Jul 12, 2024

Public workspaceCluster Counting

DOI
dx.doi.org/10.17504/protocols.io.5jyl8224rl2w/v1
  • 1Department of Clinical Neuroscience, Karolinska Institutet, 171 76 Stockholm, Sweden;
  • 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD 20815, USA
Eileen Ruth Torres
Open access
DOI: dx.doi.org/10.17504/protocols.io.5jyl8224rl2w/v1
Protocol Citation: daniel.dautan daniel, Per Svenningsson 2024. Cluster Counting. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8224rl2w/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: June 06, 2024
Last Modified: July 12, 2024
Protocol Integer ID: 101328
Keywords: ASAPCRN, alpha-synuclein, imaging, immunofluorescence
Funders Acknowledgement:
Aligning Science Across Parkinson's
Grant ID: 020608
Abstract
Used for counting clusters labeled for pSer129 alpha-synuclein in mouse upper gastrointestinal tract (intestine). Sections should be stained, mounted, and imaged with high resolution (2048 x 2048 scanning).
Stain structures for pSer129 and scan with high-resolution (pixel size of 6.25µm).
Import the images into ImageJ.
Convert to grayscale.
Adjust signal intensity to a standardized threshold of 95%. (All thresholds, exposure settings, and laser intensities were applied consistently across all scans).
Employ the "Process - Binary - Convert to Mask" function.
The mask function can be determined using the default method against a black background.
Apply a watershed filter to select clusters with similar distribution.
Execute the "Analyze Particles" function.
Size range: 0 to infinity
Display and summarize results.
This will extract all clusters including their coordinates and areas.
To extract clusters with a size equal to or smaller than 1 pixel (6.25µm²) and clusters identified as artifacts (>15000 µm²), use the Excel COUNTIF function, count the number of small clusters (6.25 to 50 pixels), medium-sized clusters (50 to 200 pixels), and large clusters (>200 pixels).
Determine relative density of clusters for the amount of tissue area analyzed.