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.
Ramos-Vara
J A
JA
Veterinary Medical Diagnostic Laboratory, University of Missouri, Columbia 65211, USA.
Beissenherz
M E
ME
D000820:Animal Diseases / Q000175:diagnosis / Q000821:virology*; D000818:Animals; D000914:Antibodies, Viral / Q000032:analysis* / Q000276:immunology; D000956:Antigens, Viral / Q000032:analysis* / Q000276:immunology; D003432:Cross-Linking Reagents; D005404:Fixatives / Q000494:pharmacology*; D005557:Formaldehyde / Q000494:pharmacology*; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods / Q000662:veterinary*; D016612:Paraffin Embedding; D012680:Sensitivity and Specificity; D016707:Tissue Fixation / Q000379:methods / Q000662:veterinary*
D000914:Antibodies, Viral; D000956:Antigens, Viral; D003432:Cross-Linking Reagents; D005404:Fixatives; D005557:Formaldehyde