The synaptosome preparation protocol was adapted from (Gylys et al., 2000). Human tissue samples were obtained at autopsy and minced, slowly frozen in 0.32 M sucrose with 10% DMSO and stored at −80 °C. To obtain a crude synaptosome fraction, tissue was thawed in a 37 °C water bath and homogenized in 10 mm Tris buffer (pH 7.4) with proteinase inhibitors (Roche) and phosphatase inhibitors (Sigma-Aldrich) using a glass/Teflon homogenizer (clearance 0.1–0.15 mm). The homogenate was centrifuged at 1000 g at 4 °C for 10 min, the supernatant was removed and centrifuged again at 10,000 g at 4 °C for 20 min. Resulting pellets were suspended in sucrose/Tris solution and stored at −80 °C. Synaptosomes were conjugated to pHrodo-Red per the manufacturer’s protocol.
