Polyclonal rabbit anti rage

The antibodies used were as follows: polyclonal rabbit anti–claudin-5 (1:500), polyclonal rabbit anti-occludin (1:1000), polyclonal rabbit anti–ZO-1, polyclonal rabbit anti-tricellulin (1:500; all from Invitrogen), polyclonal rabbit anti-LRP1 (C-terminal, 1:500; Sigma-Aldrich), polyclonal rabbit anti-RAGE (1:500), polyclonal rabbit anti-ApoE (1:500), and polyclonal rabbit anti–β-actin (1:2000; all from Abcam). AW7 antiserum recognizes multiple Aβ epitopes and aggregation states (31 (link)) and was used to detect Aβ. Briefly, membranes were incubated with primary antibody overnight at 4°C, washed with tris-buffered saline (TBS), and incubated with horseradish peroxidase (HRP)–conjugated goat anti-rabbit immunoglobulin G (IgG) secondary antibody (1:2000; Abcam) for 2 hours at room temperature. To detect HRP, immunoblots were incubated with enhanced chemiluminescence solution.

Total lysates from the liver samples were prepared using RIPA buffer (50 mM Tris-HCI, pH 7.5, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid, 1% Nonidet P-40, 0.5% sodium deoxycholic acid, 0.1% sodium dodecyl sulfate) and protease inhibitors (Pierce Biotechnology, Rockford, IL, USA). The liver tissue lysates (40 µg protein/each lane) were subjected to 12 or 15% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Merck Millipore, Lake Placid, NY, USA). The membranes were incubated with the following target primary antibodies; rabbit monoclonal anti-S100B (1:1,000; Abcam); rabbit polyclonal anti-RAGE (1:1,000; Abcam); rabbit polyclonal anti- α-SMA (1:1,000; Abcam); rabbit polyclonal anti-collagen1 (1:1,000; Abcam); and mouse monoclonal β-actin (1:5,000; Sigma). Chemiluminescent signals were detected with the ImageQuant LAS 4000 apparatus (GE Healthcare Life Sciences, Piscataway, NJ, USA).