2.4 Indirect immunofluorescence assay (IFA)
PK15 cells infected with YNG strain were fixed with 4% paraformaldehyde for 20 min and permeabilized with 0.5% Triton X-100 for 10 min at room temperature, respectively. Then the cells were blocked with 3% bovine serum albumin (BSA) for 1h, and probed with mouse monoclonal antibodies against the gB and gE proteins of PRV (1:1000) for 4 h. Following the removal of the supernatants, the cells were washed with PBS three times, the second antibodies (goat anti-mouse-FITC) (1:2500) were applied and incubated for 45 min in dark environment. The images were taken using a fluorescent microscope (Olympus, Tokyo, Japan).