Rabbit nf κb p65 antibody

The BV2 microglial cells were cultured directly on sterile glass coverslips in 24-well plates for 24 h, pretreated with ICSB for 1 h with or without LPS, fixed in 4% paraformaldehyde for 20 min at room temperature, permeabilized with 0.1% Triton X-100 in PBS, and blocked with 3% BSA. Afterwards, the cells were sequentially incubated with rabbit NF-κB(p65) antibody (Santa Cruz Biotechnology Inc., Eugene, OR, USA) at room temperature and Alexa 546-labeled goat anti-rabbit IgG (Molecular Probes, Eugene, OR, USA) at room temperature for 1 h. A Hoechst staining solution (Hoechst 33258) was used to visualize the cell nucleus. After washing with PBS, the samples were mounted with Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA), using glass coverslips, and visualized under a fluorescence microscopy (Leica Micosystems, DM2500, Wetzlar, Germany).

Translocation of NF-κB into the nucleus was detected using immunofluorescence staining as described previously^{64 (link)}. Briefly, BMDMs were treated with Rg6 (20 μM, 1 h prior to LPS addition) and LPS (100 ng/mL) for 2 h, followed by fixation in 4% paraformaldehyde in PBS for 10 min. Cells were then permeabilized with 0.25% Triton X-100 in PBS for 10 min and blocked with 10% BSA in PBS for 1 h. Rabbit NF-κB p65 antibody (Santa Cruz Biotechnology, sc-372) was diluted 1:400 and added to the cells at 4 °C O/N. After washing off the non-bound antibodies, cells were stained with anti-rabbit IgG Alexa Fluor 488 (Invitrogen, A-11008) for 1 h at RT. BMDMs on cover glass were mounted with DAPI to counterstain the nucleus and confocal images were taken using a Leica TCS SP8 confocal system.