Immunofluorescence Assay for NF-κB Activation

The immunofluorescence (IF) experiment was performed as described previously [4 (link), 19 (link)]. The MH-S cells were rinsed with DPBS and fixed for 10 min in 4% paraformaldehyde. The cells were also permeabilized for 10 min with 0.2% triton X-100 in TBS (TBST) and rinsed three times with TBST for 5 min each time. The samples were blocked for 1 h with 2% BSA before adding rabbit anti-p-NF-κB (primary antibody) overnight at 4°C. MH-S cells were rinsed three times with TBST for 5 min each time. The samples were incubated for 1 h in the dark with a secondary antibody and then washed three times with TBST before being mounted with a Prolong Gold Antifade reagent with 4,6-diamidino-2-phenylindole (DAPI) (Cell Signaling Technology) to observe the nuclei and examined using confocal microscopy (LSM700, Carl Zeiss, Jena, Germany).