Immunofluorescence Imaging of SARS-CoV-2 in HBECs

HBECs grown in transwell filters as described above were inoculated with 10⁴ PFU of ic-SARS-CoV-2-mNG [59 (link)] or a mock control. HBECs were fixed with 4% PFA for 30 minutes at RT, followed by permeabilization with 0.2% Triton X100 in 1X PBS for 10 minutes at RT. Cells were blocked with 10% normal goat serum in 1X PBS (blocking buffer) for 1 hour at RT. Primary antibodies for Ac-tubulin (Abcam, Cambridge, Massachusetts, USA) and Forkhead Box J1 (FOXJ1) (Sigma Aldrich, St. Louis, Missouri, USA) were diluted in blocking buffer at 1:500 and were incubated overnight at 4°C. Goat anti-mouse Alexa Fluor 594 (BioLegend, San Diego, California, USA) and goat anti-rabbit APC (Invitrogen) were diluted in the blocking buffer at 1:200 and were applied for 2 hours at RT and further stained with Hoechst 33342 (Life Technologies) for 30 minutes at RT. The transwell filters were then cut and placed in a glass slide and mounted with a Prolong Diamond Antifade Mountant (Life Technologies). Representative photos were taken using a Leica LSR microscope. Scale bars correspond to 25 μm.