Fluorescent inverted microscope

Cells were seeded into 6-well plates (Corning, NY, USA) and fixed with 4% paraformaldehyde (Solarbio, Beijing, China) for 20–30 min. After washing three times with PBS for 5 min each, cells were permeabilized with 0.1% Triton X-100 for 20 min at room temperature and blocked with 3% bovine serum albumin (Servicebio, Wuhan, China) for 30 min at room temperature. Cells were then immunolabeled with anti-cytokeratin 18 antibodies (1:500 mouse monoclonal, Abcam, Cambridge, MA, USA) overnight at 4 °C. Moreover, samples were washed with PBS for 5 min three times and incubated with monkey anti-mouse IgG (H+L) secondary antibody (Proteintech, Chicago, IL, USA) at room temperature for 50 min. After washing three times in PBS, cells were DAPI (Servicebio, Wuhan, China) stained for 10 min at room temperature in the dark and imaged under a fluorescent inverted microscope (Thermo Fisher, Waltham, MA, USA). The method of cell growth curves was based on a previous article [8 (link)]. Primary duck intestinal epithelial cells (P7) and IDECs (P30) were plated onto 24-well plates (Corning, NY, USA) at 2.5 × 10⁴ cells per well and incubated at 37 °C with 5% CO₂. One well of cells was calculated by a red blood cell counting board (Qiujing, Shanghai, China) every 24 h. Experiments were conducted in triplicate, and mean values were plotted for the growth curves.

To confirm viral isolation and evaluate the antigenicity of the ARV isolate SD19/11103, an immunofluorescence assay (IFA) was performed. HeB02 was used as a positive control. LMH cells were infected with 1 × 10^4.0 TCID₅₀ of SD19/11103 or HeB02. Twenty-four hours later, cells were harvested and fixed with 4% paraformaldehyde for 30 min and permeabilized with 0.1% Triton X-100 in PBS for 15 min, followed by blocking with 5% bovine serum albumin in PBS for 1 h. Then, the cells were incubated with monoclonal antibody against ARV σB or ARV-positive sera diluted in PBS for 1 h. The cells were washed five times with PBS and incubated with the fluorescein isothiocyanate-conjugated anti-mouse or anti-chicken IgG (Sigma, St. Louis, MO, USA) secondary antibodies. Finally, the cells were washed five times with PBS and observed under a fluorescent inverted microscope (Thermo Fisher Scientific, Waltham, MA, USA).