Microscopic Analysis of Synapses in Mouse Hippocampal Slices Using Immunofluorescence

Place a hippocampal slice from a mouse brain into a well containing a buffer. The slice contains a network of neurons that communicate through synapses.

Presynaptic neuron terminals contain neurotransmitter-filled vesicles with characteristic transmembrane transporters, while postsynaptic terminals feature characteristic scaffold proteins that regulate neurotransmitter receptors. Both serve as key markers for synaptic structures.

Replace the buffer with a permeabilization-blocking solution and incubate with agitation to permeabilize cellular membranes and block non-specific binding sites.

Add primary antibodies targeting the presynaptic and postsynaptic markers and incubate with agitation to allow binding. 

Wash the slice, add fluorophore-conjugated secondary antibodies specific to the primary antibodies, and incubate with agitation in the dark.

Wash the slice, then mount it onto a slide.

Using a confocal microscope, identify the region of interest and magnify to visualize synapses.

Capture images to identify the fluorescently labeled presynaptic and postsynaptic markers and evaluate their colocalization at the synapses.