Jul 11, 2024
Mouse synapse imaging and analysis
- 1Duke University
Protocol Citation: Shiyi Wang 2024. Mouse synapse imaging and analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn6mzpl5d/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 11, 2024
Last Modified: July 11, 2024
Protocol Integer ID: 103199
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s (ASAP) initiative
Grant ID: ASAP-020607
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Abstract
Mouse synapse imaging and analysis
**Tissue Sectioning and Preparation**
- Prepare coronal sections of 30 μm thickness containing the anterior cingulate cortex (ACC) and primary motor cortex (MOp) from WT and LRRK2 G2019Ski/ki mice.
**Synaptic Staining**
- Perform synaptic staining using the following antibody combinations:
- Excitatory (Intracortical): VGluT1 (pre-synaptic) and PSD95 (post-synaptic).
- Inhibitory: VGAT (pre-synaptic) and Gephyrin (post-synaptic).
**Antibody Incubation**
- Dilute primary antibodies (anti-VGluT1, anti-PSD95, anti-VGAT, anti-Gephyrin) appropriately in blocking solution.
- Incubate tissue sections overnight at 4°C with primary antibodies.
**Secondary Antibody Incubation**
- Wash sections with 1x TBS containing 0.2% Triton X-100 (TBST).
- Block with 10% normal goat serum (NGS) diluted in TBST.
- Incubate sections with Alexa Fluor-conjugated secondary antibodies (Life Technologies) for 2-3 hours at room temperature.
**Confocal Microscopy**
- Acquire high-magnification images using an Olympus FV 3000 inverted confocal microscope.
- Use a 60x objective with 1.64x optical zoom.
- Acquire z-stack images consisting of 15 optical sections spaced 0.34 μm apart.
**Image Processing**
- Convert each z-stack into 5 maximum projection images (MPI), each corresponding to a 1 μm section of the z-plane, using FIJI.
**Synaptic Puncta Analysis**
- Analyze synaptic puncta using FIJI plugin Puncta Analyzer153.
- Identify synapses by the colocalization of pre and postsynaptic puncta (VGluT1/PSD95 for excitatory synapses, VGAT/Gephyrin for inhibitory synapses).
**Data Collection**
- Analyze 15 MPIs per mouse (5 MPIs per tissue section, 3 tissue sections per mouse).
- Analyze between 4 and 5 age- and sex-matched mice per genotype and condition, as specified in the figure legends.
**Data Handling**
- Ensure all animals appear healthy at the time of collection.
- Include all data collected without exclusions, as per experimental design.
Notes:
- Maintain consistency in tissue processing and staining protocols.
- Perform all image analysis using standardized settings and procedures to ensure data reproducibility.