EFFECT OF FIXATION TIME AND MICROWAVE-OVEN HEATING TIME ON RETRIEVAL OF THE KI-67 ANTIGEN FROM PARAFFIN-EMBEDDED TISSUE

Microwave oven heating has been employed for retrieval of antigens fro m formalin-fixed, paraffin-embedded tissue for immunohistochemical sta ining. Recently, a Ki-67 equivalent murine monoclonal antibody was gen erated which can detect tumor proliferative activity in routinely proc essed tissue with microwave oven heating. We assessed the effect of fi xation time (4, 24, or 48 hr) and microwave oven heating time (7, 14, 21, 28, 35, or 49 min) on retrieval of the Ki-67 antigen from tonsil t issue. Ki-67 staining was quantitated by image analysis. Owing to the heterogeneity of Ki-67 staining within and between germinal centers, w e employed a measurement technique that averages staining across the g erminal centers of each section. Good Ki-67 immunostaining was observe d for all microwave oven heating times in tissue fixed for 4 hr. In co ntrast, poor immunostaining was observed in tissue fixed for 48 hr unl ess a heating time of at least 14 min was used. Tonsil fixed for 24 or 48 hr showed a significant increase in percentage positive nuclear ar ea after microwave times of 14 or 21 min. Prolonged heating time (up t o 49 min) had no effect on the quality of Ki-67 staining in tissue fix ed for 4 or 48 hr.