With aldehyde fixation, extended fixation times may damage antigens by forming molecular cross-links of proteins. These cross-linkages can make it more difficult for the antibody to bind to its target, a process known as "antigen masking."
Unmasking antigens from the effects of fixation is essential for optimal IHC staining. The term "unmasking" is also called epitope retrieval, antigen retrieval (AR), antigen unmasking, and others, which are usually trade names created by companies that prepare retrieval reagents. The antigen retrieval process 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)
