我想用H2DCFDA染ROS, 并用confocal来观察 (不知道是不是太浪费啦~ 但是学校萤光显微镜有点怪怪的) 但是我不晓得样品要怎麽固定, 查了一些paper和网路上的资料, 有的paper说要水镜, 有的要油镜 paper很少写详细的步骤, 目前看到写比较清楚的是 MBC Vol. 17, Issue 5, 2125-2137, May 2006 http://tinyurl.com/yymsl7 他是这麽陈述的: ROS Formation Cultured cells were loaded with 10 μM 2'7'-dichlorodihydrofluorescin diacetate (CM-H2DCFDA, Molecular Probes) for 1 h before the apoptotic stress. In cells, esterases cleave the acetate esters to release free CM-H2DCF, which is nonfluorescent. After oxidation by ROS, CM-H2DCF is converted to green- fluorescing dichlorofluorescein (DCF; Curtin et al., 2002). After brief UV exposure(这是他实验的主题, 与固定无关), the cells were left for 4 h, then washed, fixed, and permeabilized with PBS containing 20% ethanol and 0.1% Tween 20. In some experiments, 10 nM E2 was added for 4 h. Cell extracts were centrifuged, and supernatants collected. DCF fluorescence was quantified with a fluorescence plate reader using 450-nm excitation and 530-nm emission filters. Confocal microscopy provided a qualitative assessment. The study was carried out three times, and the mean and SD were analyzed by ANOVA plus Schefe's test at a p < 0.05 level of significance. 我不是很清楚作者为什麽要打洞? 感觉很像是要染核. 他的confocal图的核是蓝色的, 那是不是用Hoechst, DAPI或是红色的PI都可以? 然後接下来的步骤, 就只要加mounting solution, 封片, 就可以上机了嘛? 这种sample可以保存多久呀? 我之前的confocal sample (PI/FITC)大概在-20度保存了三 个礼拜, 还是可以上机看的蛮清楚的.. ------------------------------------------------------------------------------ 在university at buffalo讨论串中(http://tinyurl.com/yluahy), 也有人问相同的问题, 有好心人回答: 1. J.Leuk Biol 55:253, 1994 (我查了这篇, 但是没看到我想要的资料) 2. Methods in Cell Biology VOl 41 and 42 (范围好大, 我查了每篇文章的title, keyword, 但也没找到) 3. Current Protocols in Cytometry (Wiley Liss) that deals with these probes. Its in Chapter 9. (但是这只有描述流式细胞仪的样本前处里: 染ROS, 所以也不是 用confocal看) 麻烦请有兴趣的人一起讨论呀! 谢谢~~ ※ 编辑: paua 来自: 140.129.68.230 (11/04 22:12)