Intended use: 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.
Summary and explanation: 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.
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.
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.
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.
References: Shi S-R, Cote RJ, Taylor CR J Histochem Cytochem 45:327-343, 1997/Shi S-R, Key ME, Kalra KL J Histochem Cytochem 39:741-748, 1991/Nadji M and Morales AR Ann N.Y. Acad Sci 420:134-139, 1983
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