Immunofluorescence staining

Immunofluorescence staining was used to verify the presence of HAEpi cells, which were seeded in six-well plates (2x10⁵ cells/well) and fixed with 4% paraformaldehyde at room temperature for 20 min. Cell were subsequently incubated with PBS containing 0.3% Triton X-100 at 4˚C for 15 min. The cells were blocked with 10% normal goat serum (cat. no. ab7481; 1:50; Abcam) at 37˚C for 20 min and then incubated with an anti-cytokeratin (CK)-19 antibody (cat. no. ab15463; 1:100; Abcam) at 4˚C overnight (30). Cells were washed using PBS and incubated with a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (cat. no. 10285-1-AP; 1:1,000; ProteinTech Group, Inc.) at room temperature for 1 h. Nuclei were counterstained using 4',6-diamidino-2-phenylindole (DAPI) at room temperature for 10 min. An anti-fluorescence quenching agent (ProLong^® Antifade kit; cat. no. P7481; Thermo Fisher Scientific, Inc.) was then added to cells in the dark at room temperature for 30 min. Images were subsequently taken using a fluorescence microscope (magnification, x400) with NIS-Elements BR software version 4.60.