Optimization of immunohistochemical methods using two different antigen retrieval methods on formalin-fixed paraffin-embedded tissues: experience with 63 markers.

Formalin fixation produces cross-links between the proteins and the fixative that alter the ability of some antibodies to recognize antigens. We used formalin-fixed, paraffin-embedded tissues to compare two different antigen retrieval methods for 63 antibodies used in the diagnosis of infectious and neoplastic diseases of animal species. Eighty-four percent of the antibodies needed some type of antigen retrieval for optimal results. Of those antibodies, 67.7% were monoclonal and 32.3% were polyclonal. Steam heat was the method of choice for 31 antibodies. Ten antibodies reacted only with steam heat, but 9 antibodies did not react when steam heat was used. Optimal results were obtained with enzyme digestion for 22 antibodies. Only 10 antibodies yielded optimal results without antigen retrieval; 64% of these antibodies were polyclonal. All antibodies against cytokeratins were optimally retrieved with proteinese K. Antigen retrieval appears to be necessary for the majority of antibodies when used with formalin-fixed, paraffin-embedded tissues.