Preparing Cultured Neurons for Optical Analysis of Recycled Synaptic Vesicles

Begin with a coverslip containing cultured neurons expressing synaptic vesicle membrane proteins, including fluorescent-tagged synaptophysin and synaptotagmin-1.

Remove the medium, add a mix of depolarization buffer and primary antibodies, then incubate.

The buffer ions enter the cells, stimulating the fusion of synaptic vesicles with the membrane, which exposes synaptotagmin-1.

The primary antibodies selectively bind to the exposed synaptotagmin-1.

Discard the buffer and wash with the medium to remove unbound antibodies.

Introduce fresh medium and incubate to facilitate membrane internalization, forming recycled vesicles with labeled synaptophysin and antibody-bound synaptotagmin-1.

Remove the medium, fix the cells, and wash.

Introduce a detergent-based blocking buffer with fluorophore-coupled secondary antibodies.

The detergent permeabilizes the cells, allowing the secondary antibodies to interact with antibody-bound synaptotagmin-1.

Lift the coverslip, wash it with water, and dry it.

Place the coverslip face-down on a slide with an embedding medium for optical analysis of the dual-labeled recycled vesicles.