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Author:
Date:
2015-04-05
[Abstract] Terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) is a method for detecting DNA fragmentation by labelling the 3' terminal end of nucleic acids. This method can be used both in animal and plant tissues. In animal tissues, the use of Proteinase K is sufficient for permeabilizing the cells and to obtain optimal labelling, but in plant tissues, the presence of the cell wall, does not allow proper labelling. For this reason, we carried out several modifications to the original TUNEL protocol (ApoAlert® DNA Fragmentation Assay Kit, Clontech) to obtain an optimal labelling. These modifications were additional treatments with cellulase, Triton X-100 and Proteinase K. Also, we describe the optimization of the positive controls by adjusting the units of ...
[摘要] 末端脱氧核苷酸转移酶介导的dUTP缺口末端标记（TUNEL）是通过标记核酸的3'末端来检测DNA断裂的方法。该方法可以在动物和植物组织中使用。在动物组织中，蛋白酶K的使用足以使细胞透化并获得最佳标记，但是在植物组织中，细胞壁的存在不允许适当的标记。因此，我们对原始TUNEL方案（ApoAlert DNA Fragmentation Assay Kit，Clontech）进行了几个修改，以获得最佳标记。这些修饰是用纤维素酶，Triton X-100和蛋白酶K的另外处理。此外，我们通过调节所用的DNase的单位描述阳性对照的优化。用于复染的PI浓度也被特别调节以避免过度的背景噪声，并因此正确地观察标记的和未标记的核。该工作还描述了以不干扰TUNEL标记的方式收集，存储和包括样品（特别是污点臂）的附加方案。
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