The National Natural Science Foundation of China (General Program, Key Program, Major Research Plan）
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.