[問題] H2DCFDA (ROS染劑)在confocal的應用

看板Biotech (生命科學)作者paua (waiting for good news)時間19年前 (2006/11/04 14:48)推噓0(0推 0噓 0→)

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我想用H2DCFDA染ROS, 並用confocal來觀察 (不知道是不是太浪費啦~ 但是學校螢光顯微鏡有點怪怪的) 但是我不曉得樣品要怎麼固定, 查了一些paper和網路上的資料, 有的paper說要水鏡, 有的要油鏡 paper很少寫詳細的步驟, 目前看到寫比較清楚的是 MBC Vol. 17, Issue 5, 2125-2137, May 2006 http://tinyurl.com/yymsl7 他是這麼陳述的: 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(這是他實驗的主題, 與固定無關), 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. 我不是很清楚作者為什麼要打洞? 感覺很像是要染核. 他的confocal圖的核是藍色的, 那是不是用Hoechst, DAPI或是紅色的PI都可以? 然後接下來的步驟, 就只要加mounting solution, 封片, 就可以上機了嘛? 這種sample可以保存多久呀? 我之前的confocal sample (PI/FITC)大概在-20度保存了三個禮拜, 還是可以上機看的蠻清楚的.. ------------------------------------------------------------------------------ 在university at buffalo討論串中(http://tinyurl.com/yluahy), 也有人問相同的問題, 有好心人回答: 1. J.Leuk Biol 55:253, 1994 (我查了這篇, 但是沒看到我想要的資料) 2. Methods in Cell Biology VOl 41 and 42 (範圍好大, 我查了每篇文章的title, keyword, 但也沒找到) 3. Current Protocols in Cytometry (Wiley Liss) that deals with these probes. Its in Chapter 9. (但是這只有描述流式細胞儀的樣本前處裡: 染ROS, 所以也不是用confocal看) 麻煩請有興趣的人一起討論呀! 謝謝~~ ※ 編輯: paua 來自: 140.129.68.230 (11/04 22:12)