Jul 11, 2024
Tile Scan Imaging and Cell Counting
- 1Duke University
Protocol Citation: Shiyi Wang 2024. Tile Scan Imaging and Cell Counting. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6231kgqe/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: 103200
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s (ASAP) initiative
Grant ID: ASAP-020607
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Abstract
Tile Scan Imaging and Cell Counting
**Sample Preparation**
- Prepare 30 μm thick coronal sections containing the anterior cingulate cortex (ACC) and primary motor cortex (MOp) from P21 WT and LRRK2 G2019Ski/ki; Aldh1l1-eGFP mice.
**Confocal Microscopy**
- Acquire tile scan images using a confocal Leica SP8 STED microscope equipped with a galvo scanner and a 20x objective.
- Ensure consistent settings for image acquisition across all samples.
**Double Positive Cell Identification**
- Identify cells labeled with ALDH1L1-EGFP (astrocyte marker) and SOX9 (astrocyte marker) in the ACC and MOp regions.
- Use FIJI software with the cell counter tool for manual cell counting.
**Data Collection**
- Count ALDH1L1-EGFP and SOX9 double positive cells manually in 2-4 sections per brain.
- Analyze sections from 3 sex- and age-matched mice per genotype.