Rabbit anti mouse p65 antibody

DNCB, mite extract, phorbol 12-myristate 13-acetate (PMA), A23187, and carboxyfluorescein succinimidyl ester (CFSE) were purchased from Sigma (St. Louis, MO). Mouse IgG2a enzyme-linked immunosorbent assay (ELISA) kits, anti-mouse CD4 antibody conjugated with fluorescein isothiocyanate (FITC), anti-mouse interferon (IFN)γ antibody conjugated with PerCP cy5.5, and anti-mouse IL-4 antibody conjugated with phycoerythrin were obtained from eBioscience (San Diego, CA). Rabbit anti-mouse keratin5 antibody was purchased from Abcam (Cambridge, MA) and rabbit anti-mouse p65 antibody was from Cell signaling technology (Beverly, MA). Mouse IgE ELISA kits, purified anti-mouse IFNγ, anti-mouse IL-4, and anti-mouse IL-12 antibodies were purchased from BD Bioscience (San Jose, CA). Anti-mouse CD28 antibody, mouse IFNγ ELISA kits, mouse IL-4 ELISA kits, and recombinant human IFNγ and TNFα were purchased from R&D (Minneapolis, MN). Recombinant mouse IL-4 and IL-12 were obtained from Peprotech (Hamburg, Germany). 145-2C11 (mouse anti-CD3; CRL-1975) hybridoma cell line and HaCaT (human keratinocyte) were obtained from the ATCC (Manassas, VA).

RAW 264.7 cells were grown on coverslips overnight. PBS-washed cells were fixed with 4% formaldehyde for 20 min and then permeabilized with 0.1% Triton X-100 for 15 min. After blocking with 5% goat serum for 1 h, cells were incubated with 1:1000 dilution of rabbit anti-mouse p65 antibody (Cell Signaling Technology, D14E12) for 30 min. After washing, cells were incubated with a 1:1000 dilution of DyLight 594 conjugated goat anti-rabbit IgG (Thermo Scientific) for 30 min and counterstained with DAPI (Sigma) to stain cell nuclei. Microscopy (Olympus IX71) was used for observing cells and cellSens imaging software was used to capture the images.