Mouse anti dsrna

VeroE6/TMPRSS2 cells cultured on 96-well plates (Lumos multiwell 96, catalog number 94 6120 096; Sarstedt AG & Co. KG, Germany) were infected with SARS-CoV-2 (WK-521) at an MOI of 0.1 and incubated for 5 h. Next, the cells were fixed for 30 min at 4°C with 4% paraformaldehyde in phosphate-buffered saline (PBS). After washing once with PBS, the cells were permeabilized for 15 min at room temperature (RT) with PBS containing 0.1% Tween 20. The cells were then incubated with a mixture of rabbit anti-SARS-nsp4 (1:500) (catalog number ab181620; Abcam, USA) and mouse anti-dsRNA (1:1,000) (catalog number J2-1709; Scicons, Hungary) antibodies for 1 h at RT, washed three times with PBS, and incubated for 1 h at RT with a mixture of Alexa Fluor 594-conjugated anti-rabbit IgG (1:500) (catalog number A11012; Thermo Fisher) and Alexa Fluor 488-conjugated anti-mouse IgG (1:500) (catalog number A10680; Thermo Fisher). Next, the cells were washed three times with PBS, and cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (1:5,000) (catalog number D1306; Thermo Fisher). Cells were observed under an inverted fluorescence phase-contrast microscope (catalog number BZ-X810; Keyence, Japan).