As previously noted, fixation with 10% neutral buffered formalin causes the formation of hydroxymethyl groups that link to protein groups. This results in strong bonds that form between proteins and calcium ions called “methylene bridges.” These bridges create an epitope masking effect that needs to be removed in order for the antibody to effectively bind to the antigen site. Antigen detectability in formalin-fixed tissue is largely improved with the use of epitope retrieval methods. The development of epitope retrieval provides the technologist with a method to break the methylene bridges. The retrieval method of choice is usually performed before the application of the primary antibody, and it involves the use of heat and/or enzymes as mentioned previously.
