Bovine serum albumen

MDBK cells grown on 25-mm glass coverslips were infected with the vBoHV1-UL21-HA virus or mock infected. At 18 hpi, the cells were fixed in 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, and blocked with 1% bovine serum albumen (Biosharp) and 0.1% Tween 20 in PBS. To detect the interactions of UL16 with UL21, the cells were incubated with mouse anti-HA-tag antibodies (1:5,000 dilutions) or rabbit polyclonal antisera against UL16 (1:100 dilutions) as indicated. Then, they were stained with Cy3-conjugated goat anti-mouse antibodies (1:1000 dilutions; Beyotime) or fluorescein isothiocyanate-conjugated goat anti-rabbit IgG antibodies (1:1000 dilutions; Beyotime). After each step, the cells were washed three times with 1× PBS. The nuclei were visualized by DAPI staining (Beyotime). Images were captured using a Zeiss LSM 880 laser-scanning confocal microscope.

MDBK cells grown on glass coverslips were infected with the indicated viruses. At different time points, the cells were fixed in 4% paraformaldehyde at 37 C for 20 min, permeabilized with 0.2% Triton X-100, and blocked with 1% bovine serum albumen (Biosharp, Hefei, China) and 0.1% Tween 20 in PBS. The cells were incubated with mouse anti-HA antibodies (1:5,000 dilution; Beyotime) and then stained with Cy3-conjugated goat anti-mouse antibodies (1:1,000 dilution; Beyotime). Nuclei were stained blue with 4, 6-diamidino-2-phenylindole (DAPI) (Beyotime). After each step, the cells were washed three times with 1× PBS. Then, anti-fade mounting medium was used to mount the coverslips onto slides. The cells were examined, and images were captured with a Zeiss LSM 880 laser-scanning confocal microscope (Carl Zeiss, Jena, Germany).