EIER or protease digestion was the first method developed to reduce the antigen masking effects of formalin fixation. Several proteases have been used and one of the most common proteases is trypsin. Others such as proteinase K and pepsin are among the next most common.
Enzyme treatment cleaves the molecular cross-links which return the epitopes to their normal structure, thus presenting a more successful binding by the antibody. Caution to temperature and time is necessary when using an enzyme retrieval method. Each enzyme activates at a specific temperature and variances in time and/or temperature can cause extensive damage to the tissue section very quickly, thus rendering the section unusable.