Proteases inhibitors cocktail

Tau aggregates were extracted according to our protocol previously used to isolate tau aggregates in mouse models of tauopathies67 (link). This procedure uses 1% sarkosyl and is derived from one used to isolate tau aggregates from the brains of AD68 (link).
Briefly, the RIPA supernatant was adjusted to 1% sarkosyl (N-lauroylsarcosine), incubated for 30 min at room temperature with constant shaking, and centrifuged at 100,000 × g for 1 hour at 20 °C. The pellet containing sarkosyl-insoluble aggregated (SP fraction) was resuspended and diluted in Sample buffer (NuPAGE LDS) 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.
For heat stable soluble tau, the RIPA supernatant was boiled for 5 min and centrifuged at 20,000 × g for 20 min. 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) and boiled for 5 min.

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.

For immunohistochemistry, mice (n = 4−6 per group) were anesthetised with isofluorane and transcardially perfused with ice-cold saline followed by ice-cold 4% paraformaldehyde (Sigma-Aldrich, St Louis, MO, USA) in 0.1 M phosphate-buffered saline. Brains were post-fixed in 10% neutral-buffered formalin (Thermo Fisher Scientific, Mississauga, ON, Canada) for 16 h and transferred to 70% ethanol. Brains were paraffin embedded and sectioned at 4 μm.
For western blot and ELISA (n = 4−8 per group), anesthetised mice were cervically dislocated, the hippocampus and cortex dissected out and immediately frozen on dry ice. Proteins were extracted in 5× volume/weight with 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⁻¹ of Proteases Inhibitors Cocktail (Sigma-Aldrich)) on ice with a tissue homogenizer (OMNI International, Kennesaw, GA, USA). Samples were centrifuged (20,000 × g, 4 °C) for 20 min, the supernatant recovered, and protein quantified using the BCA method. The RIPA-insoluble pellet was further extracted in 70% formic acid in dH₂O, evaporated, and resolubilized in 200 mM Tris-HCl, pH 7.5.