Ultrastructural Analysis of Dopaminergic Neurons

Male animals were anesthetized, perfused and embedded in agarose as described in the mouse brain immunohistochemistry section, except that the perfusion was performed with 3% PFA complemented with 0.2% glutaraldehyde in 0.1 M phosphate buffer (PB), pH 7.4. Subsequently, 60 µm-thick sections were cut and processed for pre-embedding immunoperoxidase staining, as previously described^{94 (link)}. Briefly, immunostaining for DA neurons in the SN was performed by incubating the sections with rabbit anti-TH antibodies (Millipore ab152, 1:100) followed by goat anti-rabbit biotinylated secondary antibodies (Vector Laboratories PK-4001, 1:200) and then avidin biotin peroxidase complex (Vector Laboratories PK-4001). 3,3′-diaminobenzidine tetrachloride (DAB; Invitrogen Life Technologies 750118) was applied for 1–2 min until the stain developed. Sections were further embedded in EPON resin (Fluka) and trimmed around the labeled cells of interest. Ultrathin (60 nm) sections were produced and imaged by a Tecnai G212 electron microscope (FEI Company, Oregon, United states) equipped with a digital camera (Mega View III; Soft Imaging Systems). ~15 labeled cells from two mice were analyzed for each genotype. Mitochondria were classified blindly. Mitochondrial size and roundness were measured via Fiji software using the particle analysis plugin after a manual selection of each mitochondrion.