Rabbit anti muc5b

Anti-Cytokeratin 5 antibody [EP1601Y] (Alexa Fluor^® 647) (Abcam, ab193895). Rabbit IgG, monoclonal [EPR25A] - Isotype Control (Alexa Fluor^® 647) (Abcam, ab199093). Purified Mouse IgG2b k Isotype Ctrl (Biolegend, 401202). Purified anti-TP63 (W15093A) (Biolegend, 687202). Mouse anti-ACT (Sigma-Aldrich; T6793). Rabbit anti-MUC5B (Sigma-Aldrich; HPA008246). DAPI (Sigma-Aldrich; D9542). AF488 Donkey anti-rabbit (Life Technologies; A21206).

Fixed membranes were blocked with PBS containing BSA and Triton X‐100, then probed with primary antibodies to acetylated tubulin (mouse anti‐ACT, 1:8000; Sigma) and Mucin 5b (rabbit anti‐MUC5b, 1:500; Sigma) to visualize differentiation into ciliated and goblet epithelial cell types. Following an overnight incubation at 4 °C, membranes were washed in 1X PBS solution and probed with secondary antibodies to anti‐mouse IgG (AF488, 1:500; Invitrogen/Thermo Fisher, Waltham, MA) and anti‐rabbit IgG (AF594, 1:500; Invitrogen/Thermo Fisher, Waltham, MA). DAPI (1:1000 dilution of 1 mg/ml stock solution; Sigma) was added as a counterstain to visualize all cellular nuclei. Membranes were again washed with 1X PBS solution, then mounted on slides with Fluoromount G medium (Southern Biotech, Birmingham, AL). Images were captured on DAPI, Alexafluor‐488, and Alexafluor‐594 channels at 40× magnification (6 fields/membrane) and quantified using the ImageJ software (NIH, Bethesda, MD).