4.11. Immunofluorescence Staining

BV2 cells were seeded at 2 × 10⁴ cells/well into 60 mm dishes. After incubation for 12 h, the cells were pretreated with ALF (0.5, 1, and 2.5 μg/mL) or the positive control (NAC, 100 μM) for 2 h before exposure to 1 μg/mL LPS. After incubation for 1 h, the cells were sequentially washed with PBST (0.05% Tween 20 in PBS), fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.1% Triton X-100 for 10 min, and blocked with 1% BSA in PBST for 30 min. The cells were incubated with phospho-NF-κB p65 antibody (1:100) for 1 h at room temperature. After washing, the cells were incubated with Alexa Fluor 488-conjugated secondary antibody for 1 h in the dark. The nuclei were counterstained with DAPI solution. Fluorescent images were observed under an Axio-phot microscope (Carl Zeiss, Jena, Germany).