds to several molecules of a conjugate composed of streptavidin and alkaline phosphatase. Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The biotinylated secondary antibody in the Plus AP Kit, Mouse binds to mouse primary antibodies. Therefore this kit can detect monoclonal primary antibodies and sera obtained from mice.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Background staining caused by unspecific binding of the primary or secondary antibody is minimized by incubation with a protein blocking solution (Blocking Solution provided with the kit). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the biotinylated secondary antibody is applied and incubated. This secondary antibody functions as a link between primary antibody and the streptavidinalkaline phosphatase-conjugate (Streptavidin-AP-Conjugate). A second washing is followed by the application of this conjugate. It binds to the biotin at the secondary antibody. Any excess of unbound streptavidin-AP-conjugate is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the alkaline phosphatase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen Permanent Red (included only in kit MON-APP119) leads to a magenta-red product of reaction at the place of the target antigen. Other suitable chromogens are Permanent AP Red (magenta-red) or NBT (blue-black) with its substrate BCIP.
Reagents provided:
125 ml Blocking Solution Reagent 1 (ready-to-use) 125 ml Biotinylated Secondary Antibody, Mouse Reagent 2 (ready-to-use) 125 ml Streptavidin-AP-Conjugate Reagent 3 (ready-to-use) Substrate systems recommended (if not included in the kit): Permanent AP Red Kit, BCIP/NBT Materials required but not supplied Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O Reagents for enzyme digestion or heat pre-treatment Wash buffer PBS or TBS PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solutions should be stored at 2-8°C without further dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. They should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. Tissue sections have to be completely covered with the different reagents in order to avoid drying out. Preparation of the chromogenic substrate working solution (with MON-APP119 only): Add 2 drops (60 µl) of Permanent Red Concentrate to one bottle of Permanent Red Buffer (Substrate Buffer) and mix. This solution should be used directly after preparation.
Procedure:
1. Blocking Solution (protein block, Reagent 1) (This step is optional.) 5 min. 2. Washing with wash buffer 1 x 2 min. 3. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 4. Washing with wash buffer 3 x 2 min. 5. Biotinylated Secondary Antibody, Mouse (Reagent 2, yellow) 10-15 min. 6. Washing with wash buffer 3 x 2 min. 7. Streptavidin-AP-Conjugate (Reagent 3, red) 10-15 min. 8. Washing with wash buffer 3 x 2 min. 9. Permanent Red substrate-chromogen solution (with MON-APP119) 5 min. 10. Wash with distilled H2O 1 min. 11. Permanent Red substrate-chromogen solution (with MON-APP119) 5 min. 12. Wash with destilled water 3 x 1 min. 13. Counterstaining and blueing 14. Mounting: aqueous or permanent after dehydration * The incubation times should be adjusted, when using other substrate-chromogen systems.
Expected results:
During the reaction of the substrate with alkaline phosphatase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support . No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from mouse, but from a different species. 6. The antigen/epitope was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. If you used heat pre-treatment or enzyme digestion it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. If you are using PBS-based wash buffer: the activity of alkaline phosphatase in the reagents is blocked if too much wash buffer remains on the slides. 7. Incubation times were too short or primary antibody concentration too low. 8. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme conjugate or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous alkaline phosphatase in the tissue. This undesired activity can often be suppressed using levamisole (see section Limitations of the Procedure). 9. Non-specific binding of the secondary antibody to endogenous biotin in the tissue section. Carry out an avidin-biotin block before incubation with the primary antibody.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous alkaline phosphatase activity or the endogenous biotin content may cause non-specific staining. The enzyme activity can be blocked by incubation with levamisole. However, neither intestinal nor placental alkaline phosphatase can be blocked with levamisole. Background staining due to endogenous biotin can be blocked through an avidin-biotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the Blocking Solution instead of just draining it away as in other procedures. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. ProClin 300 and sodium azide (NaN3) are used for stabilisation. Sodium azide deposits in drainage pipes made of lead or copper can result in the formation of highly explosive metallic azides. To avoid such deposits in drainage pipes, sodium azide should be discarded in a large volume of running water. Material safety data sheets (MSDS) for the pure substances are available upon request. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear
The Plus AP Kit, Mouse is based on the streptavidin-biotin system. It is designed for the qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. The kit is developed for use in combination with monoclonal primary antibodies and sera obtained from mice. The Plus AP Kit, Mouse can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of immunohistochemical staining is to make tissue and cell antigens visible. The Plus AP Kit, Mouse is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The method is based on the streptavidin-biotin system which means that a biotinylated secondary antibody binds to several molecules of a conjugate composed of streptavidin and alkaline phosphatase. Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The biotinylated secondary antibody in the Plus AP Kit, Mouse binds to mouse primary antibodies. Therefore this kit can detect monoclonal primary antibodies and sera obtained from mice.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Background staining caused by unspecific binding of the primary or secondary antibody is minimized by incubation with a protein blocking solution (Blocking Solution provided with the kit). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the biotinylated secondary antibody is applied and incubated. This secondary antibody functions as a link between primary antibody and the streptavidinalkaline phosphatase-conjugate (Streptavidin-AP-Conjugate). A second washing is followed by the application of this conjugate. It binds to the biotin at the secondary antibody. Any excess of unbound streptavidin-AP-conjugate is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the alkaline phosphatase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen Permanent Red (included only in kit MON-APP119) leads to a magenta-red product of reaction at the place of the target antigen. Other suitable chromogens are Permanent AP Red (magenta-red) or NBT (blue-black) with its substrate BCIP.
