Intracellular neuromelanin quantification
Miquel Vila, Joan Compte, Marta Gonzalez-Sepulveda
Abstract
Protocol for quantifying intracellular neuromelanin
Steps
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
