JoVE Encyclopedia of Experiments
Neuroscience
0 views • 4:53 min • July 8th, 2025
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.
Prewarm 600 microliters of 1x depolarization buffer and 10 milliliters o
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