With aldehyde fixation, extended fixation times may damage antigens from the formation of molecular cross-links of proteins. These cross-linkages can make it more difficult for the antibody to bind to its target. This is referred to as "antigen masking."
Unmasking of antigens from the effects of fixation is important for optimal IHC staining. The term "unmasking" is also referred to as epitope retrieval, antigen retrieval (AR), antigen unmasking, and others that are usually trade names created by companies that prepare retrieval reagents. The process of antigen retrieval removes the bonds "masking" the epitope that developed during fixation. This ensures that the antibody has access to its epitope.
The two types of epitope retrieval most frequently used in IHC are:
- Heat-induced epitope retrieval (HIER)
- Proteolytic-induced epitope retrieval (PIER)
