ANTIGEN RETRIEVAL TECHNIQUES IN IMMUNOHISTOCHEMISTRY - COMPARISON OF DIFFERENT METHODS

Routine sections of normal and pathological samples fixed in 10 per ce nt buffered formalin or B5, including EDTA-decalcified bone-marrow bio psies, were tested with 61 antibodies following heating in three diffe rent fluids: 0.01 M citrate buffer (pH 6.0), 0.1 hi Tris-HCl (pH 8.0), and 1 mM EDTA-NaOH solution (pH 8.0). The sections underwent either t hree cycles of microwave treatment (5 min each) or pressure cooking fo r 1-2 min. The alkaline phosphatase/alkaline phosphatase (APAAP) techn ique was used as the standard detection method; with 16 antibodies a s lightly modified streptavidin-biotin complex (SABC)-immunoperoxidase t echnique was applied in parallel. The results obtained mere compared w ith those observed without any antigen retrieval (AR), or following se ction digestion with 0.05 per cent protease XIV at 37 degrees C for 5 min. Chess-board titration tests showed that all antibodies but one pr ofited by AR. Protease XIV digestion represented the gold standard for five antibodies, while 55 produced optimal results following the appl ication of heat-based AR. By comparison with the other fluids, EDTA ap peared to be superior in terms of both staining intensity and the numb er of marked cells. These results mere independent of tissue processin g, immunohistochemical approach, and heating device. Pressure cooking was found to be more convenient on practical grounds, as it allowed th e simultaneous handling of a large number of slides and a time sating of 1 min 30 s, representing the proper time for the treatment. (C) 199 7 by John Wiley & Sons, Ltd.