Indirect Immunofluorescence Assay for PSV

The indirect immunofluorescence assay (IFA) was conducted as previously described with slight modification [36 (link)]. Cell monolayers were inoculated with PSV at an MOI of 0.01 and incubated for 12 h. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 2% bovine serum albumin. A mAb specific for PSV-VP1 diluted 1:200 or swine serum at a 1:100 dilution was used as the primary antibody. The cells were then incubated with FITC-conjugated goat anti-mouse IgG 1:500 dilution (Thermo Fisher Scientific, Waltham, MA USA) or rabbit anti-pig IgG 1:50 dilution (Sigma, St. Louis, MO, USA). Nuclei were stained with 10 µg/mL DAPI (Solarbio, Beijing, China), and cells were then visualized under an inverted fluorescence microscope (Evos FL, Thermo-Fisher, Bothell, WA, USA).