Immunofluorescence Staining of ACE2, SARS-CoV-2 Spike, and Lung Cell Markers

Each Transwell was washed three times with PBS and fixed using 4% PFA for 15 min at room temperature before antibody staining. The following antibodies were used: anti-ACE2 antibody (Anti-ACE2 antibody)—21115-1-AP; ProteinTech (Du et al, 2021 (link)); SARS-CoV/SARS-CoV-2 (COVID-19) spike antibody [1A9] (GTX632604; GeneTex); anti-SARS-CoV-2 nucleoprotein antibody (DA114; MRC-PPU); acetylated tubulin (T7451; Sigma-Aldrich); Muc5AC (MA5-12178; Invitrogen); Hoechst dye solution (100 μg/ml) was used for nuclei staining. Confocal images were taken using Nikon Confocal Microscopes C2, magnification 40× oil. Transwell inserts were imaged as described above.