ANTIGEN UNMASKING ON FORMALIN-FIXED, PARAFFIN-EMBEDDED TISSUE-SECTIONS

Enzymatic and non-enzymatic treatments for antigen unmasking on formal in-fixed, paraffin-embedded, dewaxed sections were optimized and compa red by the use of a panel of antibodies of diagnostic relevance (anti- cytokeratins, vimentin, S-100, T- and B-cell receptors, Ki-67/MIB 1, m uscle actin). Non-enzymatic unmasking was obtained by boiling the slid es in a microwave oven in 0.01 M salt solution (pH 6) or in 6 M urea. Trypsin or pronase digestion was used for comparison and found to be n ecessary for some of the reagents. The investigation was then extended to 256 antibodies; the epitopic amino acid sequence was known for 48 of them. We found that enzymatic and non-enzymatic antigen unmasking a re not dependent on the epitope sequence, but some antigens benefit se lectively from one treatment but not from the other. Denaturation of p roteins is the likely mechanism which leads to immunodetection on micr owave oven-boiled slides; this suggestion is supported by the use of d enaturating solutions and by the observation that endogenous enzymes w ere inactivated and a few antigens were no longer immunodetectable aft er boiling. Non-enzymatic methods for antigen unmasking are a powerful new tool for broadening the use of antibodies for immunostaining form alin-fixed, paraffin-embedded sections and should be used in parallel with the traditional enzymatic methods.