Figure 1

Isolation of human neuronal synapses for optical imaging. (A) Unfixed human brain tissue is homogenized, and synaptic terminals are collected by subcellular fractionation. Synapse-enriched fractions, called synaptoneurosomes, are sparsely fixed over glass slides for optical imaging. (B) Compared to total and cytosolic extracts, synaptoneurosomes (syn) analyzed by Western blotting show enrichment in presynaptic (synaptophysin) and postsynaptic (PSD95) markers, and reduction in nuclear (histone H3) and astrocyte (GFAP) markers. Actin is a loading control. (C) Transmission electron micrograph of synaptoneurosomes, with the arrow marking an intact synaptic cleft. (D) Light microscopy of synaptoneurosomes fixed over a glass slide. Synapses and organelles appear as dark objects in brightfield images. Immunostaining against VGluT1 and MAP2 marks presynaptic and postsynaptic terminals, respectively.