摘要
目的优化细胞免疫荧光染色技术结合激光扫描共聚焦显微镜技术的实验步骤,观察并定位Merm1/Wbscr22在人肺癌细胞NCI-H1299中的表达,以期对后续深入研究Merm1/Wbscr22的分子功能提供技术支持。方法采用Western blot及细胞免疫荧光染色结合激光扫描共聚焦显微镜技术观察Merm1/Wbscr22在NCI-H1299细胞的表达与定位,改进并优化免疫荧光染色实验步骤。结果 Merm1/Wbscr22蛋白在NCI-H1299细胞内过量表达,并定位于细胞核内。细胞免疫染色步骤显著地影响Texas Red-鬼笔环肽的非特异性染色及F-actin、Merm1/Wbscr22、细胞核DNA染色荧光的强度。结论 结合细胞免疫荧光染色技术与激光扫描共聚焦显微镜技术可以清楚地观察Merm1/Wbscr22在人肺癌细胞NCI-H1299的表达与定位,该技术的最优实验步骤是细胞固定、透膜、封闭、Texas Red-鬼笔环肽标记F-actin、一抗结合、二抗结合、DAPI标记细胞核DNA,最后共聚焦显微镜成像,这样既能减少Texas Red-鬼笔环肽的非特异性染色,各组分荧光强度又足够清晰利于观察。
Objective To optimize the experimental procedures to clearly observe and localize the expression of Merml/Wbscr22 inNCI - H1299 cells using immunofluorescence and confocal laser scanning microscope （CLSM） , thus providing technical foundation for fur- ther study of the function of Merml/Wbscr22 in the context of tumorigenesis. Methods The expression and localization of Merml/Wb- scr22 in NCI - H1299 cells were determined by Western Blot and immunofluorescence staining combined with CLSM. Different conditions were tested to enhance the fluorescent signals. Results In NCI - H1299 cells, Merml/Wbscr22 proteins were overexpressed and local- ized in cell nuclei. The different staining steps could result in the non - specific staining of Texas Red - phalloidin, as well as the fluores- cent intensity of F - actin, Merml/Wbscr22 and DNA. Conclusion The optimal fluorescent staining steps are fixation, Triton X - 100 extraction, blocking, F- actin staining by Texas Red -phalloidin, primary antibody incubation, secondary antibody incubation, DNA staining by DAPI and photograph with CLSM. This procedure not only significantly decreases artificial staining caused by Texas Red - phalloidin, but also enhances the fluorescent intensity.
出处
《医学研究杂志》
2013年第3期69-73,共5页
Journal of Medical Research
基金
浙江省自然科学基金资助项目(CF1214D)
浙江省科技厅科技条件建设项目(2011F10015)
