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.

To begin immunofluorescence staining, replace the PBS in the slice wells with blocking and permeabilizing buffer, and incubate at room temperature for 4 to 6 hours on the shaker. Towards the end of the blocking step, dilute the antibody to VGLUT1 1 to 2000 in blocking and permeabilizing buffer. Please note that this dilution may differ depending on the company and lot of the antibody used. Incubate the slices in the primary antibody solution overnight at 4 degrees Celsius. Use a shaking platform with vigorous movement.

Even distribution of the primary and secondary antibodies is important for optimal staining. In our experience, vigorous shaking enables the best antibody penetration.

After the incubation in primary antibody, wash the slices three times in PBS for 10 minutes each time as before. During the last wash, dilute the appropriate secondary antibodies 1 to 500 in blocking buffer. Then incubate the slices in this solution for 2 to 3 hours at room temperature. Ensure that the slices are protected from the light, as secondary antibodies are light-sensitive.

After washing the slices as before, use a brush to carefully remove the slices from the 24-well plate and place them evenly onto pre-labeled glass slides. Add a drop of mounting medium on top of each slice. And then gently place a glass coverslip on top of the slices, being careful to avoid the formation of air bubbles.

Protect from light and allow the slides to dry for a minimum of three to four days at room temperature. Store the slides at 4 degrees Celsius in the short term. But for long-term storage, keep them at minus 20 degrees Celsius. Start by using a 10x or 20x objective to identify the region of the hippocampus to be imaged-- in this case, the synapses between the CA1 pyramidal neurons and the Schaffer collateral axons.

Change to a 40x or 63x oil immersion objective and make sure the slice anatomy is intact by identifying continuous neurites and an organized structure. Use a neuronal marker, such as MAP2, as a reference. Adjust the settings for each channel to obtain optimal signal and contrast with a 1024 by 1024 pixel resolution. Set the intensity of each laser to avoid the saturation of any pixels.

Evaluate the depth where the staining is even, and then set the software to acquire image stacks of at least eight equidistant 250-nanometer planes. Then take three adjacent representative images stacks from the same area of interest per slice. Repeat the acquisition in at least three slices per condition and from six to eight animals per treatment group.