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IHC antigen retrieval protocolHeat induced epitope retrieval and enzymatic retrieval
2IHC antigen retrieval protocolIntroductionMost formalin-fixed tissues require an antigen retrieval step before immunohistochemical staining. Methylene bridges formed during fixation cross-link proteins and mask antigenic sites. Antigen retrieval methods break these methylene bridges and expose antigenic sites, allowing antibodies to bind.The two methods for antigen retrieval are heat induced epitope retrieval (HIER) and enzymatic retrieval.Enzymatic retrieval can sometimes damage the morphology of the section, so the concentration and treatment time need to be tested.Heat-induced epitope retrieval is most often performed using a pressure cooker, a microwave, or a vegetable steamer. Some labs use a water bath set to 60°C and incubate the slides in retrieval solution overnight. This is useful when working with tissue sections that fall off the slide when heated at higher temperatures; in particular bone, cartilage, and skin. Unless the antigen retrieval method is stated on the antibody datasheet, the optimal method for each antigen must be found experimentally. This applies also to the choice of buffer used for heat-mediated retrieval. The most commonly used buffers are 10mM sodium citrate pH 6, Tris-EDTA pH 9, and EDTA pH 8. We recommend testing several methods to find the retrieval method that gives optimal staining.Buffer solutions for heat-induced epitope retrievalThe following solutions are three of the more popular buffers for HIER. In the absence of datasheet information, choice of retrieval buffer is best accomplished by trial.Sodium citrate buffer (10mM sodium citrate, 0.05% Tween 20, pH 6.0)–Tri-sodium citrate (dihydrate) 2.94 g–Distilled water 1 L–Mix to dissolve. Adjust pH to 6.0 with 1N HCl.–Add 0.5 mLTween 20 and mix well. Store at room temperature for 3 months or at 4°C for longer storage1 mM EDTA, pH 8.0–EDTA 0.37 g–Distilled water 1 L–Adjust topH 8.0 with NaOH–Store at room temperature for 3 months