摘要
目的比较不同的固定方法对细胞内胞浆蛋白Vigilin和核蛋白CTCF的免疫荧光染色结果的影响。方法在免疫染色前,分别采用四种方法对HepG2细胞进行固定及通透:①纯甲醇-20℃处理8min后,再用80%丙酮-20℃处理1min。②甲醇:丙酮的1∶1混合溶剂-20℃处理10min。③在方案2的基础上,再用0.1%Triton-X-100冰上通透10min。④4%多聚甲醛室温固定10min后,用0.1%Triton-X-100冰上通透10min。结果采用方案①、②、③、④得到的胞浆蛋白vigilin在细胞内的大致分布无明显差异,但用方案④处理后,染色效果更为清晰而能更好地观察到细胞的细微结构;另外,采用方案①和方案②处理细胞后,对核蛋白CTCF进行免疫染色,可见CTCF分布于胞浆;方案③中,在纯甲醇固定后,若用0.1%Triton-X-100通透,则CTCF显示主要分布于细胞核内,胞质内亦有阳性染色;方案④的通透条件下,CTCF特异性分布于细胞核,胞质内几乎无阳性染色。结论不同的固定及通透方法对免疫染色的结果影响很大,应针对抗原特点,采用适宜的固定方法,以得到正确的实验结果。
Objective To evaluate the effects of different fixation methods on immunostaining of cytoplasmic protein Vigilin and nuclear protein CTCF respectively.Methods HepG2 cells were fixed and permeabilized by four methods as following：①cells were soaked in pure methanol for 8min at-20℃ and in acetone at the same temperature for 1min consecutively;②Cells were soaked in the 1∶1 ratio of mixture of methanol and acetone for 10 min,-20℃.③On the basis of Method②,cells were permeablized with 0.1 ％Triton-X-100 on ice.④Cells were fixed by 4％ PFA,followed by being permeablized with 0.1 ％Triton-X-100 on ice.Results There is no significant difference among the approximately cellular location of Vigilin by methods①-④,while the subtle cellular structure could be obtained by method④.Besides,CTCF were distributed in cytoplasm after the procedure of methods① or ②.As in method③,if cells were permeabilized by 0.1％ Triton-X-100 after the fixation of pure methanol,CTCF were majorly distributed in nuclei with less positive staining in cytoplasm.After the fixation with 4 ％ PFA and permeation with 0.1 ％ Triton-X-100,CTCF was specially distributed in nuclei.Conclusion Different fixation methods have various effects on the results of immunostaining.It is expected to choose proper fixation methods aimed to the characteristic of antigens for true results.
出处
《西部医学》
2013年第11期1608-1610,1615,共4页
Medical Journal of West China
基金
国家自然科学基金资助项目(30870957)
关键词
固定方法
免疫荧光
胞浆蛋白
核蛋白
Fixation method
Immunofluorescence
Cytoplasmic protein
Nuclear protein
