Comparison of immunohistochemistry for activated caspase-3 and cleaved cytokeratin 18 with the TUNEL method for quantification of apoptosis in histological sections of PC-3 subcutaneous xenografts
Comparison of immunohistochemistry for activated caspase-3 and cleaved cytokeratin 18 with the TUNEL method for quantification of apoptosis in histological sections of PC-3 subcutaneous xenografts
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DOI：
10.1002/path.1289
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发表时间：
2003-02-01
影响因子：
7.3
通讯作者：
Blomme, EAG
中科院分区：
文献类型：
--
作者：
Duan, WR;Garner, DS;Blomme, EAG
末端脱氧核苷酸转移酶（TdT）介导的dUTP缺口末端标记（TUNEL）技术已被广泛用于检测和定量组织切片中的细胞凋亡。然而，该试验的解释和特异性一直存在争议。随着对细胞死亡的分子机制的知识的积累和半胱天冬酶作为细胞凋亡的关键介质的发现，出现了在组织切片中更直接和更早地测量细胞凋亡的方法。本研究使用特异性识别活化的caspase-3和caspase裂解的细胞角蛋白（CK）18的抗体，评估免疫组化染色是否会改善组织切片中细胞凋亡的检测和定量，与TUNEL法相比。前列腺癌细胞系PC-3的肿瘤异种移植物用作实例，因为这些组织含有大量经历凋亡的细胞。通过形态学分析、TUNEL测定、活化的caspase-3和裂解的CK 18免疫组织化学鉴定凋亡细胞后，通过计算机辅助图像分析对凋亡细胞进行定量并计算凋亡指数。结果表明，活化的caspase-3免疫组化是一种简单，敏感，可靠的方法来检测和定量细胞凋亡在这个模型中。观察到使用活化的半胱天冬酶-3和裂解的CK 18免疫染色获得的凋亡指数之间的良好相关性（R = 0.89）。活化caspase-3免疫组化法与TUNEL法检测的凋亡指数之间具有良好的相关性（R = 0.75），因此建议采用活化caspase-3免疫组化法检测和定量组织切片中的凋亡。版权所有（C）2003约翰威利父子有限公司。
The terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labelling (TUNEL) technique has been extensively used for the detection and quantification of apoptosis in histological tissue sections. However, the interpretation and specificity of this assay have been controversial. With accumulating knowledge of the molecular mechanisms of cell death and the discovery of the caspases as key mediators of apoptosis, more direct and earlier measurements of apoptosis in tissue sections have emerged. This study, using antibodies that specifically recognize activated caspase-3 and caspase-cleaved cytokeratin (CK) 18, evaluated whether immunohistochemical stains would improve the detection and quantification of apoptosis in tissue sections, compared with the TUNEL assay. Tumour xenografts of the prostate cancer cell line PC-3 were used as an example, since these tissues contain large numbers of cells undergoing apoptosis. Apoptotic cells were quantified and apoptotic indices were calculated by computer-assisted image analysis following identification of apoptotic cells by morphological analysis, the TUNEL assay, activated caspase-3 and cleaved CK18 immunohistochemistry. The results indicated that activated caspase-3 immunohistochemistry was an easy, sensitive, and reliable method for detecting and quantifying apoptosis in this model. An excellent correlation (R = 0.89) between the apoptotic indices obtained using activated caspase-3 and cleaved CK18 immunostaining was observed. A good correlation (R = 0.75) between the apoptotic indices obtained using activated caspase-3 immunostaining and the TUNEL assay was also found. Activated caspase-3 immunohistochemistry is therefore recommended for the detection and quantification of apoptosis in tissue sections. Copyright (C) 2003 John Wiley Sons, Ltd.