centrifugation (18,000 × g for 20 min at 4°C), 20% w/v N-Lauroylsarcosine (sarkosyl) stock solution was added to the RIPA-soluble supernatant fraction to adjust the final sarkosyl concentration into 1%. After incubating at room temperature with gentle rocking for 1 hour, RIPA/sarkosyl homogenates were then centrifuged for 1 hour at 150,000 × g. The pellets were then resuspended in 10 μl of PBS (RIPA-soluble/sarkosyl-insoluble fraction). The RIPA-insoluble pellets were further homogenized in H buffer containing 10mM Tris (pH 7.4), 1mM EGTA, 0.8 M NaCl, 10% w/v sucrose, a protease inhibitor mixture (Roche Molecular Biochemicals), a phosphatase inhibitor cocktail (Thermo Scientific), 1 mM NaF, 1 mM NaVO3, 2 mM PNT (EMD Millipore) and 1 mM PMSF (Sigma-Aldrich) and then centrifuged at 18,000 × g for 20 min. 20% w/v N-Lauroylsarcosine (sarkosyl) stock solution was added to adjust the final sarkosyl solution concentration into 1%. After incubating at room temperature with gentle rocking for 1 hour, H buffer/sarkosyl homogenates were then centrifuged for 1 hour at 150,000 × g and the pellets were resuspended in 10 μl of PBS (RIPA-insoluble/sarkosyl-insoluble fraction). Both RIPA-soluble/sarkosyl-insoluble and RIPA-insoluble/sarkosyl-insoluble fractions were used for immunoelectron microscopy. The same protocol was used to enrich sarkosyl-insoluble p-tau aggregates in
