*4.8. Immunofluorescence*

RAW264.7 cells plated into a 4-well plate with a density of 6 × 104 cells/well were pre-treated with 150 μM of wistin for 30 min, followed by LPS (0.1 μg/mL) treatment. After 2 h, cells were washed with PBS and fixed with 4% paraformaldehyde (Sigma-Aldrich; Merck KGaA) in PBS for 20 min. The cells were then incubated with 0.2% Triton X-100 and 0.1% citrate (Sigma-Aldrich; Merck KGaA) in PBS for 5 min, washed with PBS, and blocked with 2% bovine serum albumin (Sigma-Aldrich; Merck KGaA) for 30 min, followed by probing with rabbit anti-p65 NF-κB antibody (1:500; cat. no. 8242; Cell Signaling Technology, Inc.) for 2 h. After washing with 2% bovine serum albumin, the cells were incubated with fluorescein goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody Alexa Fluor 555 (4 μg/mL; cat. no. A-21428; Invitrogen Inc., Middlesex County, MA, USA) for 1 h. After washing with 2% bovine serum albumin, the nuclei were counterstained with DAPI solution (1 mg/mL) for 5 min in the dark, and fluorescence was visualized using a fluorescence microscope (Zeiss microscope, Carl Zeiss AC).
