Biotinylated goat anti mouse igg and fluorescein conjugated streptavidin

The effect of magnolol on the nuclear translocation of p65 was evaluated by immunofluorescence staining. Briefly, BMMCs treated with RANKL in the absence or presence of 20 µg/ml magnolol were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.1% Triton X-100 in PBS for 5 min. The cells were washed with 0.1% BSA-PBS and incubated with an anti-p65 monoclonal antibody overnight at 4°C, followed by a biotinylated goat anti-mouse IgG and fluorescein-conjugated streptavidin (Vector Laboratories, Burlingame, CA, USA) for 1 h at room temperature and Hoechst (Vector Laboratories) was used for counterstaining for 2 days. The localization of p65 was visual-ized by immunofluorescence analysis (×400 magnification) and the percentage of nuclei fluorescence intensity of p65 in 3 random fields were measured in each group with ImageJ software.