Immunofluorescent Staining of Airway Epithelial Monolayers

The immunofluorescent staining of the monolayers was carried out based on the procedures described previously^{17 (link)}. Briefly, monolayers taken off the platform were immediately fixed with 4% formaldehyde for 10 mins following a very gentle sampling of the apical medium. The samples were then permeabilized with 0.2% Triton-X for 10 minutes. After permeabilization, the wells were washed once in PBS^+/+ and immediately stained overnight with Phalloidin-iFluor 488 Reagent (1:1000, ab176753-300TEST, Abcam) and DAPI (1:1000, 62248, Thermo Scientific) in Blockaid at 4 °C. After washing the samples with PBS^+/+ for two times, the monolayers were excised and mounted on a coverslip using ProLong Gold antifade reagent (Thermo Fisher, P36930). Mounted samples were imaged with a Zeiss LSM800 confocal microscope.