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 fixativ e 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 antibodi es needed some type of antigen retrieval for optimal results. Of those anti bodies, 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 res ults were obtained with enzyme digestion for 22 antibodies. Only 10 antibod ies yielded optimal results without antigen retrieval; 64% of these antibod ies were polyclonal. All antibodies against cytokeratins were optimally ret rieved with proteinese K. Antigen retrieval appears to be necessary for the majority of antibodies when used with formalin-fixed, paraffin-embedded ti ssues.