Reagents provided:
500 ml Blocking Solution Reagent 1 (ready-to-use) 500 ml Biotinylated Secondary Antibody, Mouse Reagent 2 (ready-to-use) 500 ml Streptavidin-AP-Conjugate Reagent 3 (ready-to-use) Substrate systems recommended (if not included in the kit): Permanent AP Red Kit, BCIP/NBT Materials required but not supplied Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O Reagents for enzyme digestion or heat pre-treatment Wash buffer PBS or TBS PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solutions should be stored at 2-8°C without further dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. They should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. Tissue sections have to be completely covered with the different reagents in order to avoid drying out. Preparation of the chromogenic substrate working solution (with MON-APP119 only): Add 2 drops (60 µl) of Permanent Red Concentrate to one bottle of Permanent Red Buffer (Substrate Buffer) and mix. This solution should be used directly after preparation.
Procedure:
1. Blocking Solution (protein block, Reagent 1) (This step is optional.) 5 min. 2. Washing with wash buffer 1 x 2 min. 3. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 4. Washing with wash buffer 3 x 2 min. 5. Biotinylated Secondary Antibody, Mouse (Reagent 2, yellow) 10-15 min. 6. Washing with wash buffer 3 x 2 min. 7. Streptavidin-AP-Conjugate (Reagent 3, red) 10-15 min. 8. Washing with wash buffer 3 x 2 min. 9. Permanent Red substrate-chromogen solution (with MON-APP119) 5 min. 10. Wash with distilled H2O 1 min. 11. Permanent Red substrate-chromogen solution (with MON-APP119) 5 min. 12. Wash with destilled water 3 x 1 min. 13. Counterstaining and blueing 14. Mounting: aqueous or permanent after dehydration * The incubation times should be adjusted, when using other substrate-chromogen systems.
Expected results:
During the reaction of the substrate with alkaline phosphatase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support . No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from mouse, but from a different species. 6. The antigen/epitope was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. If you used heat pre-treatment or enzyme digestion it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. If you are using PBS-based wash buffer: the activity of alkaline phosphatase in the reagents is blocked if too much wash buffer remains on the slides. 7. Incubation times were too short or primary antibody concentration too low. 8. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme conjugate or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous alkaline phosphatase in the tissue. This undesired activity can often be suppressed using levamisole (see section Limitations of the Procedure). 9. Non-specific binding of the secondary antibody to endogenous biotin in the tissue section. Carry out an avidin-biotin block before incubation with the primary antibody.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous alkaline phosphatase activity or the endogenous biotin content may cause non-specific staining. The enzyme activity can be blocked by incubation with levamisole. However, neither intestinal nor placental alkaline phosphatase can be blocked with levamisole. Background staining due to endogenous biotin can be blocked through an avidin-biotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the Blocking Solution instead of just draining it away as in other procedures. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. ProClin 300 and sodium azide (NaN3) are used for stabilisation. Sodium azide deposits in drainage pipes made of lead or copper can result in the formation of highly explosive metallic azides. To avoid such deposits in drainage pipes, sodium azide should be discarded in a large volume of running water. Material safety data sheets (MSDS) for the pure substances are available upon request. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear
The Plus AP Kit, Mouse is based on the streptavidin-biotin system. It is designed for the qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. The kit is developed for use in combination with monoclonal primary antibodies and sera obtained from mice. The Plus AP Kit, Mouse can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of immunohistochemical staining is to make tissue and cell antigens visible. The Plus AP Kit, Mouse is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The method is based on the streptavidin-biotin system which means that a biotinylated secondary antibody binds to several molecules of a conjugate composed of streptavidin and alkaline phosphatase. Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The biotinylated secondary antibody in the Plus AP Kit, Mouse binds to mouse primary antibodies. Therefore this kit can detect monoclonal primary antibodies and sera obtained from mice.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Background staining caused by unspecific binding of the primary or secondary antibody is minimized by incubation with a protein blocking solution (Blocking Solution provided with the kit). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the biotinylated secondary antibody is applied and incubated. This secondary antibody functions as a link between primary antibody and the streptavidinalkaline phosphatase-conjugate (Streptavidin-AP-Conjugate). A second washing is followed by the application of this conjugate. It binds to the biotin at the secondary antibody. Any excess of unbound streptavidin-AP-conjugate is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the alkaline phosphatase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen Permanent Red (included only in kit MON-APP119) leads to a magenta-red product of reaction at the place of the target antigen. Other suitable chromogens are Permanent AP Red (magenta-red) or NBT (blue-black) with its substrate BCIP.
Reagents provided:
8 ml Blocking Solution Reagent 1 (ready-to-use) 8 ml Biotinylated Secondary Antibody, Mouse Reagent 2 (ready-to-use) 8 ml Streptavidin-AP-Conjugate Reagent 3 (ready-to-use) 8 x 5 ml Permanent Red Buffer (Substrate Buffer) 2 ml Permanent Red Concentrate (Chromogen) Substrate systems recommended (if not included in the kit): Permanent AP Red Kit, BCIP/NBT Materials required but not supplied Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O Reagents for enzyme digestion or heat pre-treatment Wash buffer PBS or TBS PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solutions should be stored at 2-8°C without further dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. They should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. Tissue sections have to be completely covered with the different reagents in order to avoid drying out. Preparation of the chromogenic substrate working solution (with MON-APP119 only): Add 2 drops (60 µl) of Permanent Red Concentrate to one bottle of Permanent Red Buffer (Substrate Buffer) and mix. This solution should be used directly after preparation.
