Fitc labeled anti rabbit secondary antibody

The nuclear localization of p65, c-Jun, and IRF3 was detected with Nikon A1R Eclipse Ti confocal microscope (Nikon Corp., Tokyo, Japan) as previously described[ 24 ]. Briefly, cells were seeded into chamber slides for 12 h and then treated with flavonoids of S. involucrata (100 and 200 μg/mL) for 1 h. After LPS (1 μg/mL) stimulation for 1 h, cells were fixed by 4% paraformaldehyde for 10 min and then permeabilized with Triton X-100 (0.25%) for 30 min at 37 °C. Cells were then blocked for 1 h with 2% bovine serum albumin and incubated with target antibodies including p65, c-Jun, and IRF3 diluted in cold PBS containing 2% bovine serum albumin at 4 °C overnight. Then, the cells were incubated with FITC-labeled anti-rabbit secondary antibody (Cell Signaling Technology, catalog no. 4412; 1:500 dilution) in dark under room temperature for 1 h. After incubation, cells were washed 3 times by cold PBS. 4',6-diamidino-2-phenylindole (DAPI, YESEN, Shanghai, China) was used to stain nucleus right before performing assay.