Intracellular neuromelanin quantification
Miquel Vila
Abstract
Protocol for quantifying intracellular neuromelanin in coronal sections of the rodent brain, in HE stained sections (NM-occupied area) and unstained sections (NM OD).
Steps
H&E-stained sections (NM-occupied neuronal area)
Scan H&E sections using the Pannoramic Midi II FL, HQ SCIENTIFIC 60x and section images were acquired with CaseViewer software at an objective magnification of 63x.
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, draw a neuromelanin-pigmented neuron cytoplasm (excluding the nucleus) and measure the area.
With the free hand selections tool, draw the neuromelanin pigment of the neuron and measure the area.
Calculate the percentage of the neuronal area occupied by the pigment using the formula: neuromelanin pigment area/neuronal cytoplasm area.
Unstained sections (NM optical density)
For unstained sections, take pictures of different fields in the pigmented area using the Zeiss Imager.D1microscope coupled to an AxioCamMRc camera.
Upload individual images at Image J software.
Click on Invert image.
With the free hand selections tool, draw the intraneuronal neuromelanin pigment of a neuron and measure the optical density. Measure approximately 30-50 neurons per animal, depending on the brain region.
Calculate the optical density of the neuromelanin pigment for each animal using the mean values of the pigmented neurons in the unstained sections.
