Alexa fluor 488 conjugated goat anti rabbit antibody

For immunofluorescence staining, PEDV-infected cells were washed twice with PBS and fixed with 4% paraformaldehyde for 15 min, followed by membrane permeabilization with 0.1% Triton X-100 in PBS for 15 min at room temperature. After being blocked with 5% bovine serum albumin (BSA), cells were stained with anti-PEDV M polyclonal antibody for 1 h, followed by rinsing three times with PBS and incubation with Alexa Fluor 488-conjugated goat anti-rabbit antibody (Beyotime, Shanghai, China). Nuclei were visualized by using DAPI nuclear. Pictures of immunofluorescent cells were captured using an EVOS fluorescence microscope (M7000, Thermo Fisher Scientific, Waltham, MA, USA) at 100× magnification.

PEDV-infected Vero cells were washed twice with PBS and fixed with 4% paraformaldehyde for 15 min. Cell membranes were made permeable by treating with 0.1% Triton X-100 in PBS for 15 min at room temperature. After blocking with 5% bovine serum albumin (BSA), cells were stained with anti-PEDV M polyclonal antibody for 1 h, rinsed three times with PBS, and then incubated with Alexa Fluor 488-conjugated goat anti-rabbit antibody (Beyotime, Shanghai, China). Nuclei were visualized using DAPI nuclear staining, and pictures of fluorescent cells were captured using an EVOS fluorescence microscope (M7000, Thermo Fisher Scientific, Waltham, MA, USA) at 100× magnification.

Vero cells at 90% confluence in 48-well plates were inoculated with 200 μL of recombinant virus cultures. After a 2-h incubation, the inoculum was removed, and cells were cultured in DMEM with or without trypsin (15 μg/mL). At 36 hpi, the cells were rinsed with PBS three times, fixed with 3.7% formaldehyde for 15 min at room temperature, and permeabilized with 0.25% Triton X-100 in PBS for 15 min at 37°C. Next, the cells were blocked with 5% goat serum at 37°C for 1 h and incubated with rabbit anti-PEDV M polyclonal antibody at a dilution of 1:1,000 at 37°C for 1 h. After three washes with PBS, Alexa Fluor 488-conjugated goat anti-rabbit antibody (Beyotime, Shanghai, China) was added at a dilution of 1:200, and the cells were further incubated for 1 h. Finally, the nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (1:1,000 dilution) at 37°C for 15 min, and the cells were washed three times with PBS. Pictures of immunofluorescent cells were captured using an Evos fluorescence microscope (M7000; Thermo Fisher Scientific, Waltham, MA, USA).