免疫荧光检测p53与线粒体共定位的实验方法探索  

An exploration of immunofluorescence protocol detecting co-localization of p53 and mitochondria

作　　者：龙丹[1] 冯莉[1] 陈雪璐[1] 李胜富[1] 周彦妮[1] LONG Dan;FENG Li;CHEN Xuelu;LI Shengfu;ZHOU Yanni(Key Laboratory of Transplant Engineering and Immunology of the Ministry of Health,West China Hospital,Sichuan University,Chengdu,Sichuan 610041,P.R.China)

机构地区：[1]四川大学华西医院卫生部移植工程与移植免疫重点实验室,成都610041

出　　处：《华西医学》2020年第8期948-953,共6页West China Medical Journal

基　　金：国家自然科学基金(81270552)。

摘　　要：目的探索研究p53与线粒体共定位的最佳免疫荧光条件,为检测蛋白质与线粒体共定位的研究提供参考。方法低氧处理HeLa细胞,免疫印迹检测p53表达;分别使用甲醇固定HeLa细胞、甲醇:丙酮混合液(体积比1∶1)固定HeLa细胞、4%多聚甲醛固定HeLa细胞,前二者不作通透处理,后者用0.1%聚乙二醇辛基苯基醚(Triton-X 100)通透,同时染色p53和线粒体;用4%多聚甲醛固定HeLa细胞后,降低Triton-X 100浓度至0.05%、0.025%、0.01%和0.005%,同时染色p53和线粒体;用4%多聚甲醛固定HeLa细胞后,Triton-X 100浓度降至0.01%和0.005%,通透标记p53后再次使用0.1%Triton-X 100重新通透细胞器膜染色线粒体。结果低氧后p53表达上调(P<0.01),可用于后续免疫荧光实验。使用甲醇和混合液固定组均可同时在细胞核及细胞质内观察到明显的p53信号,并可观测到其与线粒体的共定位,混合液组共定位更明显。多聚甲醛固定组用0.1%Triton-X 100通透后p53信号主要在细胞核内,未能观察到共定位;使用4%多聚甲醛固定后,降低Triton-X 100浓度至0.05%和0.025%在一定程度上削弱p53核内信号,增强共定位信号,但细胞核内信号仍强于细胞质,当降低至0.01%和0.005%,细胞质内检测到p53信号,细胞核内未检测到p53信号,提示此条件下核膜未被通透,但同样也未能通透线粒体膜,导致线粒体标记失败;二次通透完全规避p53核内信号,并成功标记线粒体,检测到p53与线粒体的共定位。结论使用甲醇或混合液固定可观察到p53与线粒体共定位,其中混合液效果更佳;使用4%多聚甲醛固定结合2次通透可有效避免核内信号,检测到p53和线粒体的共定位。Objective To establish a better immunofluorescence protocol to detect co-localization of p53 and mitochondria which may benefit studies aiming to detect mitochondrial expression of proteins.Methods HeLa cells were treated with hypoxia and the expression of p53 was detected by immunoblotting.HeLa cells were fixed with methanol,methanol:acetone(1:1,v/v)mixture,and 4%paraformaldehyde,respectively;the former two groups were not permeable,while the latter was penetrated with 0.1%Triton-X 100 and stained with p53 and mitochondria at the same time.After HeLa cells were fixed with 4%paraformaldehyde,the concentration of Triton-X 100 was reduced to 0.05%,0.025%,0.01%,and 0.005%.After the HeLa cells were fixed with 4%paraformaldehyde,the concentration of Triton-X 100 decreased to 0.01%and 0.005%for the first time,then,after staining with p53,the mitochondria were stained with 0.1%Triton-X 100 for the second time.Results The expression of p53 was up-regulated(P<0.01)after hypoxia,which could be used in the following immunofluorescence experiment.The co-localization of p53 and mitochondria was observed in the nucleus and cytoplasm in both the methanol group and the mixed solution group.The co-localization of p53 was the most obvious in the mixed solution group.After using 0.1%Triton-X 100,the p53 signal was mainly in the nucleus,but no co-localization was observed.After fixation with 4%paraformaldehyde,to some extent,the reduced concentration of0.05%and 0.025%Triton-X 100 weakened the p53 signal in nucleus and enhanced the co-localization signal.However,the signal in nucleus was still stronger than that in cytoplasm.When it was reduced to 0.01%and 0.005%,p53 signal was detected in cytoplasm but not in nucleus,suggesting that the nuclear membrane was not penetrated under this condition,but it also failed to penetrate the mitochondrial membrane,leading to the failure of mitochondrial labeling.The second permeability completely avoided the p53 signal in nucleus,and successfully labeled mitochondria,and the co-localization of p5

关 键 词：P53 线粒体 共定位 免疫荧光 

分 类 号：R446.6[医药卫生—诊断学]