不同固定方法对初级纤毛蛋白免疫荧光的影响
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南京医科大学

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国家自然科学基金项目(面上项目,重点项目,重大项目)


The comparison of different fixation methods on primary cilia immunofluorescence
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The National Natural Science Foundation of China (General Program, Key Program, Major Research Plan)

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    摘要:

    目的:比较不同固定方法对动物细胞初级纤毛组分的免疫荧光染色影响,探究初级纤毛免疫荧光成像的优化策略。方法:选择4%多聚甲醛(PFA)、甲醇(MeOH)、10%三氯乙酸(TCA)固定细胞,后续使用相同的透化、封闭、抗体孵育条件,最后比较相同拍摄参数下初级纤毛成像效果。结果:PFA固定法可使纤毛轴丝蛋白和部分纤毛膜蛋白有更好的染色效果,MeOH固定法可以更清晰地展现纤毛底部定位的蛋白。相比于前两种方法,TCA固定的细胞虽然纤毛轴丝的染色效果很差,但适用于部分纤毛膜蛋白和轴丝结合蛋白。除荧光强弱不同外,使用不同方法固定后染色得到的蛋白定位也有微小差异。结论:三种固定方法在初级纤毛免疫荧光实验中各有优劣,需要综合考虑实验目的以及目标蛋白的特点进行选择。

    Abstract:

    Objective: To compare the effects of different fixation methods on primary cilia immunofluorescence in mammalian cells and to investigate the optimized strategy of primary cilia immunofluorescence imaging. Methods: Cells were fixed with 4% paraformaldehyde (PFA), methanol (MeOH) or 10% trichloroacetic acid (TCA), followed by the same conditions of penetration, blocking and antibody incubation, and finally the primary cilia imaging results were compared under the same shooting parameters. Results: PFA fixation resulted in the better imaging of ciliary axoneme proteins and part of the ciliary membrane proteins, and MeOH fixation showed more clear imaging of the proteins localized at the bottom of cilia. Compared with other two methods, TCA fixation was suitable for some ciliary membrane proteins and axoneme binding proteins although the staining signal of ciliary axoneme was weak. In addition to the variations of fluorescence intensity, there were also minor differences in protein localization observed by staining after fixation using different methods. Conclusion: Each of the three fixation methods has their own advantages and limitations in immunofluorescent staining of primary cilia, and the appropriate fixation method needs to be selected with comprehensive consideration of the experimental purpose and the characteristics of target protein.

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  • 收稿日期:2022-11-15
  • 最后修改日期:2023-01-07
  • 录用日期:2023-03-02
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