Mouse anti mucin 5ac

HNECs were seeded into removable 8 well silicone cultivation chambers (ibidi, Planegg, Germany) and grown for 5 days in complete Airway Epithelial Cell Growth Medium (PromoCell) at 37 °C, 5%CO₂. At 80–100% confluence cells were fixed with 4% paraformaldehyde, rinsed, and permeabilized with PBS plus 0.1% Triton X and 0.02% SDS. Subsequently, cells were blocked with 10% goat serum, incubated with either rabbit anti-wide spectrum Cytokeratin (1:200, abcam), mouse anti-Mucin 5AC (1:100, abcam), mouse anti-acetylated alpha Tubulin (1:200, abcam), mouse anti-Cytokeratin 14 (Sigma Aldrich) or rabbit anti-Claudin-1 (1:50, Invitrogen), rabbit anti-ZO-1 (1:50, Invitrogen), mouse anti-Occludin (1:50, Invitrogen) and then incubated with secondary antibodies: goat anti-mouse AF488 (1:2000, Invitrogen), donkey anti-rabbit AF488 (1:2000, Invitrogen) or goat anti-rabbit PE (1:100, Santa Cruz, Dallas, US). Images were recorded on a DMi8 microscope using LAS X Life Science microscope software (Leica, Wetzlar, Germany).

After antigen retrieval step, sections were washed in PBS and then blocked in 10% normal serum containing 0.1% Triton X-100, followed by incubation with primary antibodies at 4 °C overnight. After washing in PBS, the tissue sections were incubated with Alexa Fluor conjugated secondary antibodies along with DAPI for nuclear counterstaining. The following primary antibodies were used: Goat anti-ACE2 (1:40, AF933; R&D) Mouse anti-Keratin 18 (1:500, Pierce MA1–39483), Rabbit anti-DCX (1:500, GeneTex, GTX134052), Mouse anti-Mucin 5AC (1:200, Abcam ab3649).