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目的 :观察免疫细胞化学中不同固定剂对乳癌易感基因 (brca1)和表皮生长因子 (epidermalgrowthfactor,EGF)在MCF 7细胞中的亚细胞分布的影响。方法 :采用免疫细胞化学的方法 ,按照ABC试剂盒提供的操作步骤观察用不同固定剂及不同缓冲液时BRCA1和EGF在MCF 7细胞中定位情况。结果 :如果细胞用 3.7%的甲醛 PBS溶液固定 ,抗体稀释液及细胞冲洗液皆用PBS ,则BRCA1和EGF均着色于细胞浆 ;若用 3.7%的甲醛 PBS溶液固定细胞 ,而抗体稀释液及细胞冲洗液更换为含有 0 .1%吐温 2 0或 0 .0 5 %TritonX 10 0的PBS ,则BRCA1和EGF均主要着色于细胞核。结果还表明 ,用纯甲醇、95 %乙醇或冰醋酸∶甲醇 (1∶3)固定细胞 ,无论是单用PBS或是含有化学去垢剂的PBS作为抗体稀释液及细胞冲洗液 ,BRCA1和EGF均主要着色于细胞核。结论 :用免疫细胞化学技术检测细胞核蛋白时 ,最好选用甲醇作为固定剂。如果用甲醛固定细胞 ,建议用能给核膜打孔的化学去垢剂进一步处理细胞 ,否则会导致错误结果出现
OBJECTIVE: To observe the effect of different fixatives in immunocytochemistry on the subcellular distribution of BRCA1 and epidermal growth factor (EGF) in MCF7 cells. Methods: Immunocytochemistry was used to observe the localization of BRCA1 and EGF in MCF-7 cells with different fixatives and different buffers according to the procedure provided by the ABC kit. RESULTS: If the cells were fixed with 3.7% formalin in PBS, BRCA1 and EGF were both colored in the cytoplasm of the antibody dilution and cell wash; if the cells were fixed with 3.7% formaldehyde in PBS, and the antibody diluent and Cell washings were changed to PBS containing 0.1% Tween 20 or 0.05% Triton X 10 0, then both BRCA1 and EGF were predominantly stained in the nucleus. The results also showed that cells were fixed with pure methanol, 95% ethanol or glacial acetic acid: methanol (1: 3), either as PBS or a chemical detergent containing PBS as antibody dilution and cell wash, BRCA1 and EGF Are mainly colored in the nucleus. CONCLUSIONS: When using immunocytochemistry to detect nuclear proteins, it is best to use methanol as the fixative. If cells are fixed with formaldehyde, it is advisable to further treat the cells with chemical detergents that punctuate the nuclear membrane, which can lead to erroneous results