Alexa fluor 488 conjugated goat polyclonal anti mouse igg antibody a 11029

Cells were fixed in 4% paraformaldehyde for 24 h prior to safe removal from the BSL-3 facility. Fixed cells were permeabilized in 0.2% Triton X-100 in PBS for 15 min at room temperature. After three washes in PBS, cells were blocked for 1 h at room temperature with 3% bovine serum albumin (BSA) in PBS. Cells were immunostained for dsRNA using a mouse monoclonal anti-dsRNA antibody/clone rJ2 (MABE1134; Sigma-Aldrich) overnight at 4°C. After three washes in PBS, primary antibodies were stained for with an Alexa Fluor 488-conjugated goat polyclonal anti-mouse IgG antibody (A-11029; Invitrogen) for 1 h at room temperature. After three washes in PBS, cells were stained for SARS-CoV-2 nucleocapsid with a directly conjugated Alexa Fluor 594 anti-SARS-N antibody (1C7) and for nuclear DNA using 4′,6-diamidino-2-phenylindole (DAPI) for 1 h at room temperature. After three washes in PBS, cells were imaged by fluorescence microscopy using an EVOS M5000 imaging system (Invitrogen).

Cells were fixed in 4% paraformaldehyde for 24 h prior to safe removal from the BSL-3 facility. Fixed cells were permeabilized in 0.2% Triton X-100 in PBS for 15 min at room temperature. After three washes in PBS, cells were blocked for 1 h at room temperature with 3% BSA in PBS. Cells were immunostained for SARS-CoV-2 nucleocapsid using a mouse monoclonal anti-SARS-N antibody (clone 1C7) overnight at room temperature. After three washes in PBS, cells were stained with an Alexa Fluor 488-conjugated goat polyclonal anti-mouse IgG antibody (A-11029; Invitrogen) and DAPI for 1 h at room temperature. After three washes in PBS, cells were imaged and quantified on a Celigo imaging cytometer (Nexelcom Bioscience). Graphs depict the average percentage of infected cells normalized to the control from eight biological replicates. Error bars depict the standard deviations.