TITLE: Protocol for &#34;quantification of the nigrostriatal system in non-human primate brain sections&#34;
AUTHORS: jlanciego

[DESCRIPTION]
Here we describe the method conducted to estimate (i) number of TH+ neurons in the substantia nigra pars compacta by taking advantage of a dedicated algorithm (Aiforia) and (ii) optical density of TH innervation in the putamen nucleus.
These two levels of analysis are required to properly estimate the potential damage of the nigrostriatal system in non-human primates, bot at origin (substantia nigra pars compacta) as well as at destination (putamen nucleus).

[STEPS]
SECTION: Quantification of TH+ neurons in the substantia nigra of non-human primates&quot;
1. Scan sections to be analysed in the Aperio CS scanner at a magnification of 20x.
SECTION: Quantification of TH+ neurons in the substantia nigra of non-human primates&quot;
2. Upload digitalized sections to the Aiforia cloud (www.aiforia.com).
SECTION: Quantification of TH+ neurons in the substantia nigra of non-human primates&quot;
3. Create a dedicated project for analysis.
SECTION: Quantification of TH+ neurons in the substantia nigra of non-human primates&quot;
4. Select up to 3-4 sections for training the algorithm, these sections showing different morphologies and intensity stains of tyrosine hydroxylase (TH).
SECTION: Quantification of TH+ neurons in the substantia nigra of non-human primates&quot;
5. Train the model algorithm to analyze potential errors.
SECTION: Quantification of TH+ neurons in the substantia nigra of non-human primates&quot;
6. Activate the annotation assistant tool to retrieve additional beneficial inputs that can be added by the patform itself.
SECTION: Quantification of TH+ neurons in the substantia nigra of non-human primates&quot;
7. Repeat steps 5 to 7 while increasing the number of annotations and iterations (> 1,000) until the model algorithm has learned to properly identify every single neuron showing TH stain.
SECTION: Quantification of TH+ neurons in the substantia nigra of non-human primates&quot;
8. Release the model algorithm.
SECTION: Quantification of TH+ neurons in the substantia nigra of non-human primates&quot;
9. Use the drawing tool to delineate the boundaries of the substantia nigra where TH+ neurons need to be quantified in every single section of the project.
SECTION: Quantification of TH+ neurons in the substantia nigra of non-human primates&quot;
10. Export obtained raw data to an Excel spreadsheet for statistical analysis.
SECTION: Quantification of optical density of TH innervation in the putamen of non-human primates
11. Scan sections to be analyzed in the Aperio CS scanner at a magnification of 20x
SECTION: Quantification of optical density of TH innervation in the putamen of non-human primates
12. For each section, the boundaries of the putamen nucleus (pre- and post-commissural putamen) are delineated and the optical density of TH immunoperoxidase stan measured with Fiji Image J software.
SECTION: Quantification of optical density of TH innervation in the putamen of non-human primates
13. Convert obtained values for optical densities to a logarithmic scale (Ruifrok and Johnston, 2011).
SECTION: Quantification of optical density of TH innervation in the putamen of non-human primates
14. Export obtained raw data values to an Excel spreadsheet for statistical analysis.