Immunofluorescence Staining of CCoV Infection

Monolayers of A72, pretreated or not with CH223191, were infected or not with CCoV, at an MOI of 0.05 and 5, and incubated at 37 °C. After 24 h p.i., immunofluorescence staining was evaluated as previously reported [21 (link),37 (link)], by using the following antibodies, diluted in 5% bovine serum albumin-1X Tris-Buffered Saline, 0.1% Tween^® 20 Detergent: anti-AhR (Sigma-Aldrich, St. Louis, MI, USA) (1:250), anti-NP monoclonal mouse, MAB 938 (The Native Antigen Company, Kidlington, UK), Alexa Fluor 488 goat anti-mouse (Thermo Fisher Scientific, Waltham, MA, USA) (1:1000), and Texas Red goat anti-rabbit (Thermo Fisher Scientific, Waltham, MA, USA) (1:100). Microscopy and photography were assessed by ZOE Fluorescent Cell Imager (Bio-Rad Laboratories, Hercules, CA, USA). Quantification of fluorescence signals from microscopy-generated images was performed using ImageJ (National Institutes of Health, Bethesda, MD, USA) software.