Immunofluorescence and Confocal Imaging

Human bronchial tissue or BEC-ALI cultures were fixed in 4% paraformaldehyde and embedded in paraffin blocks. The samples were sectioned and mounted on charged glass slides, then deparaffinized and treated with 10 mM sodium citrate for antigen retrieval. After blocking with 5% BSA (1 h, room temperature), the slides were incubated overnight at 4°C with primary antibody (mAb CDHR3, Abcam; mAb acetylated α-tubulin, Cell Signaling Technology; mAb E-cadherin, Abcam; mAb β-actin, Sigma-Aldrich). The following day, the slides were washed with PBS with Tween 20 and then incubated (1 h, room temperature) with fluorophore-conjugated secondary antibody (Alexa Fluor fluorophores; Pierce Biotechnology) before counterstaining with DAPI and mounting (EMS mounting medium; Electron Microscopy Sciences). Fluorescence images were captured with a Nikon Ti Eclipse microscope. For Z-stack imaging, BEC-ALI cultures were fixed, then permeabilized with 0.2% Triton X-100 before serum block and antibody incubation. Cell specimens were counterstained with DAPI and then placed on glass-bottomed plates in mounting medium.