Immunofluorescence staining

The nuclear translocation of NF-κB p65 was detected by an immunofluorescence assay. For the present study, RAW 246.7 cells were pre-treated with magnesium (5 and 10 mM) for 2 h and then simultaneously stimulated with LPS and IFN-γ for 30 min. RAW cells were fixed with 4% paraformaldehyde diluted in PBS for 20 min, permeabilized with 0.5% Triton X-100 in PBS for 15 min, and blocked with 5% BSA for 1 h. The cells were probed with an anti-p65 NF-κB antibody (1:100, Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C and then incubated with FITC-conjugated goat anti-rabbit IgG (1:1000, Abcam, Cambridge, MA, USA) for 1 h at room temperature. The position of the cell nucleus was stained with 4,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) solution (1 mg/mL) for 15 min. After washing with PBS, fluorescence was visualized using a fluorescence microscope (Leica Microsystems, Buffalo Grove, IL, USA). According to previous studies^46,47, the intensity of p65 staining in the nucleus and cytoplasm was measured to determine the nuclear:cytoplasmic ratio as a relatively quantitative measurement of NF-κB nuclear localization.