Procedure:
1. Blocking Solution (protein block, Reagent 1) (This step is optional.) 5 min. 2. Washing with wash buffer 1 x 2 min. 3. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 4. Washing with wash buffer 3 x 2 min. 5. Biotinylated Secondary Antibody, Mouse (Reagent 2, yellow) 10-15 min. 6. Washing with wash buffer 3 x 2 min. 7. Streptavidin-AP-Conjugate (Reagent 3, red) 10-15 min. 8. Washing with wash buffer 3 x 2 min. 9. Permanent Red substrate-chromogen solution (with MON-APP119) 5 min. 10. Wash with distilled H2O 1 min. 11. Permanent Red substrate-chromogen solution (with MON-APP119) 5 min. 12. Wash with destilled water 3 x 1 min. 13. Counterstaining and blueing 14. Mounting: aqueous or permanent after dehydration * The incubation times should be adjusted, when using other substrate-chromogen systems.
Expected results:
During the reaction of the substrate with alkaline phosphatase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support . No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from mouse, but from a different species. 6. The antigen/epitope was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. If you used heat pre-treatment or enzyme digestion it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. If you are using PBS-based wash buffer: the activity of alkaline phosphatase in the reagents is blocked if too much wash buffer remains on the slides. 7. Incubation times were too short or primary antibody concentration too low. 8. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme conjugate or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous alkaline phosphatase in the tissue. This undesired activity can often be suppressed using levamisole (see section Limitations of the Procedure). 9. Non-specific binding of the secondary antibody to endogenous biotin in the tissue section. Carry out an avidin-biotin block before incubation with the primary antibody.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous alkaline phosphatase activity or the endogenous biotin content may cause non-specific staining. The enzyme activity can be blocked by incubation with levamisole. However, neither intestinal nor placental alkaline phosphatase can be blocked with levamisole. Background staining due to endogenous biotin can be blocked through an avidin-biotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the Blocking Solution instead of just draining it away as in other procedures. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. ProClin 300 and sodium azide (NaN3) are used for stabilisation. Sodium azide deposits in drainage pipes made of lead or copper can result in the formation of highly explosive metallic azides. To avoid such deposits in drainage pipes, sodium azide should be discarded in a large volume of running water. Material safety data sheets (MSDS) for the pure substances are available upon request. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear
The Plus AP Kit, Mouse is based on the streptavidin-biotin system. It is designed for the qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. The kit is developed for use in combination with monoclonal primary antibodies and sera obtained from mice. The Plus AP Kit, Mouse can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of immunohistochemical staining is to make tissue and cell antigens visible. The Plus AP Kit, Mouse is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The method is based on the streptavidin-biotin system which means that a biotinylated secondary antibody binds to several molecules of a conjugate composed of streptavidin and alkaline phosphatase. Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The biotinylated secondary antibody in the Plus AP Kit, Mouse binds to mouse primary antibodies. Therefore this kit can detect monoclonal primary antibodies and sera obtained from mice.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Background staining caused by unspecific binding of the primary or secondary antibody is minimized by incubation with a protein blocking solution (Blocking Solution provided with the kit). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the biotinylated secondary antibody is applied and incubated. This secondary antibody functions as a link between primary antibody and the streptavidinalkaline phosphatase-conjugate (Streptavidin-AP-Conjugate). A second washing is followed by the application of this conjugate. It binds to the biotin at the secondary antibody. Any excess of unbound streptavidin-AP-conjugate is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the alkaline phosphatase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen Permanent Red (included only in kit MON-APP119) leads to a magenta-red product of reaction at the place of the target antigen. Other suitable chromogens are Permanent AP Red (magenta-red) or NBT (blue-black) with its substrate BCIP.
Reagents provided:
4x 15 ml Blocking Solution Reagent 1 (ready-to-use) 4x 15 ml Biotinylated Secondary Antibody, Mouse Reagent 2 (ready-to-use) 4x 15 ml Streptavidin-AP-Conjugate Reagent 3 (ready-to-use) Substrate systems recommended (if not included in the kit): Permanent AP Red Kit, BCIP/NBT Materials required but not supplied Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O Reagents for enzyme digestion or heat pre-treatment Wash buffer PBS or TBS PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solutions should be stored at 2-8°C without further dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. They should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. Tissue sections have to be completely covered with the different reagents in order to avoid drying out. Preparation of the chromogenic substrate working solution (with MON-APP119 only): Add 2 drops (60 µl) of Permanent Red Concentrate to one bottle of Permanent Red Buffer (Substrate Buffer) and mix. This solution should be used directly after preparation.
Procedure:
1. Blocking Solution (protein block, Reagent 1) (This step is optional.) 5 min. 2. Washing with wash buffer 1 x 2 min. 3. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 4. Washing with wash buffer 3 x 2 min. 5. Biotinylated Secondary Antibody, Mouse (Reagent 2, yellow) 10-15 min. 6. Washing with wash buffer 3 x 2 min. 7. Streptavidin-AP-Conjugate (Reagent 3, red) 10-15 min. 8. Washing with wash buffer 3 x 2 min. 9. Permanent Red substrate-chromogen solution (with MON-APP119) 5 min. 10. Wash with distilled H2O 1 min. 11. Permanent Red substrate-chromogen solution (with MON-APP119) 5 min. 12. Wash with destilled water 3 x 1 min. 13. Counterstaining and blueing 14. Mounting: aqueous or permanent after dehydration * The incubation times should be adjusted, when using other substrate-chromogen systems.
