After MRI scanning, the tissue blocks were taken out of the tube and immersed in a 4% formaldehyde solution. Tissue blocks were placed in a solution of 30% sucrose in phosphate-buffered (PB) saline for several days until they sank to the bottom of the vessel. They were then removed from the sucrose bath, frozen in powdered dry ice, and stored at −80 °C. The tissue block was trimmed and sectioned at a thickness of 50 μm parallel to the block face. The histological sections were stored in individual wells in a PB solution containing 0.1% sodium azide at 4 °C. Of ten serial histological slides corresponding to each MRI slice (thickness 0.5 mm), four adjacent slides were stained serially with Perl’s Prussian blue (which is sensitive to ferric iron) without a counter stain, Kluver-Barrera (KB) (luxol fast blue stain for myelin with Nissl counterstain for neurons), tyrosine hydroxylase (TH) (to identify dopamine cells and fibres), and calbindin D28K (to subdivide the SN), using methods previously described121. The sections for Perl’s iron staining were selected to visualize NM, which appear as unstained brown pigments11.