Ab218714

Inserts were fixed in 4% paraformaldehyde, washed with PBS and then the fixed membranes were removed from the inserts. Cells were permeabilized with 0.1% Triton in PBS for 15 min and then blocked overnight with 10% BSA–0.5% Tween 20 (both: Sigma-Aldrich, St. Louis, MO, USA) in PBS to reduce binding of unspecific antibodies. Subsequently, the inserts were washed with PBS 0.5%–Tween 20 after which the primary antibodies (diluted in 3% BSA–0.5% Tween 20 in PBS) were added and incubated overnight. The next day inserts were washed in PBS 0.5% Tween and secondary antibodies (diluted in 3% BSA and 0.5% Tween) were added. Cells were stained with DAPI (cell nuclei), ciliated cells (anti-b tubulin 1:1000, recombinant with Alexa Fluor 647 Abcam Catalog ab204034), goblet cells (anti-MUC5AC, 1:50, recombinant with Alexa Fluor 555 Abcam Catalog ab218714) and for RSV (primary antibody goat anti- RSV 1:100, Abcam Catalog ab20745, with secondary donkey anti-goat Alexa Fluor 488, 1:1000, Abcam Catalog ab150129). Finally, inserts were washed and mounted on glass slides.

To detect goblet cells and tight junctions, we used antibodies targeting Muc5Ac and ZO-1, respectively. The cultures were first fixed with 4% (w/v) paraformaldehyde (Sigma, Saint Louis, United States) for 15 min and then blocked for 1 h in a blocking solution (0.5% Triton X-100, 5% normal goat serum, and 2% bovine serum albumin; all reagents from Thermo Fisher Scientific, Waltham, MA, United States) in 1× Dulbecco’s phosphate-buffered saline (D-PBS; without calcium, magnesium, or Phenol red; STEMCELL Technologies). The cultures were stained with a Muc5AC antibody conjugated to Alexa 550 (1:250, ab218714, Abcam) or a ZO-1 antibody (1:250, 339194, Thermofisher), diluted in D-PBS with 2% normal goat serum (Thermo Fisher Scientific, Waltham, MA, United States) and 1% bovine serum albumin (Thermo Fisher Scientific). Nuclei were counterstained by using ProLong™ Diamond Antifade Mountant with DAPI (4′,6-diamidino-2-phenylindole; Thermo Fisher Scientific). Images were acquired with the CellInsight™ CX7 HCS platform (Thermo Fisher).