Immunofluorescence and Cell Growth Assays

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.