Anti nf κb p65 mouse monoclonal antibody

Human NP cells were plated on a glass-bottom confocal dish and exposed to MCM for 48 h. The cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100 in PBS for 15 min at room temperature, blocked with 5% bovine serum albumin (BSA; Millipore) in PBS, and then incubated with the primary antibodies overnight at 4 °C in 5% BSA. Anti-NF-κB p65 mouse monoclonal antibody (Santa Cruz) was used to detect the NF-κB p65 protein. Goat anti-mouse Alexa 555 secondary antibodies (Invitrogen) and 5% BSA were used for the secondary incubation in PBS for 1 h at room temperature. After washing in PBS, the plate was counterstained with 4′,6-diamidino-2-phenylindol (DAPI, Invitrogen). Images were acquired using the EVOS FL Auto cell imaging system (Thermo Fisher Scientific Inc., USA).

Microglial cells on the microfluidic chip were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100 in phosphate-buffered saline (PBS; Gibco-BRL, Gaithersburg, MD, USA) for 15 min at room temperature (RT). Blocking was performed using 3% bovine serum albumin (BSA; Millipore, MN, USA) in PBS (1% w/v) for approximately 2 h at RT. Subsequently, the cells were incubated with CD11b (Millipore, MN, USA) and CD206 (Novus biologicals, CO, USA) primary antibodies for 2 h at 37 °C at a dilution ratio of 1:100 and washed with PBS containing 1% BSA. Thereafter, the cells were incubated with Alexa 555 secondary antibody (1:200; Invitorgen, Waltham, MA, USA) for 2 h at 37 °C.
Human NP cells were treated with recombinant IL-1β for 48 h and fixed and blocked with proprietary reagents. Anti-NF-κB p65 mouse monoclonal antibody (Santa Cruz, Dallas, TX, USA) was used to detect NF-κB p65 protein. Goat anti-mouse Alexa 555 secondary antibodies (Invitrogen, Waltham, MA, USA) and 5% BSA were used for secondary incubation in PBS for 1 h at room temperature. After staining with 4′,6-diamidino-2-phenylindole (DAPI) for 10 min, fluorescence images were acquired using a confocal microscope (Zeiss, LSM 900).

DCs were seeded in an eight-well microtiter plate, cultured with or without Pmp18.1 for 24 h, harvested by digestion with accutase detachment solution (Thermo Fisher Scientific, Rockford, IL) and centrifuged at 1,500 g for 10 min. After washing thrice with FACS Buffer, cells were fixed and permeabilized using BD Cytofix/Cytoperm™ Plus Fixation/Permeabilization Kit with BD GolgiStop™ according to the manufacture's protocol. The permeabilized cells were stained for 30 min on ice with mouse anti-NF-κB p65 monoclonal antibody (Santa Cruz, Dallas, Texas) washed with FACS Buffer, incubated with FITC-conjugated (green) goat anti-mouse IgG secondary antibody (BD Pharmingen, San Diego, CA) for 1 h on ice in the dark and counterstained with the nuclear stain, DAPI (Blue). The cells were washed with FACS Buffer, centrifuged to a slide, examined and photographed by immunofluorescence microscopy as previously described (Ekong et al., 2009 (link)).