Monoclonal rabbit anti occludin

Dulbecco’s modified Eagle’s medium (DMEM), 0.25% trypsin-EDTA solution and foetal bovine serum (FBS) were purchased from Gibco-BRL (Grand Island, NY, USA). Mouse IL-17A and isotype-control Abs were purchased from R&D Systems (Minneapolis, MN, USA). Monoclonal mouse anti-MMP-2, monoclonal mouse anti-MMP-9 and monoclonal rabbit anti-occludin were purchased from Abcam (Hong Kong, China). Monoclonal mouse anti-claudin-5 antibody was purchased from Invitrogen (Invitrogen, USA). Recombinant IL-17A protein and Fluoroshield mounting medium with 4,6-diamidino-2-phenylindole (DAPI) were purchased from Abcam (Hong Kong, China). The mouse IL-17A ELISA kit, IL-6 ELISA kit and IL-1β ELISA kit were obtained from R&D Systems (Minneapolis, MN, USA).

Hippocampal tissues were homogenized in RIPA lysis buffer and centrifuged for 20 min at 12000 g (4°C). Then, the supernatants were harvested for immunoblot analysis and the protein concentration was measured by the BCA kit. The protein samples were denatured with sodium dodecyl sulfate (SDS) sample buffer, separated by 10% SDS polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore). Subsequently, the membranes were blocked with 5% skim milk at room temperature for 1 h and were incubated with corresponding antibodies overnight at 4°C. The following primary antibodies were used: monoclonal rabbit anti-Iba1 (1 : 1000, Abcam, USA), monoclonal rabbit anti-GFAP (1 : 500, Abcam, USA), monoclonal rabbit anti-albumin (1 : 1000, Invitrogen, USA), monoclonal rabbit anti-occludin (1 : 1000, Abcam, USA), monoclonal mouse anti-claudin-5 (1 : 500, Invitrogen, USA), monoclonal rabbit anti-MMP-2 (1 : 1000, Invitrogen, USA), and monoclonal rabbit anti-MMP-9 (1 : 500, Abcam, USA). After incubation with an anti-rabbit or anti-mouse secondary antibody (1 : 5000) for 1 h, the protein on the membranes was finally visualized with an ECL kit (Thermo Fisher Scientific, Rockford, IL, USA).