EFFECTS OF PREFIXATION AND FIXATION TIMES ON APOPTOSIS DETECTION BY IN-SITU END-LABELING OF FRAGMENTED DNA

Objective.-Apoptosis is considered to play an important role in the pa thogenesis and progression of neoplasia. An in situ 3'-end DNA labelin g (TUNEL) method was recently developed and has been widely used to id entify apoptotic cells in tissue sections. However, sometimes the TUNE L method labels many more cells than expected. We investigated the eff ects of prefixation time and fixation time on the apoptotic index dete cted by this method. Materials and Methods.-Using the spleen and thymu s of rats, the effects of prefixation time (0, 1, 2, 4, 6, 12, 24, 48, and 72 hours) at 4 degrees C and fixation time (6, 12, 24, 48, 72, an d 96 hours; i, 2, and 3 weeks) on the apoptotic index were examined by the TUNEL method. Agarose gel electrophoresis of extracted DNA from t he specimens of each prefixation time was also performed. Results.-In comparison with control tissue (no prefixation time), which showed sca ttered positive cells with distinct staining restricted to the nucleus , the splenic tissue unfixed for 2 hours or more and the thymic tissue unfixed for 4 hours or more showed cytoplasmic staining in the positi ve cells. Moreover, as the prefixation time was prolonged, the number of positive cells gradually increased. Agarose gel analysis of DNA ext racted from tissue sections left unfixed longer than 24 hours showed a ladder pattern consisting of multiples of about 200 base pairs. Concl usions.-Two hours was the limit of prefixation time for the precise id entification of apoptosis by the TUNEL method. The false-positive cell s in tissue sections left unfixed for longer time intervals may have b een due to internucleosomal DNA cleavage following necrosis. In contra st, the length of fixation time in buffered formalin seemed to have no effect on the results obtained by this method.