Immunofluorescence microscopy

Cultures were fixed in 4% (w/v) paraformaldehyde for 15 min at room temperature and rinsed with PBS. The samples were incubated in 0.5 ml permeabilization buffer (0.5% [v/v] Triton X-100 in PBS containing 100 mg/ml sucrose, 4.8 mg/ml HEPES, 2.9 mg/ml NaCl and 600 μg/ml MgCl₂, pH 7.2) for 10 min, washed three times with PBS and blocked for 1 h in PBST (PBS with 0.05% [v/v] Tween-20) containing 10% (v/v) normal goat serum and 1% (w/v) bovine serum albumin. The cultures were incubated with primary antibodies diluted in blocking buffer for 1 h at room temperature. The primary antibodies included mouse anti-ZO-1 antibody (Thermo Fisher; #33-9100) used at a 1:50 dilution to identify tight junction formation and rabbit anti-β-tubulin antibody (Abcam; #ab6046) used at a 1:200 dilution to identify cilia. The samples were subsequently washed three times in PBST for 2 min and incubated with secondary antibodies at a dilution of 1:400 in blocking buffer for 1 h at room temperature in the dark. The secondary antibodies included goat anti-mouse-Alexa Fluor 488 (Thermo Fisher; #A-11001) and goat anti-rabbit-Alexa Fluor 488 (Thermo Fisher; #A-11034). Mucus was visualized by incubation with a 1:200 dilution of fluorescein-labelled Jacalin for 1 h (Vector Laboratories; #FL-1151)⁷⁵. After washing three times with PBST, nuclei were stained with 300 nM 4′,6 diamidino-2-phenylindole (DAPI) in PBS for 10 min and F-actin visualized by incubation with a 1:40 dilution of rhodamine phalloidin (Thermo Fisher; #R415) for 20 min. The samples were washed three times with PBST after each of these stains and the membranes cut from the inserts and placed onto glass slides. A drop of Vectashield mounting medium (Vector Laboratories) was added to the surface of each sample and a coverslip sealed in place using clear nail varnish. Images were acquired with a Leica DMi8 microscope for standard fluorescence microscopy. Analysis of captured images was performed using ImageJ software.