Expected results:
During the reaction of the substrate with alkaline phosphatase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support . No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from mouse, but from a different species. 6. The antigen/epitope was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. If you used heat pre-treatment or enzyme digestion it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. If you are using PBS-based wash buffer: the activity of alkaline phosphatase in the reagents is blocked if too much wash buffer remains on the slides. 7. Incubation times were too short or primary antibody concentration too low. 8. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme conjugate or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous alkaline phosphatase in the tissue. This undesired activity can often be suppressed using levamisole (see section Limitations of the Procedure). 9. Non-specific binding of the secondary antibody to endogenous biotin in the tissue section. Carry out an avidin-biotin block before incubation with the primary antibody.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous alkaline phosphatase activity or the endogenous biotin content may cause non-specific staining. The enzyme activity can be blocked by incubation with levamisole. However, neither intestinal nor placental alkaline phosphatase can be blocked with levamisole. Background staining due to endogenous biotin can be blocked through an avidin-biotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the Blocking Solution instead of just draining it away as in other procedures. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. ProClin 300 and sodium azide (NaN3) are used for stabilisation. Sodium azide deposits in drainage pipes made of lead or copper can result in the formation of highly explosive metallic azides. To avoid such deposits in drainage pipes, sodium azide should be discarded in a large volume of running water. Material safety data sheets (MSDS) for the pure substances are available upon request. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear
The Plus AP Kit, Rabbit is based on the streptavidin-biotin system. It is designed for the qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. The kit is developed for use in combination with mono- and polyclonal primary antibodies and sera obtained from rabbit. The Plus AP Kit, Rabbit can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus AP Kit, Rabbit is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The method based on the streptavidin-biotin system which means that a biotinylated secondary antibody binds to several molecules of a conjugate composed of streptavidin and alkaline phosphatase. Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The biotinylated secondary antibody in the Plus AP Kit, Rabbit binds to rabbit primary antibodies. Therefore this kit can detect mono- and polyclonal primary antibodies and sera obtained from rabbit.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Background staining caused by unspecific binding of the primary or secondary antibody is minimized by incubation with a protein blocking solution (Blocking Solution provided with the kit). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the biotinylated secondary antibody is applied and incubated. This antibody functions as a link between primary antibody and streptavidin-alkaline phosphatase-conjugate (Streptavidin-AP-Conjugate). A second washing is followed by the application of this conjugate. It binds to the biotin at the secondary antibody. Any excess of unbound streptavidin-AP-conjugate is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the alkaline phosphatase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen Permanent Red (included only in kit MON-APP128) leads to the formation of a magenta-red product of reaction at the place of the target antigen. Other suitable chromogens are Permanent AP Red (magenta-red) or NBT (blue-black) with its substrate BCIP.
Reagents provided:
125 ml Blocking Solution Reagent 1 (ready-to-use) 125 ml Biotinylated Secondary Antibody, Rabbit Reagent 2 (ready-to-use) 125 ml Streptavidin-AP-Conjugate Reagent 3 (ready-to-use) Substrate systems recommended (if not included in the kit) Permanent AP Red kit, BCIP/NBT Materials required but not supplied: Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O Reagents for enzyme digestion or heat pre-treatment Wash buffer PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solutions should be stored at 2-8°C without further dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. They should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. Tissue sections have to be completely covered with the different reagents in order to avoid drying out. Preparation of the chromogenic substrate working solution (with MON-APP128 only): Add 2 drops (60 µl) of Permanent Red Concentrate to one bottle of Permanent Red Buffer (Substrate Buffer) and mix. This solution should be used directly after preparation.
Procedure:
1. Blocking Solution (protein block, Reagent 1) (This step is optional.) 5 min. 2. Washing with wash buffer 1 x 2 min. 3. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 4. Washing with wash buffer 3 x 2 min. 5. Biotinylated Secondary Antibody, Rabbit (Reagent 2, yellow) 10-15 min. 6. Washing with wash buffer 3 x 2 min. 7. Streptavidin-AP-Conjugate (Reagent 3, red) 10-15 min. 8. Washing with wash buffer 3 x 2 min. 9. Permanent Red substrate-chromogen solution (with AP008RED-RB) 5 min. 10. Wash with distilled H2O 1 min. 11. Permanent Red substrate-chromogen solution (with AP008RED-RB) 5 min. 12. Wash with destilled water 3 x 1 min. 13. Counterstaining and blueing 14. Mounting: aqueous or permanent after dehydration * The incubation times should be adjusted, when using other substrate-chromogen systems
Expected results:
During the reaction of the substrate with alkaline phosphatase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support. No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from rabbit, but from a different species. 6. The antigen/epitope was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. If you used heat pre-treatment or enzyme digestion it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. If you are using PBS-based wash buffer: the activity of alkaline phosphatase in the reagents is blocked if too much wash buffer remains on the slides. 7. Incubation times were too short or primary antibody concentration too low. 8. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme conjugate or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous alkaline phosphatase in the tissue. This undesired activity can often be suppressed using levamisole (see section Limitations of the Procedure). 9. Non-specific binding of the secondary antibody to endogenous biotin in the tissue section. Carry out an avidin-biotin block before incubation with the primary antibody.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific binding.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous alkaline phosphatase activity or the endogenous biotin content may cause non-specific staining. The enzyme activity can be blocked by incubation with levamisole. However, neither intestinal nor placental alkaline phosphatase can be blocked with levamisole. Background staining due to endogenous biotin can be blocked through an avidin-biotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the Blocking Solution instead of just draining it away as in other procedures. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. ProClin 300 and sodium azide (NaN3) are used for stabilisation. Sodium azide deposits in drainage pipes made of lead or copper can result in the formation of highly explosive metallic azides. To avoid such deposits in drainage pipes, sodium azide should be discarded in a large volume of running water. Material safety data sheets (MSDS) for the pure substances are available upon request. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear.
