2.7. Immunofluorescence Staining

Prior to culture, 1 × 10⁴ RAW264.7 cells/well were treated with CoQ₀ (10 μM) for 1 h and then stimulated with LPS (1 μg/mL) for 5 h followed by ATP (5 mM) for 1 h and then seeded in an eight-well glass Tek chamber. Post culture using 2% paraformaldehyde, the cells were fixed for 15 min and then permeabilized with 0.1% Triton X-100 for 10 min and then washed and blocked with 10% FBS in PBS. Primary antibodies of anti-NLRP3 and anti-LC3B were incubated with 1.5% FBS for 2 h and then with fluorescein (FITC) (488 nm)-conjugated secondary antibody for 1 h in 6% bovine serum albumin (BSA). Cells were later stained with 1 μg/mL of DAPI for 5 min and washed with PBS and observed using a confocal microscope (630x magnification) (Leica TCS SP2, Heidelberg, Germany).