Mucin 5ac

Cells were fixed in either 95% (w/v) chilled methanol (Thermo Fisher, UK) or 4% (w/v) paraformaldehyde (Sigma-Aldrich, UK) and stored under PBS at 4 °C until staining. Cells were permeabilized with 0.1% (w/v) Triton X for 10 min and then incubated with 5% (w/v) BSA for 1 h at room temperature before overnight incubation with primary antibody diluted in PBS at 4° C (vimentin, 1 in 500 (ab92547); pan-cytokeratin, 1 in 100 (ab86734); alpha-smooth muscle actin, 1 in 100 (ab5694); Ecadherin, 1 in 50 (ab15148); P63, 1 in 300 (ab124762); cytokeratin 5, 1 in 100 (ab52635); mucin 5AC, 1 in 200 (ab3649); β IV tubulin, 1 in 500 (ab179509); mucin 5AC (Abcam, UK); mucin 5B, 1 in 50 (sc-21768) (Santa Cruz Biotechnology, US). Primary antibody was then removed, and cells were washed in 3 changes of PBS prior to incubation with secondary antibody (DyLight 488 anti-rabbit IgG (ab96881) or DyLight 594 anti-mouse IgG (ab96881) (Abcam, UK)) for 1 h at room temperature in the dark. The secondary antibody was then removed, and cells were washed in 2 changes of PBS before incubation with DAPI (Sigma-Aldrich, UK) for 5 min. Cells were then washed once, covered with PBS, and imaged on a Nikon Eclipse T1 fluorescence microscope with a Photometrix Prime Mono camera or an Olympus FV1200 confocal microscope.