The Plus AP Kit, Rabbit is based on the streptavidin-biotin system. It is designed for the qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. The kit is developed for use in combination with mono- and polyclonal primary antibodies and sera obtained from rabbit. The Plus AP Kit, Rabbit can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus AP Kit, Rabbit is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The method based on the streptavidin-biotin system which means that a biotinylated secondary antibody binds to several molecules of a conjugate composed of streptavidin and alkaline phosphatase. Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The biotinylated secondary antibody in the Plus AP Kit, Rabbit binds to rabbit primary antibodies. Therefore this kit can detect mono- and polyclonal primary antibodies and sera obtained from rabbit.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Background staining caused by unspecific binding of the primary or secondary antibody is minimized by incubation with a protein blocking solution (Blocking Solution provided with the kit). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the biotinylated secondary antibody is applied and incubated. This antibody functions as a link between primary antibody and streptavidin-alkaline phosphatase-conjugate (Streptavidin-AP-Conjugate). A second washing is followed by the application of this conjugate. It binds to the biotin at the secondary antibody. Any excess of unbound streptavidin-AP-conjugate is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the alkaline phosphatase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen Permanent Red (included only in kit MON-APP128) leads to the formation of a magenta-red product of reaction at the place of the target antigen. Other suitable chromogens are Permanent AP Red (magenta-red) or NBT (blue-black) with its substrate BCIP.
Reagents provided:
500 ml Blocking Solution Reagent 1 (ready-to-use) 500 ml Biotinylated Secondary Antibody, Rabbit Reagent 2 (ready-to-use) 500 ml Streptavidin-AP-Conjugate Reagent 3 (ready-to-use) Substrate systems recommended (if not included in the kit) Permanent AP Red kit, BCIP/NBT Materials required but not supplied: Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O Reagents for enzyme digestion or heat pre-treatment Wash buffer PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solutions should be stored at 2-8°C without further dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. They should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. Tissue sections have to be completely covered with the different reagents in order to avoid drying out. Preparation of the chromogenic substrate working solution (with MON-APP128 only): Add 2 drops (60 µl) of Permanent Red Concentrate to one bottle of Permanent Red Buffer (Substrate Buffer) and mix. This solution should be used directly after preparation.
Procedure:
1. Blocking Solution (protein block, Reagent 1) (This step is optional.) 5 min. 2. Washing with wash buffer 1 x 2 min. 3. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 4. Washing with wash buffer 3 x 2 min. 5. Biotinylated Secondary Antibody, Rabbit (Reagent 2, yellow) 10-15 min. 6. Washing with wash buffer 3 x 2 min. 7. Streptavidin-AP-Conjugate (Reagent 3, red) 10-15 min. 8. Washing with wash buffer 3 x 2 min. 9. Permanent Red substrate-chromogen solution (with AP008RED-RB) 5 min. 10. Wash with distilled H2O 1 min. 11. Permanent Red substrate-chromogen solution (with AP008RED-RB) 5 min. 12. Wash with destilled water 3 x 1 min. 13. Counterstaining and blueing 14. Mounting: aqueous or permanent after dehydration * The incubation times should be adjusted, when using other substrate-chromogen systems
Expected results:
During the reaction of the substrate with alkaline phosphatase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support. No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from rabbit, but from a different species. 6. The antigen/epitope was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. If you used heat pre-treatment or enzyme digestion it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. If you are using PBS-based wash buffer: the activity of alkaline phosphatase in the reagents is blocked if too much wash buffer remains on the slides. 7. Incubation times were too short or primary antibody concentration too low. 8. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme conjugate or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous alkaline phosphatase in the tissue. This undesired activity can often be suppressed using levamisole (see section Limitations of the Procedure). 9. Non-specific binding of the secondary antibody to endogenous biotin in the tissue section. Carry out an avidin-biotin block before incubation with the primary antibody.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific binding.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous alkaline phosphatase activity or the endogenous biotin content may cause non-specific staining. The enzyme activity can be blocked by incubation with levamisole. However, neither intestinal nor placental alkaline phosphatase can be blocked with levamisole. Background staining due to endogenous biotin can be blocked through an avidin-biotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the Blocking Solution instead of just draining it away as in other procedures. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. ProClin 300 and sodium azide (NaN3) are used for stabilisation. Sodium azide deposits in drainage pipes made of lead or copper can result in the formation of highly explosive metallic azides. To avoid such deposits in drainage pipes, sodium azide should be discarded in a large volume of running water. Material safety data sheets (MSDS) for the pure substances are available upon request. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear.
The Plus AP Kit, Rabbit is based on the streptavidin-biotin system. It is designed for the qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. The kit is developed for use in combination with mono- and polyclonal primary antibodies and sera obtained from rabbit. The Plus AP Kit, Rabbit can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus AP Kit, Rabbit is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The method based on the streptavidin-biotin system which means that a biotinylated secondary antibody binds to several molecules of a conjugate composed of streptavidin and alkaline phosphatase. Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The biotinylated secondary antibody in the Plus AP Kit, Rabbit binds to rabbit primary antibodies. Therefore this kit can detect mono- and polyclonal primary antibodies and sera obtained from rabbit.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Background staining caused by unspecific binding of the primary or secondary antibody is minimized by incubation with a protein blocking solution (Blocking Solution provided with the kit). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the biotinylated secondary antibody is applied and incubated. This antibody functions as a link between primary antibody and streptavidin-alkaline phosphatase-conjugate (Streptavidin-AP-Conjugate). A second washing is followed by the application of this conjugate. It binds to the biotin at the secondary antibody. Any excess of unbound streptavidin-AP-conjugate is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the alkaline phosphatase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen Permanent Red (included only in kit MON-APP128) leads to the formation of a magenta-red product of reaction at the place of the target antigen. Other suitable chromogens are Permanent AP Red (magenta-red) or NBT (blue-black) with its substrate BCIP.
