Nov 17, 2025

Public workspaceQuantification of human mDA/progenitor cells in grafted animal brain tissue

 Forked from Stereological estimation of human TH+ cells in grafted animal brain tissue
DOI
https://dx.doi.org/10.17504/protocols.io.14egnrwopl5d/v1
  • Roberto Garcia Swinburn1,
  • Ernest Arenas1
  • 1Karolinska Institute Stockholm
  • SOX6 mDA differentiation
Roberto Garcia Swinburn
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DOI: https://dx.doi.org/10.17504/protocols.io.14egnrwopl5d/v1
Protocol Citation: Roberto Garcia Swinburn, Ernest Arenas 2025. Quantification of human mDA/progenitor cells in grafted animal brain tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.14egnrwopl5d/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: September 16, 2025
Last Modified: November 17, 2025
Protocol Integer ID: 227399
Keywords: grafted animal brain tissue, th positive cells within grafted animal, animal brain tissue, grafted animal, progenitor cell, human nuclei, positive cell
Abstract
We used this Qupath-based quantification for the percentage of human nuclei and TH positive cells within grafted animals with several other markers of interest.
Protocol materials
ReagentOCT (Optimal Cutting Temperature compound)Sakura FinetekCatalog #4583
ReagentSheep Tyrosine HydroxylasePel-FreezCatalog #P60101-0
ReagentAnti-Nuclei Antibody clone 235-1Merck Millipore (EMD Millipore)Catalog #MAB1281
ReagentDAPIMerck MilliporeSigma (Sigma-Aldrich)Catalog #10236276001
Troubleshooting
Imaging
Slice the fixed frozen brain in 5 series at 20 µm using a Cryostat at Temperature-20 °C . The brain can be embedded in ReagentOCT (Optimal Cutting Temperature compound)Sakura FinetekCatalog #4583 to stabilize better. Each slice could be either in cold PBS1X or a cryoprotective solution for storage.

We ended with 15-25 slices of striatum with grafts.

Stain slices against TH, human nuclei and another marker of interest for mDA.
We used ReagentSheep Tyrosine HydroxylasePel-FreezCatalog #P60101-0 , ReagentAnti-Nuclei Antibody clone 235-1Merck Millipore (EMD Millipore)Catalog #MAB1281 and ReagentDAPIMerck MilliporeSigma (Sigma-Aldrich)Catalog #10236276001 for the TH and human nuclei. The remaining channel was used for a marker of interest
Mount slices on glass slides.
Image human cells at 20X magnification. TH cells, human nuclei and DAPI-stained nuclei should be discernible.
We used Zeiss LSM880 confocal microscopes
Imaging
Analysis
Creat a New Project on Qupath.
Software
QuPath
NAME
Pete Bankhead
DEVELOPER
http://qupath.github.io
SOURCE LINK

Load images onto QuPath project
Create a Full Image annotation.
Use the Cell Detection tool (Analyze>Cell Detection>Cell Detection) to detect Human Nuclei.
Adjust the detection channel to the human nuclei and threshold to optimize detection.
Use the Classify tool (Classify>Object Classification>Create composite classifier). Adjust channel filter to TH channel, and threshold for optimized classification.
Above threshold should be TH, below should display nothing.
We recommend activating Live Preview to optimize Threshold.

Save the classifier.
Repeat step 9 with another channel with associated marker
Create a composite classifier (Classify>Object Classification>Create composite classifier) with your relevant markers.
Once you have saved the Cell Detection parameters and the Classification, re-use on all images.
Export the Image measurements in CSV
Calculate the percentage of double positive cells