Diamond antifade mountant with 4 6 diamidino 2 phenylindole dapi

VeroE6 cells seeded on glass coverslips were infected with SARS-CoV-2 isolates at a moi of 0.1. At 8 hours post-infection, cells were fixed in 4% paraformaldehyde, permeabilized with 0.3% Triton X-100 and blocked in 10% fetal calf serum. SARS-CoV-2 N- and spike-specific primary antibodies (dilution 1:1000 and 1:500, respectively) and AF568-labeled goat-anti-rabbit (Invitrogen, #A11011, 1:400) secondary antibody as well as AF488-labeled Phalloidin (Hypermol, #8813-01, 1:250) were used for staining. The coverslips were embedded in Diamond Antifade Mountant with 4′,6-diamidino-2-phenylindole (DAPI) (ThermoFisher, #P36971). Fluorescence images were generated using an LSM800 confocal laser-scanning microscope (Zeiss) equipped with a 63X, 1.4 NA oil objective, and Airyscan detector and processed with Zen blue software (Zeiss) and ImageJ/Fiji.

VeroE6 cells seeded on glass coverslips were either infected with SARS-CoV-2 isolates at a moi of 0.1 or left uninfected. At 8 h post infection, cells were fixed in 4% paraformaldehyde in PBS, permeabilized with 0.3% Triton X-100 and blocked in 10% FCS. SARS-CoV-2 N- (Rockland #200-401-A50, 1:1000), S- (Rockland #600-401-MS8, 1:250) and ORF3a-specific primary antibodies (https://mrcppu-covid.bio/, 1:100) and AF568-labeled goat-anti-rabbit (Invitrogen, #A11011, 1:400) secondary antibody as well as AF488-labeled Phalloidin (Hypermol, #8813-01, 1:400) were used for staining. The coverslips were embedded in Diamond Antifade Mountant with 4′,6-diamidino-2-phenylindole (DAPI) (ThermoFisher, #P36971). Fluorescence images were generated using a LSM800 confocal laser-scanning microscope (Zeiss) equipped with a 63X, 1.4 NA oil objective and Airyscan detector and processed with Zen blue software (Zeiss) and ImageJ/Fiji.