Phosphatase and protease inhibitor mixtures

Half brain hemispheres, that were stored at − 80°C, were crushed on dry ice using mortar and pestle, then homogenized in T-PER solution (Thermo Scientific, Waltham, MA) and phosphatase and protease inhibitor mixtures (Thermo Scientific, MA and Roche, CA) and processed as previously described (Marsh et al., 2016 (link)). Quantitative biochemical analysis of human Aβ was conducted using commercially available electro-chemiluminescent multiplex assay system [Meso Scale Discovery (MSD)]. Human Aβ duplex (6E10 capture antibody) was used for simultaneous measurement of Aβ40 and Aβ42 in both soluble and insoluble protein fractions (Marsh et al., 2016 (link)).

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 described^{62 (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 described^{62 (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.