Reagents provided:
8 ml Blocking Solution Reagent 1 (ready-to-use) 8 ml Biotinylated Secondary Antibody, Rabbit Reagent 2 (ready-to-use) 8 ml Streptavidin-AP-Conjugate Reagent 3 (ready-to-use) 8 x 5 ml Permanent Red Buffer (Substrate Buffer) 2 ml Permanent Red Concentrate (Chromogen) Substrate systems recommended (if not included in the kit) Permanent AP Red kit, BCIP/NBT Materials required but not supplied: Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O Reagents for enzyme digestion or heat pre-treatment Wash buffer PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solutions should be stored at 2-8°C without further dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. They should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. Tissue sections have to be completely covered with the different reagents in order to avoid drying out. Preparation of the chromogenic substrate working solution (with MON-APP128 only): Add 2 drops (60 µl) of Permanent Red Concentrate to one bottle of Permanent Red Buffer (Substrate Buffer) and mix. This solution should be used directly after preparation.
Procedure:
1. Blocking Solution (protein block, Reagent 1) (This step is optional.) 5 min. 2. Washing with wash buffer 1 x 2 min. 3. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 4. Washing with wash buffer 3 x 2 min. 5. Biotinylated Secondary Antibody, Rabbit (Reagent 2, yellow) 10-15 min. 6. Washing with wash buffer 3 x 2 min. 7. Streptavidin-AP-Conjugate (Reagent 3, red) 10-15 min. 8. Washing with wash buffer 3 x 2 min. 9. Permanent Red substrate-chromogen solution (with AP008RED-RB) 5 min. 10. Wash with distilled H2O 1 min. 11. Permanent Red substrate-chromogen solution (with AP008RED-RB) 5 min. 12. Wash with destilled water 3 x 1 min. 13. Counterstaining and blueing 14. Mounting: aqueous or permanent after dehydration * The incubation times should be adjusted, when using other substrate-chromogen systems
Expected results:
During the reaction of the substrate with alkaline phosphatase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support. No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from rabbit, but from a different species. 6. The antigen/epitope was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. If you used heat pre-treatment or enzyme digestion it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. If you are using PBS-based wash buffer: the activity of alkaline phosphatase in the reagents is blocked if too much wash buffer remains on the slides. 7. Incubation times were too short or primary antibody concentration too low. 8. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme conjugate or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous alkaline phosphatase in the tissue. This undesired activity can often be suppressed using levamisole (see section Limitations of the Procedure). 9. Non-specific binding of the secondary antibody to endogenous biotin in the tissue section. Carry out an avidin-biotin block before incubation with the primary antibody.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific binding.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous alkaline phosphatase activity or the endogenous biotin content may cause non-specific staining. The enzyme activity can be blocked by incubation with levamisole. However, neither intestinal nor placental alkaline phosphatase can be blocked with levamisole. Background staining due to endogenous biotin can be blocked through an avidin-biotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the Blocking Solution instead of just draining it away as in other procedures. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. ProClin 300 and sodium azide (NaN3) are used for stabilisation. Sodium azide deposits in drainage pipes made of lead or copper can result in the formation of highly explosive metallic azides. To avoid such deposits in drainage pipes, sodium azide should be discarded in a large volume of running water. Material safety data sheets (MSDS) for the pure substances are available upon request. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear.
The Plus AP Kit, Rabbit is based on the streptavidin-biotin system. It is designed for the qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. The kit is developed for use in combination with mono- and polyclonal primary antibodies and sera obtained from rabbit. The Plus AP Kit, Rabbit can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus AP Kit, Rabbit is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The method based on the streptavidin-biotin system which means that a biotinylated secondary antibody binds to several molecules of a conjugate composed of streptavidin and alkaline phosphatase. Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The biotinylated secondary antibody in the Plus AP Kit, Rabbit binds to rabbit primary antibodies. Therefore this kit can detect mono- and polyclonal primary antibodies and sera obtained from rabbit.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Background staining caused by unspecific binding of the primary or secondary antibody is minimized by incubation with a protein blocking solution (Blocking Solution provided with the kit). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the biotinylated secondary antibody is applied and incubated. This antibody functions as a link between primary antibody and streptavidin-alkaline phosphatase-conjugate (Streptavidin-AP-Conjugate). A second washing is followed by the application of this conjugate. It binds to the biotin at the secondary antibody. Any excess of unbound streptavidin-AP-conjugate is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the alkaline phosphatase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen Permanent Red (included only in kit MON-APP128) leads to the formation of a magenta-red product of reaction at the place of the target antigen. Other suitable chromogens are Permanent AP Red (magenta-red) or NBT (blue-black) with its substrate BCIP.
Reagents provided:
4x 15 ml Blocking Solution Reagent 1 (ready-to-use) 4x 15 ml Biotinylated Secondary Antibody, Rabbit Reagent 2 (ready-to-use) 4x 15 ml Streptavidin-AP-Conjugate Reagent 3 (ready-to-use) Substrate systems recommended (if not included in the kit) Permanent AP Red kit, BCIP/NBT Materials required but not supplied: Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O Reagents for enzyme digestion or heat pre-treatment Wash buffer PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solutions should be stored at 2-8°C without further dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. They should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. Tissue sections have to be completely covered with the different reagents in order to avoid drying out. Preparation of the chromogenic substrate working solution (with MON-APP128 only): Add 2 drops (60 µl) of Permanent Red Concentrate to one bottle of Permanent Red Buffer (Substrate Buffer) and mix. This solution should be used directly after preparation.
