Brain Tissue Homogenization and Aβ/Tau Extraction

Before extraction of Aβ and tau from brain tissue, 10% (w/v) homogenates were prepared in tissue homogenization buffer (20 mM Tris base, pH 7.4, 250 mM sucrose, 1 mM EDTA, 1 mM EGTA) with 100 mM phenylmethylsulphonyl fluoride, protease inhibitors [protease inhibitors cocktail Complete (Roche Diagnostic GmbH, Mannheim, Germany) plus pepstatin A (Sigma-Aldrich Inc., St. Louis, MO)] and phosphatase inhibitors (1 mM NaF, 1 mM Na₃VO₄ and 0.5 mM okadaic acid) added immediately before homogenization, as we have previously published [18 ,19 (link)]. Subsequently, brain homogenates were aliquoted, frozen, and stored at −80°C until used for extraction of soluble and insoluble fractions of both Aβ and tau, and their biochemical analyses (ELISA, Western blot). All quantitative biochemical analyses were performed in brain homogenate fractions of the entire left hemisphere.