Protocol for preparing post-mortem tissue for intracellular neuromelanin quantification in H&E-stained sections.

Formalin-fixed paraffin-embedded tissue blocks from the pontine and midbrain regions were sectioned at 5µm, collected onto adhesive microscope slides and allowed to dry at room temperature for 24 hours.

Slides were incubated in the oven at 60°C for 30 minutes to melt the paraffin.

To remove the paraffin, sections were submerged in Xylene (Panreac Applichem 211769.2714) for 3 x 3 minutes, followed by rehydration in decreasing ethanol concentrations (100% ethanol for 2 x 5 minutes, 95% ethanol for 2 x 5, 70% ethanol for 2x5 minutes) and distilled H₂O for 5 minutes.

Standard hematoxylin-eosin (H&E) staining.

Sections were scanned using 20x objective (NA=0.8) with pre-set focusing and exposure parameters for optimal neuromelanin signal quality with an automated Slide Scanner Olympus (SLIDEVIEW VS200, Tokyo, Japan).

Identical parameters were applied to each scanned section to ensure consistency in capturing neuromelanin.