Stähelin B J, Marti U, Solioz M, Zimmermann H, Reichen J
Department of Clinical Pharmacology, University of Berne, Switzerland.
Mol Pathol. 1998 Aug;51(4):204-8. doi: 10.1136/mp.51.4.204.
The terminal transferase uridyl nick end labelling (TUNEL) assay allows the easy demonstration of cell death as a result of apoptosis. However, when this assay is applied to liver tissue, the number of TUNEL positive cells is dependent on the time of incubation with proteinase K.
To test whether false positive results are the result of the release of endogenous endonucleases by proteinase K and can be abolished by pretreatment with diethyl pyrocarbonate (DEPC).
Involution of hyperplastic ductules in bile duct ligated rats after biliary decompression by Roux-en-Y anastomosis and acute CCl4 intoxication were studied as models of apoptosis and necrosis, respectively. A standard TUNEL assay was applied to formalin fixed tissue sections mounted with cement. To inhibit putative endogenous endonucleases, tissue slides were pre-incubated with DEPC.
In the standard TUNEL assay, the number of positive nuclei was highly dependent upon the length of time that sections were incubated with proteinase K. After pretreatment with DEPC, only cells that also exhibited morphological features of apoptosis stained positive. DEPC pretreatment abolished false positive staining in CCl4 induced hepatocyte necrosis and blocked interference by endogenous alkaline phosphatase in intestine. The method of gluing the tissue section to the glass slide was found to be of utmost importance because the effect of DEPC was abolished on silanised slides.
False positive staining in the TUNEL assay in the liver is caused by the release of endogenous endonucleases as a result of proteinase treatment. This can be abolished by pretreatment of tissue slides with DEPC.
末端脱氧核苷酸转移酶介导的缺口末端标记（TUNEL）检测可轻松显示凋亡导致的细胞死亡。然而，将该检测应用于肝组织时，TUNEL阳性细胞的数量取决于与蛋白酶K孵育的时间。
测试假阳性结果是否是由于蛋白酶K释放内源性核酸内切酶所致，以及是否可通过焦碳酸二乙酯（DEPC）预处理消除。
分别以胆管结扎大鼠经Roux-en-Y吻合术胆道减压后增生性小胆管的退化和急性四氯化碳中毒作为凋亡和坏死的模型进行研究。将标准TUNEL检测应用于用水泥固定的福尔马林固定组织切片。为抑制假定的内源性核酸内切酶，组织切片先用DEPC预孵育。
在标准TUNEL检测中，阳性细胞核的数量高度依赖于切片与蛋白酶K孵育的时间。用DEPC预处理后，只有同时表现出凋亡形态特征的细胞染色呈阳性。DEPC预处理消除了四氯化碳诱导的肝细胞坏死中的假阳性染色，并阻断了肠道内源性碱性磷酸酶的干扰。发现将组织切片粘贴到载玻片上的方法至关重要，因为在硅烷化载玻片上DEPC的作用被消除。
肝脏TUNEL检测中的假阳性染色是由于蛋白酶处理导致内源性核酸内切酶释放所致。这可通过用DEPC预处理组织切片来消除。
