Antigen retrieval


Tissues that have been preserved with formaldehyde, a highly reactive compound, contain a variety of chemical modifications that can reduce the detectability of proteins in biomedical procedures such as immunohistochemistry. Antigen retrieval is an approach to reducing or eliminating these chemical modifications. The two primary methods of antigen retrieval are heat-mediated epitope retrieval and proteolytic induced epitope retrieval.

Protease-induced epitope retrieval (PIER)

In PIER, enzymes including Proteinase K, Trypsin, and Pepsin have been used to restore antibody binding to its epitope. The purported mechanism of action is cleavage of peptides masking the epitope, restoring antigenicity.
Disadvantages of PIER include a low success rate for restoring immunoreactivity, and the potential destruction of tissue morphology and the antigen of interest.

Heat-induced epitope retrieval (HIER)

HIER is believe to reverse some cross-links, and allows for restoration of secondary of tertiary structure of the epitope. The protocol requires optimisation for each tissue, fixation method, and antigen to be studied.
In general, HIER has a much higher success rate than PIER. HIER can be performed with microwave ovens, pressure cookers, vegetable steamers, autoclaves, or water baths. HIER is time, temperature, buffer, and pH-sensitive.

Room Temperature Epitope Retrieval (RTER)

RTER involves reversal of cross-links using acids such as hydrochloric acid or formic acid.