TUNEL apoptotic cell detection in tissue sections: Critical evaluation and improvement
TUNEL apoptotic cell detection in tissue sections: Critical evaluation and improvement
复制标题
DOI：
10.1177/002215549804600306
复制
发表时间：
1998-03-01
影响因子：
3.2
通讯作者：
Negoescu, A
中科院分区：
文献类型：
--
作者：
Labat-Moleur, F;Guillermet, C;Negoescu, A
TUNEL，即，末端脱氧核苷酸转移酶介导的dUTP缺口末端标记，已经成为一种广泛使用的染色方法，以帮助检测组织切片中的凋亡细胞。然而，尽管其表面上的简单，这种技术已经导致了相当大的失望，因为它的严重限制的灵敏度，甚至更多，在特异性。我们回顾了TUNEL的局限性和伪影，并设计了一个全面的协议，以重新评估各种程序中使用的五个交联和/或沉淀固定剂。通过在极端pH值溶液（福尔马林pH 3和布因固定剂pH 10.6）中引入微波加热并结合蛋白水解，我们获得了存档组织块上70-80%的凋亡细胞和体的强烈染色，几乎没有或没有背景。由于增强的敏感性，早期阶段的细胞凋亡可以可视化，并可能扩大我们的视野的凋亡细胞超越单纯的图像萎缩坏死。我们的结论是，TUNEL仍然是一种有用的技术，因为它是微妙的，需要严格的解释染色。这项研究指出，在档案组织，尽管我们提出的技术改进，没有协议可以是所有问题的最终答案。必须根据组织处理的任何变化重新调整技术。然而，一步一步的进展，使这种方法不仅适用，而且在档案外科病理学标本的限制内执行。
TUNEL, i.e., terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling, has become a widely used staining method to assist in detection of apoptotic cells in tissue sections. However, despite its apparent simplicity, this technique has led to considerable disappointment because of its serious limitations in sensitivity and, even more, in specificity. We reviewed the limitations and artifacts of TUNEL and designed a comprehensive protocol to reassess the various procedures in use for five crosslinking and/or precipitating fixatives. By introducing microwave heating in extreme pH-value solutions (pH 3 for formalin and pH 10.6 for Bouin's fixative) coupled with proteolysis, we obtained an intense staining of 70-80% of apoptotic cells and bodies on archival tissue blocks, with little or no background. Owing to the enhanced sensitivity, early stages of apoptosis could be visualized and may enlarge our vision of the apoptotic cell beyond the mere image of shrinkage necrosis. We conclude that TUNEL remains a technique as useful as it is delicate, requiring critical interpretation of the staining. This study points out that, on archival tissues, despite the technical improvements we propose no protocol can be the final answer to all problems. Technique must be readjusted for any variation in tissue processing. However, step-by-step progress has rendered this method not only applicable but also performable within the constraints of archival surgical pathology specimens.