影响共聚焦荧光法检测单细胞中蛋白表达的因素探讨

Factors Affecting the Location of Protein in Single Cells by Immunofluorescence with Laser Scanning Confocal Microscopy

采用免疫荧光技术联合共聚焦技术研究不同培养细胞中蛋白的表达及定位.结果发现:①同样以丙酮固定293细胞,不用0.5%Triton X-100处理,着丝粒蛋白E亚型(CENP-E)在分裂期细胞核周浓聚,核内未见;而用0.5%TritonX-100处理后,CENP-E呈散点状分布于分裂期细胞的核内.②分别以甲醇、95%乙醇和4%多聚甲醛固定并以1%TritonX-100处理k562细胞,不同固定剂处理组间结果无明显差异,信号转导与转录激活因子(STAT3)均定位于胞浆.共聚焦系统PMT=511时,显示胞核染色适中,核仁染色明显,而PMT=593时,整个核呈饱和染色状,微细结构不清楚.③以2%甲醛联合0.5%TritonX-100处理平滑肌细胞,固醇调节元件结合蛋白裂解激活蛋白(SCAP)在整个细胞中呈弥散性分布,核内含量较高,高尔基体上SCAP无特异性高表达.结论:是否使用TritonX-100处理对293细胞中核蛋白CENP-E的定位具有较大影响;k562细胞对醛类固定剂及醇类固定剂无特殊选择性,图片采集时共聚焦参数PMT对蛋白定位结果也有影响;醛类固定剂和Triton联用可准确定位SCAP在平滑肌细胞中的表达.

The expression and location of proteins in single cells cultured under different conditions were studied with a laser scanning eonfocal microscope. CENP-E appeared mainly around the nucleus in HEK293 cells by fixing with acetone without 0.5% Triton X-100 treatment, but localized mainly in the nucleus when the cells were fixed with acetone plus further 0.5 %Triton X-100-PBS solution and scattered in the nucleus with condensed points. Plus 1% Triton treatment, K562 cells fixed with 4% paraformaldehyde, methanol or 95% ethanol all showed that STAT3 protein mainly distributed in cytoplasm at the edge of the cell body, the nucleus occupied a greater part of the cell body with PI staining, nucleolus staining seemed stronger under PMT= 511, but seemed the same as the other part of the nucleus under PMT= 593. SCAP （SREBP cleavage-activating protein） could be detected in cytoplasm and was more accumulated in nucleus in smooth muscle cells fixed with 2 % formaldehyde and 0.5 % Triton penetrating, and Golgi apparatus were found to scatter around the nucleus. In conclusion, it is important to locate CENP-E protein in HEK293 cells with TritonX-100 by immunofluorescence microscopy. SCAP in smooth muscle cells and STAT3 expression in K562 cell can be exactly located with the combination of formaldehyde and Triton penetrating. STAT3 expression in k562 cells fixed with formaldehyde or spirits fixative has no difference. The confocal system itself, such as PMT gain, can also affect the location of proteins in single cells.