Immunocytochemistry for PEDV Antigen Detection

To detect the PEDV antigen, immunocytochemistry (ICC) was performed as described previously [15 (link)]. Briefly, Vero cells infected with iPEDVPT-P96 showing typical CPE were fixed with 80% ice-cold acetone, air-dried, and incubated with an in-house anti-PEDV N antibody at room temperature (RT) for 1 h. After washing three times with PBS, a polyclonal anti-rabbit/mouse immunoglobulin, EnVision-DAB⁺ system (Dako, Carpinteria, CA, USA), was applied for 1 h at RT. Following three washes with PBS, the cells were incubated with 3, 3′-diaminobenzidine (DAB) chromogen from a peroxidase DAB substrate kit (Dako, Carpinteria, CA, USA) according to the manufacturer’s instructions. Positive signals were visualized under an inverted light microscope (Nikon, Tokyo, Japan).