In the majority of cases, tissue specimens for immunohistochemical (IHC) staining are routinely fixed in formalin and subsequently embedded in paraffin.Most formalin-fixed tissues require an antigen retrieval step before immunohistochemical staining can proceed. The major drawback of FFPE tissue is that formalin-induced molecular modification of proteins (antigens) may result in loss of the ability of the antibody to react with the antigen, a loss that can only be corrected by the restoration (retrieval) of the formalin-modified' antigen molecular structure.The two methods of antigen retrieval are as heat-induced antigen retrieval (HIER), and Protease-induced Epitope Retrieval(PIER).
Heat-induced antigen retrieval (HIER) is believed to reverse some cross-links and allows for restoration of secondary or tertiary structure of the epitope. In general, HIER has a much higher success rate than PIER. 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 at 60°C and incubate the slides in retrieval solution overnight. The optimal method for each antigen must be found experimentally. Microwaves are an increasingly popular appliance for HIER. These protocols tend to involve 5 minute periods of heat followed by replacement of the buffer. For all HIER methods, slides must be cooled before commencing IHC/ICC incubations. HIER is especially time-, temperature-, buffer-, and pH-sensitive, and the best method must be determined empirically.
In the Protease-induced Epitope Retrieval(PIER) method, enzymes including Proteinase K, Trypsin, and Pepsin have been used successfully to restore the binding of an antibody to its epitope. The mechanism of action is thought to be the cleavage of peptides that may be masking the epitope. The disadvantages of PIER are the low success rate for restoring immunoreactivity and the potential for destroying both tissue morphology and the antigen of interest. Enzymatic retrieval can sometimes damage the morphology of the section, so the concentration and treatment time need to be tested.