Procedure:
1. Blocking Solution (protein block, Reagent 1) (This step is optional.) 5 min. 2. Washing with wash buffer 1 x 2 min. 3. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 4. Washing with wash buffer 3 x 2 min. 5. Biotinylated Secondary Antibody, Rabbit (Reagent 2, yellow) 10-15 min. 6. Washing with wash buffer 3 x 2 min. 7. Streptavidin-AP-Conjugate (Reagent 3, red) 10-15 min. 8. Washing with wash buffer 3 x 2 min. 9. Permanent Red substrate-chromogen solution (with AP008RED-RB) 5 min. 10. Wash with distilled H2O 1 min. 11. Permanent Red substrate-chromogen solution (with AP008RED-RB) 5 min. 12. Wash with destilled water 3 x 1 min. 13. Counterstaining and blueing 14. Mounting: aqueous or permanent after dehydration * The incubation times should be adjusted, when using other substrate-chromogen systems
Expected results:
During the reaction of the substrate with alkaline phosphatase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support. No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from rabbit, but from a different species. 6. The antigen/epitope was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. If you used heat pre-treatment or enzyme digestion it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. If you are using PBS-based wash buffer: the activity of alkaline phosphatase in the reagents is blocked if too much wash buffer remains on the slides. 7. Incubation times were too short or primary antibody concentration too low. 8. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme conjugate or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous alkaline phosphatase in the tissue. This undesired activity can often be suppressed using levamisole (see section Limitations of the Procedure). 9. Non-specific binding of the secondary antibody to endogenous biotin in the tissue section. Carry out an avidin-biotin block before incubation with the primary antibody.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific binding.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous alkaline phosphatase activity or the endogenous biotin content may cause non-specific staining. The enzyme activity can be blocked by incubation with levamisole. However, neither intestinal nor placental alkaline phosphatase can be blocked with levamisole. Background staining due to endogenous biotin can be blocked through an avidin-biotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the Blocking Solution instead of just draining it away as in other procedures. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. ProClin 300 and sodium azide (NaN3) are used for stabilisation. Sodium azide deposits in drainage pipes made of lead or copper can result in the formation of highly explosive metallic azides. To avoid such deposits in drainage pipes, sodium azide should be discarded in a large volume of running water. Material safety data sheets (MSDS) for the pure substances are available upon request. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear.
The Plus AP Kit, Broad Spectrum is based on the streptavidin-biotin system. It is designed for the qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. The kit is developed for use in combination with mono- and polyclonal primary antibodies and sera obtained from mouse, rabbit, rat, and guinea pig. The Plus AP Kit, Broad Spectrum can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus AP Kits, Broad Spectrum is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The method is based on the streptavidin-biotin system which means that a biotinylated secondary antibody binds to several molecules of a conjugate composed of streptavidin and alkaline phosphatase. Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The biotinylated secondary antibody in the Plus AP Kit, Broad Spectrum is polyvalent. With this kit it is therefore possible to detect mono- and polyclonal primary antibodies and sera obtained from mouse, rabbit, rat, and guinea pig.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Background staining caused by unspecific binding of the primary or secondary antibody is minimized via incubation with a protein blocking solution (Blocking Solution provided with the kit). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the biotinylated secondary antibody is applied and incubated. This secondary antibody functions as a link between primary antibody and streptavidin-alkaline phosphatase-conjugate (Streptavidin-AP-Conjugate). A second washing is followed by the application of this conjugate. It binds to biotin at the secondary antibody. Any excess of unbound streptavidin-AP-conjugate is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the alkaline phosphatase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen Permanent Red (included only in kit MON-APP110) leads to the formation of a magenta-red product of reaction at the place of the target antigen. Other suitable chromogens are Permanent AP Red (magenta-red) or NBT (blue-black) with its substrate BCIP.
Reagents provided:
125 ml Blocking Solution Reagent 1 (ready-to-use) 125 ml Biotinylated Secondary Antibody, polyvalent Reagent 2 (ready-to-use) 125 ml Streptavidin-AP-Conjugate Reagent 3 (ready-to-use) Substrate systems recommended (if not included in the kit): Permanent AP Red Kit, BCIP/NBT Materials required but not supplied Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O Reagents for enzyme digestion or heat pre-treatment Wash buffer PBS or TBS PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solution should be stored at 2-8°C without further dilution. Please store the reagent in a dark place and do not freeze it. Under these conditions the solution is stable up to the expiry date. It should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support .
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion.Tissue sections have to be completely covered with the different reagents in order to avoid drying out. Preparation of the chromogenic substrate working solution (with MON-APP110 only): Add 2 drops (60 µl) of Permanent Red Concentrate to one bottle of Permanent Red Buffer (substrate buffer) and mix. This solution should be used directly after preparation.
Procedure:
1. Blocking Solution (protein block, Reagent 1) (This step is optional.) 5 min. 2. Washing with wash buffer 1 x 2 min. 3. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 4. Washing with wash buffer 3 x 2 min. 5. Biotinylated Secondary Antibody, polyvalent (Reagent 2, yellow) 10-15 min. 6. Washing with wash buffer 3 x 2 min. 7. Streptavidin-AP-Conjugate (Reagent 3, red) 10-15 min. 8. Washing with wash buffer 3 x 2 min. 9. Permanent Red substrate-chromogen solution (with MON-APP110) 5 min. 10. Wash with distilled H2O 1 min. 11. Permanent Red substrate-chromogen solution (with MON-APP110) 5 min. 12. Wash with distilled H2O 3 x 1 min. 13. Counterstaining and blueing 14. Mounting: aqueous or permanent after dehydration * The incubation times should be adjusted, when using other substrate-chromogen systems.
