Tateyama H, Tada T, Hattori H, Murase T, Li W X, Eimoto T
Department of Pathology, Nagoya City University Medical School, Nagoya, Japan.
Arch Pathol Lab Med. 1998 Mar;122(3):252-5.
Apoptosis is considered to play an important role in the pathogenesis and progression of neoplasia. An in situ 3'-end DNA labeling (TUNEL) method was recently developed and has been widely used to identify apoptotic cells in tissue sections. However, sometimes the TUNEL method labels many more cells than expected. We investigated the effects of prefixation time and fixation time on the apoptotic index detected by this method.
Using the spleen and thymus 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, and 96 hours; 1, 2, and 3 weeks) on the apoptotic index were examined by the TUNEL method. Agarose gel electrophoresis of extracted DNA from the specimens of each prefixation time was also performed.
In comparison with control tissue (no prefixation time), which showed scattered 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 positive cells. Moreover, as the prefixation time was prolonged, the number of positive cells gradually increased. Agarose gel analysis of DNA extracted from tissue sections left unfixed longer than 24 hours showed a ladder pattern consisting of multiples of about 200 base pairs.
Two hours was the limit of prefixation time for the precise identification of apoptosis by the TUNEL method. The false-positive cells in tissue sections left unfixed for longer time intervals may have been due to internucleosomal DNA cleavage following necrosis. In contrast, the length of fixation time in buffered formalin seemed to have no effect on the results obtained by this method.
细胞凋亡被认为在肿瘤的发生发展过程中起重要作用。一种原位3'-末端DNA标记（TUNEL）方法最近被开发出来，并已广泛用于识别组织切片中的凋亡细胞。然而，有时TUNEL方法标记的细胞比预期的要多得多。我们研究了预固定时间和固定时间对该方法检测凋亡指数的影响。
使用大鼠的脾脏和胸腺，通过TUNEL方法检测4℃下预固定时间（0、1、2、4、6、12、24、48和72小时）和固定时间（6、12、24、48、72和96小时；1、2和3周）对凋亡指数的影响。还对每个预固定时间的标本提取的DNA进行了琼脂糖凝胶电泳。
与对照组织（无预固定时间）相比，对照组织显示散在阳性细胞，染色仅限于细胞核且清晰，未预固定2小时或更长时间的脾脏组织和未预固定4小时或更长时间的胸腺组织在阳性细胞中出现细胞质染色。此外，随着预固定时间延长，阳性细胞数量逐渐增加。对未固定超过24小时的组织切片提取的DNA进行琼脂糖凝胶分析，显示出由约200个碱基对倍数组成的梯状条带模式。
对于通过TUNEL方法精确识别凋亡而言，2小时是预固定时间的极限。未固定较长时间间隔的组织切片中的假阳性细胞可能是由于坏死导致的核小体间DNA裂解。相比之下，在缓冲福尔马林中的固定时间长度似乎对该方法获得的结果没有影响。
