Right hemispheres of mouse brain, previously frozen on dry ice and stored at −80 °C, were crushed on dry ice using mortar and pestle, then homogenized in solution of T-PER (Thermo Scientific, MA) and phosphatase and protease inhibitor mixtures (Thermo Scientific, MA and Roche, CA) and processed as previously described62 (link),78 (link),79 (link). Concentrations of human total and phosphorylated Tau in samples (soluble and insoluble brain extracts) were determined by Tau (total) Human ELISA kit, Tau [pS396] Human ELISA Kit, Tau [pS199] Human ELISA Kit, and Tau [pT231] Human ELISA Kit (all from ThermoFisher Scientific, MA), according to the manufacturer’s instructions.
Soluble SDS-PAGE WB was performed following standard protocols as previously described62 (link),78 (link),79 (link). Primary antibodies used for WB analysis included the following: Armanezumab (1:2000; Institute for Molecular Medicine, Huntington Beach, CA), anti-GFAP (1:400; Millipore-Sigma, MO), IBA-1 (1:200; FUJIFILM Wako Chemicals U.S.A. Corp, VA) and anti-CD45 (1:400; Bio-Rad, CA). All blot membranes were also labeled with anti-β-actin antibodies (1:1000; Millipore-Sigma, MO) as loading control.
Soluble SDS-PAGE WB was performed following standard protocols as previously described62 (link),78 (link),79 (link). Primary antibodies used for WB analysis included the following: Armanezumab (1:2000; Institute for Molecular Medicine, Huntington Beach, CA), anti-GFAP (1:400; Millipore-Sigma, MO), IBA-1 (1:200; FUJIFILM Wako Chemicals U.S.A. Corp, VA) and anti-CD45 (1:400; Bio-Rad, CA). All blot membranes were also labeled with anti-β-actin antibodies (1:1000; Millipore-Sigma, MO) as loading control.
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