Rabbit anti mouse nrf2 polyclonal antibody

RAW 264.7 macrophages were cultured in the presence of EPDENs (10 µg/ml) or PBS for 24 hr. Ten µg/ml of EPDENs was chosen and is based on the concentration of EPDENs in the extracted juice, (in addition we assume after passing through the stomach, the concentration of EPDENs in the juice would have been diluted 50-fold in the small intestine). The supernatants from 24 hr cultured cells were harvested for ELISA determination of IL-10 and IL-6. The cells were either lysed for western blot analysis for HO-1 or fixed in cold 4% paraformaldehyde for immunohistological staining of Nrf2. Paraformaldehyde fixed cells were first permeabilized with 1% Triton-X 100 in PBS for 10 min, followed by blocking with 5% BSA in PBS containing 0.1% Triton-X 100 for 1 h, and then stained with a rabbit anti-mouse Nrf2 polyclonal antibody (Santa Cruz Biotechnology) at 22°C for 2hr. After washing, cells were stained with a goat anti-rabbit to fluorescein isothiocyanate–Alexa Fluor 488 (Invitrogen Life Sciences). Slides were mounted with Slow Fade Gold Antifade plus DAPI (4,6-diamidino-2-phenylindole; S36938; Molecular Probes and Invitrogen Life Sciences). Staining of cells was assessed using a Nikon A1R Confocal system.