Immunofluorescence Analysis of CCL20 Expression

Immunofluorescence analysis was performed as reported previously [61 (link)]. The cells were cultured in 4-well slide chambers (1.5 × 10⁵ cells/well) (Lab-Tek, Rochester, NY, USA) in accordance with the culture methods described herein. Then, the cell sheets were scratched and incubated for 6 h at 37 °C in 5% CO₂. The cell sheets were washed with PBS 3 times for 5 min each and fixed in cold acetone for 10 min at room temperature. The cell sheets were blocked with 10% BSA (Roche Diagnostics, Basel, Switzerland) and incubated with rabbit anti-human CCL20 polyclonal antibody or control normal rabbit polyclonal IgG. Goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody with Alexa Fluor 488 dye was used as the secondary antibody. The nucleus was stained with 4′,6-diamino-2-phenylindole (DAPI). Slides were then mounted with Ultra Cruz mounting medium (Santa Cruz Biotechnology, Dallas, TX, USA) and were observed using a D-Eclipse confocal laser scanning microscope (Nikon, Tokyo, Japan).