Multiparametric Imaging of SARS-CoV-2 Infection

WT or D614G-infected LAE ALI cultures were fixed twice for 20 minutes in 4% formaldehyde in PBS and stored in PBS. The SARS-CoV-2 N antigen was stained with polyclonal rabbit anti-SARS-CoV N protein (Invitrogen PA1–41098, 0.5 ug/mL),and using species-specific secondary antibodies as previously described (4 (link)). The cultures were also imaged for a-tubulin (Millipore MAB1864; 3ug/mL) and MUC5AC (Thermo Scientific 45M1; 4ug/mL) as indicated. Filamentous actin was localized with phalloidin (Invitrogen A22287), and nuclei was visualized with Hoechst 33342 staining (Invitrogen). An Olympus FV3000RS confocal microscope in Galvo scan mode was used to acquire 5-channel Z stacks by 2-phase sequential scan. Representative stacks were acquired and are shown as Z-projections and XZ cross sections to distinguish individual cell features and to characterize the infected cell types. ImageJ was used to measure the relative apical culture surface covered by multiciliated cells.