Expected results:
During the reaction of the substrate with alkaline phosphatase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support. No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from mouse, rabbit, rat or guinea pig. 6. The antigen was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. If you are using PBS-based wash buffer: the activity of alkaline phosphatase in the reagents is blocked if too much wash buffer remains on the slides. 7. Incubation times were too short or primary antibody concentration too low. 8. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme conjugate or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolized by endogenous alkaline phosphatase in the tissue. This undesired activity can often be suppressed using levamisole (see also Limitations of the procedure). 9. Non-specific binding of the secondary antibody to endogenous biotin in the tissue section. Carry out an avidin-biotin block before incubation with the primary antibody.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous alkaline phosphatase activity or the endogenous biotin content may cause non-specific staining. The enzyme activity can be blocked by incubation with levamisole. However, neither intestinal nor placental alkaline phosphatase can be blocked with levamisole. Background staining due to endogenous biotin can be blocked through an avidin-biotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the Blocking Solution instead of just draining it away as in other procedures. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. ProClin 300 and sodium azide (NaN3), used for stabilisation. Sodium azide deposits in drainage pipes made of lead or copper can result in the formation of highly explosive metallic azides. To avoid such deposits in drainage pipes, sodium azide should be discarded in a large volume of running water. Material safety data sheets (MSDS) for the pure substances are available upon request. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear.
The Plus AP Kit, Broad Spectrum is based on the streptavidin-biotin system. It is designed for the qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. The kit is developed for use in combination with mono- and polyclonal primary antibodies and sera obtained from mouse, rabbit, rat, and guinea pig. The Plus AP Kit, Broad Spectrum can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus AP Kits, Broad Spectrum is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The method is based on the streptavidin-biotin system which means that a biotinylated secondary antibody binds to several molecules of a conjugate composed of streptavidin and alkaline phosphatase. Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The biotinylated secondary antibody in the Plus AP Kit, Broad Spectrum is polyvalent. With this kit it is therefore possible to detect mono- and polyclonal primary antibodies and sera obtained from mouse, rabbit, rat, and guinea pig.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Background staining caused by unspecific binding of the primary or secondary antibody is minimized via incubation with a protein blocking solution (Blocking Solution provided with the kit). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the biotinylated secondary antibody is applied and incubated. This secondary antibody functions as a link between primary antibody and streptavidin-alkaline phosphatase-conjugate (Streptavidin-AP-Conjugate). A second washing is followed by the application of this conjugate. It binds to biotin at the secondary antibody. Any excess of unbound streptavidin-AP-conjugate is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the alkaline phosphatase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen Permanent Red (included only in kit MON-APP110) leads to the formation of a magenta-red product of reaction at the place of the target antigen. Other suitable chromogens are Permanent AP Red (magenta-red) or NBT (blue-black) with its substrate BCIP.
Reagents provided:
500 ml Blocking Solution Reagent 1 (ready-to-use) 500 ml Biotinylated Secondary Antibody, polyvalent Reagent 2 (ready-to-use) 500 ml Streptavidin-AP-Conjugate Reagent 3 (ready-to-use) Substrate systems recommended (if not included in the kit): Permanent AP Red Kit, BCIP/NBT Materials required but not supplied Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O Reagents for enzyme digestion or heat pre-treatment Wash buffer PBS or TBS PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solution should be stored at 2-8°C without further dilution. Please store the reagent in a dark place and do not freeze it. Under these conditions the solution is stable up to the expiry date. It should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support .
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion.Tissue sections have to be completely covered with the different reagents in order to avoid drying out. Preparation of the chromogenic substrate working solution (with MON-APP110 only): Add 2 drops (60 µl) of Permanent Red Concentrate to one bottle of Permanent Red Buffer (substrate buffer) and mix. This solution should be used directly after preparation.
Procedure:
1. Blocking Solution (protein block, Reagent 1) (This step is optional.) 5 min. 2. Washing with wash buffer 1 x 2 min. 3. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 4. Washing with wash buffer 3 x 2 min. 5. Biotinylated Secondary Antibody, polyvalent (Reagent 2, yellow) 10-15 min. 6. Washing with wash buffer 3 x 2 min. 7. Streptavidin-AP-Conjugate (Reagent 3, red) 10-15 min. 8. Washing with wash buffer 3 x 2 min. 9. Permanent Red substrate-chromogen solution (with MON-APP110) 5 min. 10. Wash with distilled H2O 1 min. 11. Permanent Red substrate-chromogen solution (with MON-APP110) 5 min. 12. Wash with distilled H2O 3 x 1 min. 13. Counterstaining and blueing 14. Mounting: aqueous or permanent after dehydration * The incubation times should be adjusted, when using other substrate-chromogen systems.
Expected results:
During the reaction of the substrate with alkaline phosphatase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support. No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from mouse, rabbit, rat or guinea pig. 6. The antigen was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. If you are using PBS-based wash buffer: the activity of alkaline phosphatase in the reagents is blocked if too much wash buffer remains on the slides. 7. Incubation times were too short or primary antibody concentration too low. 8. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme conjugate or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolized by endogenous alkaline phosphatase in the tissue. This undesired activity can often be suppressed using levamisole (see also Limitations of the procedure). 9. Non-specific binding of the secondary antibody to endogenous biotin in the tissue section. Carry out an avidin-biotin block before incubation with the primary antibody.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous alkaline phosphatase activity or the endogenous biotin content may cause non-specific staining. The enzyme activity can be blocked by incubation with levamisole. However, neither intestinal nor placental alkaline phosphatase can be blocked with levamisole. Background staining due to endogenous biotin can be blocked through an avidin-biotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the Blocking Solution instead of just draining it away as in other procedures. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. ProClin 300 and sodium azide (NaN3), used for stabilisation. Sodium azide deposits in drainage pipes made of lead or copper can result in the formation of highly explosive metallic azides. To avoid such deposits in drainage pipes, sodium azide should be discarded in a large volume of running water. Material safety data sheets (MSDS) for the pure substances are available upon request. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear.
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