不同打孔条件对BGC-823中MAC30和F-actin免疫荧光检测精度的影响

Effect of different perforating conditions on the detection accuracy of MAC30 and F-actin in gastric cancer cell line BGC-823 by immunofluorescence technique

目的探讨不同浓度和时间条件下,打孔试剂Triton X-100对胃癌细胞系BGC-823中MAC30和F-actin表达和定位精度的影响。方法应用0.1%、0.2%或0.5%含量的打孔试剂Triton X-100,处理BGC-823细胞5、10或15min,然后利用MAC30和F-actin抗体进行免疫荧光染色。结果当应用0.1%Triton X-100与固定的胃癌细胞系BGC-823作用5min,MAC30蛋白在BGC-823细胞质中高表达,F-actin染色清晰。随着打孔试剂含量从0.1%提高到0.5%,或作用时间从5min延长到15 min,MAC30在细胞质和细胞核中表达也随之上调,尤在细胞质中高表达,而F-actin染色则开始变得模糊,直至无法分辨肌丝和伪足。结论应用细胞免疫荧光染色揭示某蛋白与胃癌发生发展相关性研究中,应考虑不同打孔时间和含量所产生的染色结果差异,注重实验条件的选择与优化。

OBJECTIVE Immunofluoresence technique was utilized to explore the effect of various concentrations and incubation periods of Triton X-100 on the detection accuracy of cellular localization and expression of MAC30 and F-actin in gastric cancer cell line BGC-823. METHODS BGC-823 cells were treated with 0.1%, 0.2% or 0.5% Triton X-100 for 5 rain, 10 min or 15 min, followed by immunofluorescence staining with antibodies against MAC30 and F-actin. RESULTS Upon exposure to 0.1% Triton X-100 for 5 min, MAC30 protein was abundantly expressed in the cytosol of fixed gastric cancer cell line BGC-823, and the staining of F-actin was clear. When the concentration of the perforating re- agent was increased from 0. 1% to 0.5%, or the incubation period increased from 5 min to 15 min, the expression of MAC30 was accordingly upregulated in nucleus, and more prominently in cytoplasm. In contrast, the F-actin staining became blurred, and the filaments and pseudopodia could not be distinguished. CONCLUSIONS When investigating the correlation between proteins and development of gastric carcinoma by immunofluorescence, the different effects resulting from distinct concentrations and incubation periods of perforating reagents should be considered. Great efforts should be dedicated to the selection and optimization of experimental conditions.