不同固定液对胃癌细胞骨架及连接蛋白荧光染色标记效果的比较  

Comparison of the Effect of Different Fixatives on Cytoskeleton and Connectin Fluorescence Staining in SGC-7901 Cells

作　　者：高建[1] 郑倩倩[2] 李彦姝[2] 周末[1] 

机构地区：[1]中国医科大学实验技术中心三部,沈阳110001 [2]中国医科大学基础医学院细胞生物学教研室,沈阳110001

出　　处：《中国医科大学学报》2012年第10期870-872,876,共4页Journal of China Medical University

基　　金：国家自然科学基金资助项目(30800415);辽宁省科学计划项目(2009415005);沈阳市科学技术项目(F11-241-00)

摘　　要：目的通过激光共聚焦技术,观察SGC-7901胃癌细胞系在不同固定液作用下的细胞骨架及连接蛋白荧光染色。方法接种SGC-7901细胞24 h后,分别用4%多聚甲醛、2.5%戊二醛及95%乙醇室温固定20 min,5%BSA(含0.2%Triton X-100)封闭透膜1 h,直标荧光一抗避光37℃孵育1 h,DAPI室温染核15 min,激光共聚焦扫描显微镜扫描。结果 4%多聚甲醛对细胞骨架微丝结构及连接蛋白Zo-1细胞膜定位均有较好的固定作用,2.5%戊二醛对微丝结构基本起到固定作用,而95%乙醇对于微丝的固定效果略差。后两种固定液固定细胞均出现连接蛋白胞质表达的非特异染色现象。结论 4%多聚甲醛对细胞骨架及细胞间连接蛋白的固定效果较优,荧光染色标记结果为后续研究提供依据。Objective To compare the results of cytoskeleton and connectin fluorescence staining in SGC-7901 cells pre-treated with different fixatives by laser scanning confocal technology.Methods SGC-7901 cells were fixed by 4% paraformaldehyde（4% PFA）,2.5% glutaraldehyde（2.5% GA） or 95% ethanol at room temperature for 20 min.Antigen was blocked and the cells membrane were penetrated by 5% BSA（containing 0.2% Triton X-100） for 1 h.The samples were incubated with first antibody at 37 ℃ for 1 h avoiding light.The nuclei were stained with DAPI at room temperature for 15 min.Results The construction of microfilament and the position of Zo-1 were fixed better by 4% PFA than 2.5% GA,while the fixation effect（95%） was the worst of all.Conclusion 4% PFA showed good fixation effect,which provides a basis for the further research.

关 键 词：固定液 细胞骨架 连接蛋白 激光共聚焦 

分 类 号：Q291[生物学—细胞生物学]