三种固定液对两种氧化应激细胞模型Bclin1和LC3蛋白免疫荧光染色的影响

摘要 目的：比较采用三种不同的固定液对两种氧化应激细胞模型Beclin1和LC3蛋白免疫荧光染色的影响。方法：本研究使用丙酮/甲醇（1:1）固定液、甲醇固定液和4%多聚甲醛三种固定液分别对氧化应激细胞模型大鼠原代心肌成纤维细胞和MCF-7乳腺癌细胞株进行固定，然后再分别进行免疫荧光双染实验，对比三种固定液固定后对自噬关键调控蛋白Beclin1和LC3染色效果。结果：三种固定液对氧化应激细胞模型Beclin1和LC3蛋白免疫荧光染色结果存在较大差异。丙酮/甲醇（1:1）固定液固定后免疫荧光染色效果最佳，细胞结构清晰可见，两种蛋白定位表达清晰，甲醇固定液次之，4%多聚甲醛固定液效果欠佳。结论：在对大鼠原代心肌成纤维细胞和MCF-7乳腺癌细胞进行自噬相关蛋白免疫荧光双染色实验中，在使用其它固定液染色效果不佳的情况下，可以选择应用丙酮/甲醇（1:1）固定液固定，再进行免疫荧光染色；根据不同实验需求相应选择更适宜的固定液，以达到最佳的荧光染色结果。

Effects of Three Kinds of Fixatives on Immunofluorescence Staining of Beclin1 and LC3 Proteins in Two Hypoxic Cell Models

ABSTRACT Objective: To compare the effects of three different fixation solutions on immunofluorescence staining of Beclin1 and LC3 proteins in two hypoxic cell models. Methods: In this study, primary myocardial fibroblasts and MCF-7 breast cancer cell lines of hypoxic cell models were fixed with acetone/methanol (1:1), methanol and 4% paraformaldehyde, respectively, and then double immunofluorescence staining were carried out to compare the staining effects of the three fixatives on the key autophagy regulatory proteins Beclin1 and LC3. Results: There were significant differences in the results of immunofluorescence staining of Beclin1 and LC3 proteins in two hypoxic cell models with three kinds of fixed solutions. The best effect of immunofluorescence staining was fixed by acetone/methanol (1:1). The localization and expression of the two proteins could be seen clearly, while methanol, and 4% paraformaldehyde appeared poor effect. Conclusion: In the autophagy-associated protein immunofluorescence double staining experiment of cardiac fibroblasts and MCF-7 breast cancer cells, when other fixatives are used and the staining effect is worse, acetone/methanol (1:1) fixation solution is a good choice before immunofluorescence staining. Moreover, choosing a more suitable fixation solution according to different experimental requirements could help to achieve the best fluorescence staining results.