The first step in any immunostaining procedure is fixation; it’s a necessary part of the process that helps preserve the tissue structure that can, unfortunately, occasionally obscure the epitope of interest. Overcoming epitope masking is one of the major challenges involved in a successful stain and there are a number of different techniques, collectively referred to as “antigen retrieval”, that have been developed to do just that.
Antigen retrieval techniques depend on a large number of variables. This includes everything from the tissue type, the fixation method, the primary antibody, the antibody diluent; essentially everything that goes into a staining experiment. There are two broad categories of antigen retrieval methods: Protease-Induced Epitope Retrieval (PIER) and Heat-Induced Epitope Retrieval (HIER). Finding the one that can work for your specific tissue/fixation/antibody combination can mean the difference between getting results and not getting them.
R&D Systems has developed a number of different protocols for PIER and HIER as well as provides tips for optimization and limitations of these methods.
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