Scan midbrain section using the Pannoramic Midi II FL, HQ Scientific 60x

Acquire SNpc images at 63x with CaseViewer

Upload individual images at Image J software

Click on Adjust canvas size and adjust it at 1596x1198

Click on Invert image

With the free hand selections tool, draft a neuromelanin-pigmented neuron (excluding the nucleus) and measure the optical density (pixel brightness) and the area

With the free hand selections tool, draft the neuromelanin pigment of the neuron and measure the optical density (pixel brightness) and the area

With the free hand selections tool, draft 15-25 non-pigmented neurons (excluding the nucleus), measure the optical density and calculate the mean value

Normalize (i.e., substract) the values of the neuromelanin-pigmented neuron’s optical density with the mean value of the optical density of the non-pigmented neurons

Extra measurement: calculate the percentage of the neuronal area occupied by the pigment diving the neuromelanin pigment area by the neuron’s area