| 林国豪,蒋 旭,孙丽纳,王 昊,姚 茜,韦正波,谢 莹.不同固定液对体外细胞系荧光蛋白淬灭及对胞内蛋白荧光染色的影响[J].,2019,19(11):2072-2075 |
| 不同固定液对体外细胞系荧光蛋白淬灭及对胞内蛋白荧光染色的影响 |
| Effects of Different Fixatives on the Quenching of Fluorescent Proteins in Vitro Cell Lines and Intracellular Protein Fluorescence Staining |
| 投稿时间：2019-01-08 修订日期：2019-01-30 |
| DOI：10.13241/j.cnki.pmb.2019.11.014 |
| 中文关键词: 荧光蛋白 猝灭 免疫荧光 固定方法 |
| 英文关键词: Fluorescent protein Quenching Immunofluorescence Fixation method |
| 基金项目:国家自然科学基金项目（81760361）；广西壮族自治区自然科学基金项目（2017GXNSFAA198064，2016GXNSFAA380096） |
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| 中文摘要: |
| 摘要 目的：比较不同细胞固定液对荧光蛋白淬灭情况以及核内、胞浆蛋白免疫荧光染色的影响。方法：分别对融合了RFP和GFP基因的鼻咽癌HK1细胞采用95 %乙醇、75 %乙醇、甲醇、丙酮：甲醇=1：1、5 %冰乙酸、Carnoy固定液进行固定，然后采用免疫荧光法对细胞进行免疫染色。结果：六种固定液均能使荧光蛋白猝灭。免疫荧光染色方面，对于核蛋白染色，75 %乙醇、95 %乙醇、丙酮：甲醇=1：1、Carnoy固定液固定后核区获得明显的荧光染色，而采用甲醇、5 %冰乙酸固定后荧光染色不明显。对于胞质蛋白染色，按荧光染色的清晰程度分为固定于Carnoy固定液> 丙酮：甲醇=1：1>甲醇>5 %冰乙酸>75 %乙醇>95 %乙醇，前四者固定可见分布于胞质，75 %乙醇或95 %乙醇固定的目标蛋白定位不清。结论：六种不同的固定液在有效失活荧光蛋白的情况下对核蛋白及胞浆蛋白抗原性的影响略有不同，可根据研究目的蛋白表达的部位及特点来选用合适的固定液。 |
| 英文摘要: |
| ABSTRACT Objective: Comparing the effects of different cell fixatives on the quenching of fluorescent proteins and the nuclear and cytoplasmic protein antigenicity. Methods: Nasopharyngeal carcinoma HK1 cells fused with RFP and GFP genes were fixed with 95% ethanol, 75% ethanol, methanol, acetone: methanol=1:1, 5% glacial acetic acid, Carnoy’ fixative, respectively. All cells were stained by immunofluorescence method. Results: Six fixation buffers can quench the fluorescent protein. For immunofluorescence staining, for nucleoprotein staining, 75 % ethanol, 95 % ethanol acetone: methanol = 1:1 fixation, Carnoy's fixative fixed nuclear staining after obtaining significant fluorescent staining. The fluorescence signal was weakened by fixation with methanol, 5 % glacial acetic acid. For cytoplasmic protein staining, the performed according to the clarity of fluorescent staining was Carnoy's fixative > acetone: methanol = 1:1 > methanol fixation > 5 % glacial acetic acid > 75 % ethanol > 95 % ethanol, the first four were fixedly distributed in the cytoplasm, 75 % ethanol and 95 % ethanol was fixedly distributed in the nucleus. Conclusion: Six different fixatives have slightly different effects on the antigenicity of nuclear protein and cytosolic protein in the case of effective inactivation of fluorescent protein. The appropriate fixative can be selected according to the location and characteristics of the protein expression of the research object. |
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