Jul 11, 2024
Assessing Astrocyte Territory Volume and 3D Sholl Analysis
- 1Duke University
Protocol Citation: Shiyi Wang 2024. Assessing Astrocyte Territory Volume and 3D Sholl Analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzj734lx1/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: 103198
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s (ASAP) initiative
Grant ID: ASAP-020607
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Abstract
Assessing Astrocyte Territory Volume and 3D Sholl Analysis
**Section Collection**
- Collect 100 μm-thick floating sections containing the anterior cingulate cortex (ACC) and primary motor cortex (MOp) from mice.
- Ensure astrocytes are sparsely labeled via PALE with mCherry-CAAX.
**Imaging**
- Use an Olympus FV 3000 microscope with a 60x objective to acquire high-magnification images (50-60 μm z-stack) of individual astrocytes.
- Criteria for inclusion: Capture the entire astrocyte within a single brain section, located in layer 2/3 of ACC or MOp.
**Surface Reconstructions**
- Analyze imaged astrocytes using Imaris Bitplane software.
- Generate surface reconstructions of the astrocytes.
**Territory Volume Measurement**
- Use Imaris Xtensions “Visualize Surface Spots” and “Convex Hull” to create additional surface renders representing the territory of each astrocyte.
- Record the volume of each astrocyte territory.
**Statistical Testing**
- Analyze astrocyte territory sizes from biological replicates across experimental conditions.
- Use a nested two-way ANOVA followed by the Bonferroni posthoc test for statistical comparisons.
**Surface and Filament Creation**
- Load astrocyte images onto Imaris and create a surface representation for each astrocyte.
**Filament Generation**
- Create filaments using ‘Add new filament (leaf icon)’ in Imaris.
**Quantification of Complexity**
- Use the gear tool on Imaris to display Sholl intersections for each astrocyte.
- Quantify the complexity of astrocytes based on Sholl intersections.
#### Experimental Details
### Notes:
- Maintain consistency in imaging and analysis methodologies across experimental conditions.
- Consider biological variability and replicate experiments to ensure robust conclusions.