Microdissected Tissue Fractionation

For the PBS-soluble fraction, 40–50 mg of frozen microdissected tissue was thawed on wet ice and then immediately homogenized in 500 μl of PBS spiked with protease (Roche, Basel, Switzerland) and phosphatase inhibitors (Thermo Scientific, Waltham, MA) in a gentle-MACS Octo Dissociator (Miltenyi BioTec, Auburn, CA). The homogenate was transferred to a 1.5-ml low protein-binding tube (Eppendorf) and centrifuged at 10,000 g for 10 min at 4 °C, as previously described [21 (link)]. Then, the supernatant was collected and aliquoted in 0.5-ml low protein-binding tubes (Eppendorf) to avoid excessive freeze–thaw cycles. Sarkosyl-insoluble material was extracted using 1 g of frozen brain tissue from three brain regions (cerebellum, putamen and frontal cortex) of individuals with MSA, as previously described [9 (link)]. Bicinchoninic acid protein (BCA) assay (Thermo Scientific) was performed to determine the total protein concentrations of all aliquots.