Streptozotocin-Induced Tau Hyperphosphorylation

Four weeks after STZ injections, the mice were killed by decapitation without anaesthesia, as anaesthesia can lead to hypothermia-induced tau hyperphosphorylation65 (link). Brains were immediately removed and the tissues dissected on ice, frozen on dry ice, and kept at −80 °C until they were processed as described66 (link). Briefly, dissected brain structures (hippocampus and cortex) were homogenized, without thawing, in 5 times volume/weight of radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl, pH 7.4, 1% NP-40, 150 mM NaCl, 0.25% Na-deoxycholate, 1 mM EDTA, 1 mM Na₃VO₄, 1 mM NaF, 1 mM PMSF, 10 μl/ml of Proteases Inhibitors Cocktail (P8340, Sigma-Aldrich, St. Louis, MO)), using a mechanical homogenizer (TH, Omni International, Marietta, GA). Samples were then centrifuged for 20 min at 20,000 g at 4 °C. The supernatant was recovered, diluted in sample buffer (NuPAGE LDS; Invitrogen, Carlsbad, CA) containing 5% of 2-β-mercapto-ethanol, 1 mM Na₃VO₄, 1 mM NaF, 1 mM PMSF, 10 μl/ml of Proteases Inhibitors Cocktail (P8340; Sigma-Aldrich), boiled for 5 min. and kept at −20 °C.