Indirect immunofluorescence assay and confocal microscopy.

PK15 monolayer cells grown on chamber slides (BD) at 72 h after PCV3 strain LY infection were washed with phosphate-buffered saline (PBS) and then fixed in 4% paraformaldehyde (PFA). The cells were incubated with anti-PCV3 polyclonal antibody diluted in 3% bovine serum albumin (BSA)-PBS at room temperature (RT) for 1 h, followed by incubation with fluorescein isothiocyanate (FITC)-conjugated anti-mouse immunoglobulin G (Sigma-Aldrich) at RT for 1 h. Nuclei were stained with DAPI (2,4-diamidino-2-phenylindole) for 30 min at 37°C. The cells were washed with PBS, rinsed in distilled water, dried, mounted with fluorescence mounting medium, and examined under a Nikon AIR confocal laser microscope system.