2.9. Immunocytochemistry

Vero E6 cells were seeded (1.5 × 10⁴ cells) in an 8-well chamber and incubated for 24 h. PEDV and HCoV-OC43 were infected at an MOI of 0.002 and 0.2, respectively, for 2 h. Approximately 5 μM (185 μg/mL) purified 3D8 and 1% Pen/Strep antibiotic were added to DMEM supplemented with 10% FBS and incubated at 37 °C in a 5% CO₂ incubator. The cells were washed with PBS and fixed for 15 min in ice-cold methanol at room temperature. The cells were permeabilized with permeabilization buffer (421002, Biolegend^®, San Diego, CA) for 10 min at room temperature. After blocking with 1% BSA and 0.3 M (22.52 mg/mL) glycine in PBST buffer for 1 h at room temperature, primary antibodies for detecting PEDV (mouse anti-PEDV monoclonal Ab, MBS313516, Mybio), HCoV-OC43 (mouse anti-OC43 monoclonal Ab, LS-C79764, LS-bio), and 3D8 (polyclonal rabbit IgG serum Ab) were incubated overnight at 4 °C. PEDV and HCoV-OC43 were incubated with TRITC-conjugated anti-mouse Ab (ab6786, Abcam) and 3D8 was incubated with Alexa 488-conjugated anti-rabbit Ab (ab150077, Abcam). The nuclei were stained with Hoechst (62249, Thermo Fisher Scientific Inc., Rockford, IL, USA) during the last 10 min of incubation at RT. Cells were mounted in mounting medium (H-1200 Vectashield, Vector Laboratories, Burlingame, CA, USA) and observed with a NIKON A1R (Eclipse A1Rsi and Eclipse Ti-E).