Air confocal laser microscope system

Sf9 monolayer cells seeded in chamber slides were infected with recombinant baculovirus. At the indicated times, the cells were washed with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde (PFA). After three washes, the cells were incubated with rabbit anti-Cap antibody, which was derived from sera of rabbits after immunization with recombinant Cap protein three times, diluted in 3% bovine serum albumin (BSA)-PBS at room temperature (RT) for 1 h. After three further washes, cells were incubated with fluorescein isothiocyanate (FITC)-conjugated anti-rabbit antibodies (DAKO) at 37°C for 1 h. Nuclei were visualized by staining with 4’,6’-diaminido-2-phenylindole (DAPI) at a concentration of 1 μg/ml for 30 min at 37°C. Cells were washed three times with PBS, rinsed in dH₂O, dried and mounted with fluorescence mounting media, and examined under Nikon AIR confocal laser microscope system.

PK15 monolayer cells grown in chamber slides (BD) were infected with PCV2 strain BJW. At the indicated times postinfection, the cultured cells were washed with phosphate-buffered saline (PBS) and then fixed in 4% paraformaldehyde (PFA) diluted in PBS. The cells were co-incubated with guinea pig anti-ORF1 antibody and relevant DDR signal antibodies diluted in 3% bovine serum albumin (BSA)-PBS at room temperature (RT) for 1 h followed by incubation with fluorescein isothiocyanate (FITC)- and rhodamine-conjugated antibodies (DAKO) at 37 °C for 1 h. Nuclei were stained with 4′,6′-diaminido-2-phenylindole (DAPI). The cells were then rinsed in dH₂O, dried and mounted with fluorescence mounting media, and observed under Nikon AIR confocal laser microscope system. In addition, immunofluorescence images were quantitatively determined in over 100 cells on each of 3 occasions.
For BrdU incorporation, PK15 cells were grown on a chamber slide and inoculated with PCV2 at an MOI of 1 TCID₅₀. At 18 h postinfection, BrdU was supplemented into the cell culture medium at a final concentration of 5 μM. At 24 h postinfection, cells were fixed and coimmunostained with guinea pig anti-PCV2 ORF1 and mouse anti-BrdU antibody to mark the PCV2 replication centers.