Ultrastructural detection of DNA strand breaks in apoptotic neural cells by in situ end‐labelling techniques
Ultrastructural detection of DNA strand breaks in apoptotic neural cells by in situ end‐labelling techniques
复制标题
通过原位末端标记技术超微结构检测凋亡神经细胞中的 DNA 链断裂
作者：
A. Migheli;A. Attanasio;D. Schiffer
最近开发的基于DNA链断裂“原位末端标记”（ISEL）的技术可能有助于识别组织切片中的凋亡细胞。我们在电子显微镜（EM）水平上应用了ISEL技术，以验证细胞凋亡的超微结构特征是否确实与DNA断裂的证据有关，以及尚未进入细胞死亡阶段的细胞是否也被标记。应用末端转移酶和DNA聚合酶测定Araldite和LR金包埋的髓母细胞瘤和胚胎小鼠背根神经节的薄片。使用地高辛标记的核苷酸；免疫金染色证实掺入。在两种组织中均能很容易地标记出处于死亡过程各阶段的凋亡细胞。此外，DNA片段在初始染色质凝聚的细胞中被证实，但与邻近未染色的细胞无法区分。我们的研究结果表明，EM - ISEL技术有效地证明了神经组织中凋亡细胞和可能的“凋亡前”细胞中DNA链断裂的发生。由于标签很容易在常规处理的组织上获得电子显微镜，因此该技术可以对档案材料进行回顾性研究。
Recently developed techniques based on ‘in situ end‐labelling’ (ISEL) of DNA strand breaks may help to identify apoptotic cells in tissue sections. We have applied ISEL techniques at the electron microscopic (EM) level, in order to verify if ultrastructural features of apoptosis are indeed associated with evidence of DNA fragmentation, and whether cells committed to, but which have not yet entered the stage of cell death are also labelled. Terminal transferase and DNA polymerase assays were applied to thin sections of Araldite and LR Gold‐embedded medulloblastomas and embryonic mouse dorsal root ganglia. Digoxigenin‐labelled nucleotides were used; incorporation was demonstrated by immunogold staining. Apoptotic cells in various stages of the death process were easily labelled in both tissues. In addition, DNA fragmentation was demonstrated in cells with initial chromatin condensation, but otherwise indistinguishable from adjacent unstained cells. Our results show that EM‐ISEL techniques effectively demonstrate the occurrence of DNA strand breaks in apoptotic and possibly ‘pre‐apoptotic’ cells in neural tissues. Since the labelling is easily obtained on tissue that is routinely processed for electron microscopy, this technique may allow retrospective studies on archival material.
DOI：
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发表时间：
1993-03
期刊：
The American journal of pathology
影响因子：
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作者：
K. Fukuda;M. Kojiro;Jen-Fu Chiut
通讯作者：
K. Fukuda;M. Kojiro;Jen-Fu Chiut
DOI：
10.1073/pnas.90.3.980
发表时间：
1993-02-01
影响因子：
11.1
作者：
SCHWARTZ, LM;SMITH, SW;OSBORNE, BA
通讯作者：
OSBORNE, BA
影响因子：
56.9
作者：
WHITE, K;GRETHER, ME;STELLER, H
通讯作者：
STELLER, H