An Enhanced Antigen-Retrieval Protocol for Immunohistochemical Staining of Formalin-Fixed, Paraffin-Embedded Tissues

Abstract

Formalin is the most commonly used fixative for light microscopy because of its preservation of ­morphological details. A major adverse effect of formalin fixation is formation of cross-linkages between epitopes (amino acid residues) and unrelated proteins by formaldehyde groups. The great majority of monoclonal and polyclonal antibodies used for immunohistochemical (IHC) staining of formalin-fixed, paraffin-embedded (FFPE) tissues necessitate unmasking antigens for antigen retrieval. There are currently two major antigen-retrieval procedures based on treatment of deparaffinized tissue sections with heat or, less commonly, with enzymatic digestion. The use of various antigen-retrieval solutions and heating sources does not allow standardization of IHC staining and minimalization of interlaboratory discrepancies. We developed a novel modified antigen-retrieval protocol for reversing the effect of ­formalin fixation. The key feature of this protocol is treatment of deparaffinized tissue sections at reduced constant heat (97^oC in a water bath) for 40 min in 25 mM Tris–HCl (pH 8.5), 1 mM EDTA, and 0.05% SDS (Tris–EDTA–SDS) buffer. Sections are then immunostained with primary and secondary antibodies conjugated with polymer-labeled Horse Radish Peroxidase. Compared to conventional antigen-retrieval procedures, this protocol more efficiently reverses the effect of formalin fixation of a wide variety of cellular antigens and in most instances decreases the use of primary antibody by 2–40 times, resulting in cost savings. Moreover, this protocol eliminates the need for using different antigen-retrieval methods in the laboratory, which reduces both time and labor for medical technologists.