Fig. 2

Permeabilization before or after PFA fixation affects microtubule immunolabeling in IMCD3 and RPE cells. Cells were either (1) washed with a permeabilizing solution before being fixed with PFA-S, or (2) fixed with PFA-S, and then permeabilized with solutions of different strength. Cilia immunolabeling was assessed with a β-tubulin antibody (green) and Hoechst dye for nuclear staining (blue). No permeabilization resulted in little or absent cilia labeling and sporadic microtubule cytoskeleton staining. Permeabilization of any strength resulted in robust cilia labeling and microtubule staining. Permeabilization strength was negatively correlated with the preservation of microtubule labeling, although CB which contains 0.5% Triton X-100 appears to offer some benefit to microtubule preservation. Permeabilization done after cells were already fixed did not affect microtubule or cilia immunolabeling. Scale bars 10 µm