Heat Mediated Antigen Retrieval

The rationale behind these heat pretreatment methods is unclear and several theories have been postulated. One theory is that heavy metal salts (such as the heavy metal solutions described by Shi. et al. act as a protein precipitant, forming insoluble complexes with polypeptides and that protein precipitating fixatives frequently display better preservation of antigens than do cross-linking aldehyde fixatives. Another theory is that during formalin fixation inter- and intra-molecular cross linkages are formed by methylene bridges and weak Schiff bases. These cross linkages alter the protein conformation of the antigen such that it may not be recognized by a specific antibody. It is postulated that heat mediated antigen retrieval removes the weaker Schiff bases but does not affect the methylene bridges so that the resulting protein conformation is intermediate between fixed and unfixed.

Antigens masked during routine fixation and processing can be revealed by using one of the following heat mediated antigen retrieval techniques; microwave oven irradiation, combined microwave oven irradiation and proteolytic enzyme digestion, pressure cooker heating, autoclave heating, water bath heating, Steamer heating, or high temperature incubator.

At MDS we prefer to use microwave irradiation and a combined trypsinisation and microwave irradiation method.