Nov 17, 2025
Stereological estimation of human TH+ cells in grafted animal brain tissue
- Roberto Garcia Swinburn1,
- Ernest Arenas1
- 1Karolinska Institute Stockholm
- SOX6 mDA differentiation
Protocol Citation: Roberto Garcia Swinburn, Ernest Arenas 2025. Stereological estimation of human TH+ cells in grafted animal brain tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov11zbkvr2/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: 227393
Keywords: grafted animal brain tissue, th positive cells within grafted animal, animal brain tissue, grafted animal, total number of human nuclei, stereological estimation, modified stereological estimation, human nuclei, positive cell
Abstract
We used this modified stereological estimation to quantify the total number of human nuclei and TH positive cells within grafted animals.
Protocol materials
OCT (Optimal Cutting Temperature compound)Sakura FinetekCatalog #4583
Anti-Nuclei Antibody clone 235-1Merck Millipore (EMD Millipore)Catalog #MAB1281
Donkey Anti-mouse AF647Invitrogen - Thermo FisherCatalog #A31571
DAPIMerck MilliporeSigma (Sigma-Aldrich)Catalog #10236276001
Sheep Tyrosine HydroxylasePel-FreezCatalog #P60101-0
Donkey anti-Sheep IgG (H L) Cross-Adsorbed Secondary Antibody Alexa Fluor 488Thermo Fisher ScientificCatalog #A-11015
Troubleshooting
Imaging
Slice the fixed frozen brain in 5 series at 20 µm using a Cryostat at -20 °C . The brain can be embedded in OCT (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.
We used Sheep Tyrosine HydroxylasePel-FreezCatalog #P60101-0 and Donkey anti-Sheep IgG (H L) Cross-Adsorbed Secondary Antibody Alexa Fluor 488Thermo Fisher ScientificCatalog #A-11015 , Anti-Nuclei Antibody clone 235-1Merck Millipore (EMD Millipore)Catalog #MAB1281 and Donkey Anti-mouse AF647Invitrogen - Thermo FisherCatalog #A31571 DAPIMerck MilliporeSigma (Sigma-Aldrich)Catalog #10236276001
Mount slices on glass slides from most rostral to most caudal.
We mounted several series with TH-stained slices (with other markers as well).
Image whole graft at 10X magnification. TH cells, human nuclei and DAPI-stained nuclei should be discernible.
We used Zeiss Axio Scan.Z1 Slide Scanner for a High-throughput analysis, and several series were imaged
Analysis
Creat a New Project on Qupath.
Load images onto QuPath project
Encircle the graft (detected by human nuclei) with either the Polygon tool or the Brush tool.
Use the Cell Detection tool (Analyze>Cell Detection>Cell Detection) to detect Human Nuclei.
Adjust the detection channel to the human nuclei (in our case, AF647) and threshold to optimize detection.
Use the Classify tool (Classify>Object Classification>Create single measurement classifier). Adjust channel filter to TH channel (in our case AF488), and threshold for optimized classification.
Above threshold should be TH, below should display nothing.
We recommend activating Live Preview to optimize Threshold.
Once you have saved the Cell Detection parameters and the Classification, re-use on all images.
Export the Image measurements in CSV
Calculate the density of TH cells with human nuclei by dividing TH-positive objects per volume (area of graft * 20 µm)
Estimate total number: