710 inverted confocal microscope

The transferrin (Tf) uptake essay was modified from that previously described^{50 (link)}. Syt11-carrying lentivirus was unilaterally injected into the SNpc of TH-GFP mice (provided by Minmin Luo, National Institute of Biological Sciences, China). The SNpc-containing slices were placed on Millicell^® CM (0.4 μm pore size; Millipore) culture plates and incubated with DMEM supplemented with 10% FBS at 37 °C in a humidified incubator (5% CO₂). After 4-h incubation, the slices were serum-starved by replacing the medium with serum-free DMEM containing 20 mM HEPES and 1 mg ml⁻¹ BSA for 45 min at 37 °C. They were then incubated with serum-free medium containing 25 μg ml⁻¹ human Tf conjugated to Alexa Fluor 594 (Invitrogen) for 30 min at 37 °C. The unbound Tf was washed off with ice-cold PBS containing 0.3 mM CaCl₂ and 0.3 mM MgCl₂. The slices were subsequently fixed in an ice-cold solution of 4% formaldehyde and mounted on slides with 50% glycerol. A ×42 oil lens on a Zeiss 710 inverted confocal microscope was used to scan z-stack of 1-μm optical sections. Identical settings were applied to all samples in each experiment. The average fluorescence intensity of the Tf signal in GFP-positive cell bodies in the SNpc was calculated with ImageJ.

Immunohistochemistry was performed as described previously with slight modifications^{32 (link)–34 (link)}. Briefly, mice were anesthetized and perfused with 0.9% saline followed by 4% paraformaldehyde in PBS, and the brain was removed and post-fixed overnight in 4% paraformaldehyde in PBS at 4 °C. After dehydration in 10, 20, and 30% sucrose, a series of coronal sections (40 μm thick) across the striatum and midbrain were cut on a Leica cryostat (every fourth section was used for counting dopaminergic neurons). The sections were permeabilized with 0.3% Triton X-100 in PBS containing 2% bovine serum albumin (BSA) for 5 min at room temperature. After blocking with 2% BSA in PBS, the samples were incubated with primary antibodies at 4 °C overnight. After three washes with blocking solution, samples were incubated for 1 h with secondary antibodies. The sections were subjected to TUNEL staining (Cat. No. 12 156 792 910; Roche) following the manufacturer’s protocols. Nuclei were visualized by DAPI staining and samples were mounted on slides with 50% glycerol. Fluorescence images were captured using a Zeiss 710 inverted confocal microscope. All paired images were captured at the same gain and offset settings and post-collection processing was applied uniformly to all paired images using ImageJ (National Institutes of Health, Bethesda, MD).