2.6. Immunocytofluorescence Assay

For immunocytofluorescence staining, cells were washed twice with PBS and fixed with 4% formaldehyde (Merck, Darmstadt, Germany) in PBS, followed by membrane permeabilization with 0.1% Triton X-100 (Sigma, Burlington, MA, USA) in PBS for 10 min at room temperature. Fixed cells were blocked using 5% (w/v) bovine serum albumin (BSA, Sigma, USA) in PBS for 1 h followed by incubation with PDCoV polyclonal antibody (prepared in our lab, 1:100 dilution) for 1 h. After washing three times with PBS, cells were incubated with fluorescent-labeled polyclonal goat anti-pig IgG antibody (Sigma, 1:100 dilution) for 1 h. Cells were washed 5 times with PBS, and further counterstained with DAPI (Sigma, Burlington, MA, USA) for another 5 min at room temperature. After being washed 5 times with cold PBS, the culture plate was observed under a fluorescence microscope (Zeiss, Oberkochen, Germany).