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All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Acute Hippocampal Slice Preparation
- Remove the mouse brain as quickly as possible, using a spatula and sharp scissors to cut the skull, and place it in ice-cold, oxygenated (95% O2, 5% CO2), high-sucrose artificial cerebrospinal fluid (ACSF, ~100 mL).
- Place the mouse brain on a Petri dish and remove the cerebellum and a small section of the frontal cortex with a scalpel before hemisecting down the midline of the brain.
- Place the two hemispheres on the side that was just cut and glue each hemisphere onto the stage of a vibratome.
- Cut 200 - 300 µm-thick sections of the complete region of interest. In the case of the hippocampus, approximately 3 - 4 slices per hemisphere should be obtained.
- Using a plastic Pasteur pipette, transfer the slices to a chamber submerged in oxygenated (95% O2, 5% CO2) ACSF. Maintain at 34 °C for 30 min.
NOTE: At this stage, slices can be treated with active pharmacological agents, such as recombinant Dickkopf-1 (Dkk1) proteins, by adding the agents to the slice chamber. - Using a paintbrush, remove the slices from the chamber, place them into a 24-well plate, and fix them for 20 min to 1 h in 4% paraformaldehyde (PFA)/4% sucrose at room temperature (RT).
Caution: PFA is toxic, wear appropriate protection. - Wash the slices three times in 1X phosphate-buffered saline (PBS, 10 min each).
NOTE: The protocol can be paused here for 1 - 2 days, as long as the slices are kept at 4 °C in 1X PBS.
2. Immunofluorescence for Synaptic Markers
NOTE: The recipes of all buffers used can be found in Table 1.
- In the slice wells, replace the PBS with blocking/permeabilizing buffer (Table 1) and incubate at RT for 4 - 6 h.
- Dilute the polyclonal guinea pig antibody against vesicular glutamate transporter 1 (vGlut1) at a 1:2,000 dilution in blocking/permeabilization buffer.
NOTE: vGlut1 is a marker for pre-synaptic excitatory terminal visualization. For post-synaptic excitatory synapse identification, use a polyclonal antibody against postsynaptic density-95 (PSD-95) and dilute 1:500 in the same solution. Use a minimum volume of 300 µL in each well. To identify the anatomical location of the hippocampus, use an antibody that can also identify the neuronal structure, such as microtubule associated protein 2 (MAP2) or Tubulin. Work out the correct concentrations of all primary antibodies for the synaptic markers of choice (pre- and post-synaptic) and dilute in fresh blocking/permeabilizing buffer. - Incubate the slices in the primary antibody solution overnight (or for 1 - 2 days) at 4 °C. Use a shaking platform with vigorous movement.
- Wash the slices three times in PBS (10 min each).
- Dilute the appropriate secondary antibodies 1:500 in the blocking buffer. For example, when using the vGlut1 guinea pig antibody, apply a secondary antibody that recognizes the guinea pig component (e.g., donkey anti-guinea pig) conjugated to a specific fluorophore. When using the PSD-95 rabbit antibody, apply a secondary antibody that recognizes the rabbit component (e.g., donkey anti-rabbit) conjugated to a different specific fluorophore.
- Incubate the slices in this solution for 2 - 3 h at RT. Ensure that the slices are protected from the light, as secondary antibodies are light-sensitive.
- Wash the slices three times in PBS (10 min each).
- Carefully remove the slices from the 24-well plate using a paintbrush 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. Avoid the formation of air bubbles. Take care to use enough mounting medium (300-400 µL), as an insufficient amount may lead to dry slices.
- Leave to dry for a minimum of 1 - 2 days at RT and keep the slides protected from light.
- Store the slides at 4 °C in the short-term, but for long-term storage, keep them at -20 °C.
3. Confocal Image Acquisition and Analysis
- Imaging using confocal laser scanning microscopy.
- Identify the region of the hippocampus to be imaged (e.g., the cornu ammonis 1 and 3 (CA1, CA3) or the dentate gyrus (DG)) by means of a 10x or 20x objective.
- Change to a 40x or 63x oil-immersion objective to make sure the slice anatomy is intact by identifying continuous neurites and organized structure. Use a neuronal marker, such as MAP2, as a reference (Figure 1A).
- Subsequently, switch to a 60x oil-immersion objective (numerical aperture [NA] = ~1.3 - 1.4) and adjust the settings for each channel to obtain optimal signal and contrast. Use a high-/low-contrast option to set the intensity of each laser to avoid the saturation of any pixels.
NOTE: These settings will depend on the microscope used, but refer to the Table of Materials for the laser power settings. For best results, use a 1024 x 1024-pixel resolution. The settings should remain constant throughout the different conditions of the same experiment. Additional zoom can be applied if required. - Evaluate the depth where the staining is even and acquire image stacks of at least 8 equidistant (250 nm) planes. Then take 3 adjacent representative image stacks from the same area of interest per slice.
NOTE: The depth may vary from one slice to another but should always be approximately 2 - 5 µm from the surface of the slice. - Repeat the acquisition in at least 3 slices per condition and from 6 - 8 animals per treatment group.
Table 1: Buffers used for immunofluorescent staining.
| Blocking/permeabilizing Buffer | Concentration | Notes |
| Donkey serum | 10% | |
| Triton-X | 0.50% | |
| PBS | 1x | |
| 10x PBS | | pH to 7.4 |
| NaCl | 1.37 M | |
| KCl | 27 mM | |
| NaH2PO4 | 100 mM | |
| KH2PO4 | 18 mM | |
| 4% PFA/4% Sucrose | | pH to 7.4 |
| PBS | 1x | Dilute from 10x |
| PFA | 1.33 M | |
| Sucrose | 117 mM | |