Quantitative Analysis of Tau Protein in AD

Preparation of brain homogenates, Western blot (WB) and Dot blot (DB) analysis were performed as previously described^{30 (link),62 (link)}. Briefly, 0.2 g of brain tissue from four different AD cases were homogenized in 0.4 ml TBS buffer with Halt™ Protease and Phosphatase Inhibitor Cocktail (100X, Thermo Scientific, CA), then centrifuged at 6400xg for 15 minutes at +4 °C. Supernatants (soluble fractions) were collected and stored at −80 °C for further analysis. For WB soluble fractions applied to electrophoresis on NuPAGE 4–12% Bis-Tris gel in MES buffer under reducing conditions (Invitrogen, CA) and electrotransferred onto nitrocellulose membrane (GE Healthcare, NJ). Tau were visualized by incubating with sera (dilution at 1:1000) from mice immunized with AV-1980R/A and injected with Advax^CpG only followed by HRP-conjugated anti-mouse IgG (Santa Cruz Biotechnology, CA). For DB assay the same extracts were applied to membrane (1 μg). Proteins were detected using sera from mice immunized with AV-1980R/A and control mice injected with Advax^CpG only, TNT-1 (Millipore, MA), HT7 (Life Technology, CA) antibodies. All primary antibodies were used at concentration of 1 μg/ml, serum was used at dilution 1:2500. Bovine anti-mouse HRP-conjugated secondary antibody was used (Santa Cruz Biotechnology, CA).