Transmission Electron Microscopic Imaging of Synaptic Vesicle Endocytosis in Mouse Hippocampal Neurons

Begin by treating mouse hippocampal neurons with a high-potassium buffer containing horseradish peroxidase or HRP.

High potassium depolarizes presynaptic neurons, triggering synaptic vesicle exocytosis. The neurons then retrieve the synaptic vesicle membranes through endocytosis, by incorporating HRP into the newly formed vesicles. Fix the neurons with glutaraldehyde and wash to remove excess glutaraldehyde.

Add hydrogen peroxide and a chromogenic substrate, which is oxidized by HRP to form electron-dense precipitates. Wash to remove any unreacted substrate.

Treat with osmium tetroxide to enhance membrane contrast, then wash off the excess reagent. Next, add uranyl acetate to enhance organelle contrast.

Dehydrate the neurons using increasing ethanol concentrations and embed them in resin.

Microscopically identify cell-dense regions and excise them. Then, prepare ultra-thin sections.

Transfer a section onto a copper grid and counterstain them with contrast agents.

Using transmission electron microscopy, vesicles containing HRP appear as electron-dense structures.