Nuclear translocation of NF-κB

The NF-κB-p65 in THP-1 cells was detected by indirect immunofluorescence assay using confocal microscopy. THP-1 cells were cultured on glass dishes. After the respective treatments, the cells were fixed using 4% paraformaldehyde, permeabilized with 0.2% Triton X-100 (w/v), and blocked with 3% bovine serum albumin (Sigma-Aldrich, Germany). Subsequently monoclonal antibodies against NF-κB-p65 (1:100) were applied for 12 h followed by 1 h incubation with anti-rabbit IgG conjugated to Texas Red antibody (1:400). Excitation and emission maxima used were 578 nm 602 nm respectively. The nuclei were visualized by staining with DAPI (excitation wavelength 359 nm and emission wavelength 461 nm).