MICROWAVE PROCESSING FOR IMMUNOHISTOCHEMICAL DEMONSTRATION OF PROLIFERATING CELL NUCLEAR ANTIGEN (PCNA) IN FORMALIN-FIXED AND PARAFFIN-EMBEDDED TISSUE

The effect of microwave irradiation on the immunoreactivity of proliferating cell nuclear antigen (PCNA) in five primary breast carcinomas, a tonsil with hyperplasia, and a seminoma of the testis was evaluated in formalin-fixed and paraffin-embedded sections using the monoclonal antibody PC-10. The tissues were fixed in 4% buffered formaldehyde for 6, 24, 28, 72 h, and 1 week. Before incubation with the primary antibody, sections were microwaved for x 25 min in distilled water, in citrate buffer, or they were processed routinely without microwave irradiation. In sections microwaved in distilled water the immunoreactivity was found to be independent of the length of fixation. PCNA immunoreactivity in sections microwaved twice in citrate buffer was not satisfactory. The PCNA immunoreactivity in sections which were routinely processed without microwave irradiation was markedly decreased after 48 h of fixation, and was nearly absent in sections fixed for up to 1 week. In sections processed without microwave irradiation the optimal dilution of the primary antibody was 1 : 10, whereas in sections microwaved twice in distilled water the optimal dilution was 1: 600. The morphology of the tissue was well preserved after microwave processing. We therefore recommend microwave processing in distilled water for x 2 5 min for demonstration of PCNA in formalin-fixed and paraffin-embedded sections using the monoclonal antibody PC-10, since this technique yields optimal staining reactions independent of the length of fixation.