Rabbit anti p nf κb p65

Amlexanox was purchased from Tokyo Chemical Industry (Tokyo, Japan). STAT3 activation inhibitor SPI was purchased from BioVision (Milpitas, CA, USA). LPS from Escherichia coli O111:B4 was obtained from Sigma–Aldrich (St. Louis, MO, USA). The following primary antibodies, which were diluted at 1:1000 ratios in EveryBlot Blocking Buffer (Bio-Rad Laboratories), were used for Western blotting (WB): rabbit anti-COX2 (Cell Signaling Technology), mouse anti-iNOS (Cell Signaling Technology), rabbit anti-p-AKT (Ser473, Cell Signaling Technology), rabbit anti-AKT (Cell Signaling Technology), rabbit anti- IκBα (Cell Signaling Technology), rabbit anti-p- IκBα (Ser32, Cell Signaling Technology), rabbit anti-IKKε (Cell Signaling Technology), rabbit anti-ERK (Cell Signaling Technology), rabbit anti-p-ERK (Thr202/Tyr204, Cell Signaling Technology), rabbit anti-STAT3 (Cell Signaling Technology), rabbit anti-p-STAT3 (Tyr705, Cell Signaling Technology), rabbit anti-NF-κB p65 (Cell Signaling Technology), rabbit anti-p-NF-κB p65 (Ser536, Cell Signaling Technology), rabbit anti-JNK, rabbit anti-p-JNK (Thr183/Tyr185, Cell Signaling Technology), rabbit anti-p38 (Cell Signaling Technology), and rabbit anti-p-p38 (Thr180/Tyr182, Cell Signaling Technology), and mouse anti-β-actin (Santa Cruz Biotechnology). Other chemicals for Western blotting were obtained from Bio-Rad Laboratories.

Rats were anesthetized with 10% chloral hydrate (0.3 ml/100 g, i.p.) and perfused transcardially with 200 ml of 0.9% saline followed by 300 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The brain tissues were removed and postfixed in 4% paraformaldehyde overnight and cryoprotected in 30% sucrose. Thirty-micron-thick frozen sections from the rat brains were cut using a freezing microtome and serially collected throughout the hippocampus. Free-floating tissue sections were rinsed three times with PBS-T. The tissue sections were incubated with 10% normal donkey serum in PBS-T for 2 h, followed by incubation with rabbit anti-p-NF-κB p65 (1:500, Cell Signaling Technology, Inc., Beverly, MA) and mouse anti-glial fibrillary acidic protein (GFAP) monoclonal antibody (1:600, Abcam, Cambridge, UK) at 4 °C for 24 h. After three 5-min rinses in PBS, the sections were incubated with donkey anti-rabbit IgG conjugated to Alexa Fluor® 488 and donkey anti-mouse IgG conjugated to Alexa Fluor® 594 (1:500, Life Technologies, Carlsbad, CA, USA) in the dark for 2 h at 37 °C. Fluorescence intensity was visualized under a confocal microscope (FV1000, Olympus Corp., Tokyo, Japan). The intensity on four slides (three to four sections per slide) was averaged for each animal and then normalized by that of the control group.