Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation.
Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation.
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DOI：
10.1083/jcb.119.3.493
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发表时间：
1992-11
期刊：
影响因子：
--
通讯作者：
Ben-Sasson SA
中科院分区：
文献类型：
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作者：
Gavrieli Y;Sherman Y;Ben-Sasson SA
程序性细胞死亡（PCD）在发育生物学和维持持续更新组织的稳态中起着关键作用。目前，它的存在主要是从合并的DNA提取物的凝胶电泳推断，因为PCD被证明与DNA片段化有关。基于这一观察结果，我们在这里描述了一种方法的发展，在单细胞水平的PCD原位可视化，同时保留组织结构。用蛋白酶预处理的常规组织切片用生物素化的聚脱氧尿苷缺口末端标记，通过末端脱氧转移酶引入，然后使用抗生物素蛋白缀合的过氧化物酶染色。该反应是特异性的，仅位于预期PCD的位置处的细胞核被染色。初步筛查包括：小肠和大肠、表皮、淋巴组织、卵巢和其他器官。详细的分析表明，该过程是在核周边启动的，它是相对较短的（1-3小时，从启动到细胞消除），PCD出现在组织中的集群。这种方法揭示的组织PCD的程度大大大于细胞核形态学检测到的凋亡，从而为各种研究开辟了道路。
Programmed cell death (PCD) plays a key role in developmental biology and in maintenance of the steady state in continuously renewing tissues. Currently, its existence is inferred mainly from gel electrophoresis of a pooled DNA extract as PCD was shown to be associated with DNA fragmentation. Based on this observation, we describe here the development of a method for the in situ visualization of PCD at the single-cell level, while preserving tissue architecture. Conventional histological sections, pretreated with protease, were nick end labeled with biotinylated poly dU, introduced by terminal deoxy- transferase, and then stained using avidin-conjugated peroxidase. The reaction is specific, only nuclei located at positions where PCD is expected are stained. The initial screening includes: small and large intestine, epidermis, lymphoid tissues, ovary, and other organs. A detailed analysis revealed that the process is initiated at the nuclear periphery, it is relatively short (1-3 h from initiation to cell elimination) and that PCD appears in tissues in clusters. The extent of tissue-PCD revealed by this method is considerably greater than apoptosis detected by nuclear morphology, and thus opens the way for a variety of studies.