Immunofluorescence staining for nuclear translocation of NF-κB and formation of LPS/TLR4 complexes

After cells (5×10⁵ cells/ml) were pretreated with or without 200 µM isorhamnetin for 1 h, they were treated with 100 ng/ml LPS for 1 h. The cells were then washed twice with PBS, fixed in 3.7% paraformaldehyde for 15 min at 25°C, permeabilized with 0.2% Triton X-100 in PBS for 15 min, and blocked for 10 min at 20°C with PBS containing 5% bovine serum albumin (Sigma-Aldrich; Merck KGaA). The cells were then incubated with a primary antibody against NF-κB p65 (dilution, 1:100; cat. no. sc-71675; Santa Cruz Biotechnology, Inc.) at 4°C overnight, followed by incubation with a fluorescein-conjugated anti-mouse immunoglobulin G secondary antibody (dilution, 1:100; cat. no. 62-6511; Molecular Probes; Thermo Fisher Scientific, Inc.) in the dark at 37°C for 40 min. In addition, BV2 cells were pretreated with or without 200 µM isorhamnetin for 30 min, followed by treatment with Alexa Fluor^® (AF) 488-conjugated LPS (AF-LPS; 100 ng/ml; Invitrogen; Thermo Fisher Scientific, Inc.) for 6 h, in order to analyze the formation of LPS/TLR4 complexes. Fixed cells were also incubated with anti-TLR4 antibody (1:100; cat. no. ab8376; Abcam, Cambridge, UK) at 4°C for 90 min and were then incubated with AF 594-conjugated secondary antibody (1:100; cat. no. A-11032; Invitrogen; Thermo Fisher Scientific, Inc.) at room temperature for 1 h. Nuclei were sequentially stained with DAPI (Sigma-Aldrich; Merck KGaA) solution (2.5 µg/ml). Slides were mounted and fluorescence images were captured under a fluorescence microscope (Zeiss AG, Oberkochen, Germany).