The overall goal of this immunostaining protocol is to evaluate synaptic density in ex vivo brain slices. This method can help answer key questions in the neuroscience field and is useful when studying synapse density changes, as observed in neurodegenerative diseases. The main advantage of this technique is that it provides a semiquantitative estimation of synapse density, which can be compared between experimental condition prepared at the same time.
Begin by placing the mouse brain on a Petri dish. Then, remove the cerebellum and a small section of the frontal cortex with a scalpel, before hemisecting down on the midline of the brain. Turn one hemisphere onto the side that was just cut, and glue it onto the stage of a vibratome.
Pour ice-cold ACSF into the microtome chamber, and oxygenate the solution. Cut 200 to 300 micron thick sections of the complete region of interest. For the hippocampus, approximately three to four slices should be obtained per hemisphere.
Use a plastic Pasteur pipette to transfer the slices to a temperature-controlled chamber submerged in oxygenated ACSF. Maintain at 34 degrees Celsius for 30 minutes. After 30 minutes have elapsed, transfer the slices into a 24-well plate containing 4%paraformaldehyde with 4%sucrose, and fix for 20 minutes to one hour at room temperature.
Following fixation, wash the slices three times in 1x PBS for 10 minutes each time. To begin immunofluorescent staining, replace the PBS in the slice wells with blocking and permeabilizing buffer, and incubate at room temperature for four to six hours on the shaker. Towards the end of the blocking step, dilute the antibody to vGlut1 one to 2, 000 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 four 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 one to 500 in blocking buffer. Then, incubate the slices in this solution for two to three 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 prelabeled 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 four 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 1, 024 by 1, 024 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. Excitatory synapses can be identified by colocalization between vGlut1 and PSD95, as indicated by the white arrows in the higher magnification image.
Blockade of Wnt signaling in the adult hippocampus by inducing the Wnt antagonist Dkk1 triggers excitatory synapse loss, specifically in the CA1 stratum radiatum. The percentage of excitatory synapses between control and iDkk1 mice were quantified and found to be significantly lower in iDkk1 transgenic animals. Inhibitory synapses can be identified by the colocalization between vGat and Gephyrin.
The percentage of inhibitory synapses between control and iDkk1 mice were quantified, and no significant difference was detected. While attempting this procedure, it's important to remember to take as much care as possible with the slices. After watching this video, you should have a good understanding of how to evaluate synaptic density in ex vivo brain slices.
