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

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1h,直标荧光一抗避光37℃孵育1h,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% glu-taraldehyde (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 DAP! 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.