FFPE tissues form hydroxymethyl groups that link to protein groups, which result in strong bonds that form between proteins and calcium ions called “methylene bridges.” These bridges create an epitope masking affect which needs to be removed or destroyed. This condition is easily remedied using epitope/antigen retrieval techniques. Because of epitope/antigen retrieval methods that are currently available, it is better to slightly "over-fix” a tissue than to “under-fix” a tissue for IHC staining. However, because over-fixation of tissue in formalin can cause an antibody not to have access to its epitope, false-negative staining can result.
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 prior to application of the primary antibody and it involves the use of heat and/or enzymes as mentioned above.
