Sweeney Sean T, Hidalgo Alicia, de Belle J Steven, Keshishian Haig
Cold Spring Harb Protoc. 2012 Sep 1;2012(9):1013-6. doi: 10.1101/pdb.prot068379.
The terminal deoxynucleotide transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) method for monitoring targeted cell ablation is based on the in situ labeling of DNA fragmentation sites in nuclei of intact fixed cells. Unlike other methods of detecting dying cells, the use of fixed material allows antigen expression to be monitored at the same time that apoptosis is confirmed in the targeted cells. Double-labeling of Drosophila embryos using the TUNEL reaction and fluorescently tagged antibodies can be adapted to the selected antigen. For some antigens, it is preferable that the TUNEL reaction be performed first, whereas for others, the TUNEL reaction should follow antigen detection. This may be because some antigens may not survive the 37°C incubation or the conditions of the reaction. Similarly, increased fixation times yield better results for some antigens, but not for others. This protocol describes a TUNEL reaction adapted for use on Drosophila embryos in conjunction with fluorescently labeled antibodies.
用于监测靶向细胞消融的末端脱氧核苷酸转移酶（TdT）介导的dUTP缺口末端标记（TUNEL）方法基于对完整固定细胞细胞核中DNA片段化位点的原位标记。与其他检测死亡细胞的方法不同，使用固定材料可以在确认靶向细胞发生凋亡的同时监测抗原表达。使用TUNEL反应和荧光标记抗体对果蝇胚胎进行双重标记可适用于所选抗原。对于某些抗原，最好先进行TUNEL反应，而对于其他抗原，TUNEL反应应在抗原检测之后进行。这可能是因为某些抗原可能无法在37°C孵育或反应条件下存活。同样，延长固定时间对某些抗原可产生更好的结果，但对其他抗原则不然。本方案描述了一种适用于果蝇胚胎并结合荧光标记抗体的TUNEL反应。
