Rat il 1β il 1f2 duoset elisa

For hippocampus and cortex homogenate, rats were anaesthetized and transcardially perfused only with saline. Brains were carefully removed and dissected to separate hippocampus and cortex, which were then split with RIPA Lysis Buffer. Protein concentration of homogenate was measured by BCA Protein Assay Kit (Beyotime) to prepare samples at the same concentration for Western blot (10% SDS-PAGE, 40 μg total proteins). Targeted strips were incubated with primary antibodies (anti-NF-κB p65 antibody, ab16502, Abcam; anti-NF-κB pp65 antibody, ab86299, Abcam; β-actin mouse monoclonal antibody, Beyotime) and then secondary antibodies (biotin-labeled goat anti-rabbit IgG, Beyotime; biotin-labeled goat anti-mouse IgG, Beyotime), followed by visualization with Immobilon Western HRP Substrate (Merck, Darmstadt, Germany). IL-1β and TNF-α in the homogenate were analyzed by ELISA (Rat IL-1β/IL-1F2 DuoSet ELISA, DY501, R&D, Minneapolis, MN, USA; rat TNF-α DuoSet ELISA, DY510, R&D) according to kit instructions.