Alphaeasefc software version 4

Fresh-frozen brain tissue was homogenized with a glass-Teflon homogenizer at 500 rpm in 10 volumes (wt/vol) of ice-cold tissue homogenization buffer. The buffer contained 20 mM Tris, pH 7.4, 250 mM sucrose, 1 mM EDTA, 1mM EGTA and Halt protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). For dephosphorylated samples, a buffer containing 0.5% Triton-X, 50mM Tris, pH 8.0, 1mM ZnSO₄, 1mM MgCl₂, Halt protease inhibitor cocktail (Thermo Fisher Scientific) and Bacterial Alkaline Phosphatase (Takara Bio) was used instead. For each sample, 10 or 30 μg of proteins were separated in 10% PROTEAN TGX Precast Gels (Bio-Rad), blotted to nitrocellulose membranes, and stained with HT7 (1:3000, Thermo Fisher Scientific) for total tau. A tau ladder (RPeptide) was used as a control. HRP-labeled secondary anti-mouse antibody (Vector Labs) was detected by Pierce ECL substrate (Thermo Fisher Scientific). To quantify and standardize protein levels without reliance on specific housekeeping proteins, total protein was detected with Amido Black as previously described (Sigma-Aldrich) [35 (link)]. Chemiluminescence was measured in an LAS-4000 Intelligent Dark Box imager (Fuji Film), and relative optical densities were determined by using AlphaEaseFC software, version 4.0.1 (Alpha Innotech), normalized to total protein loaded.