Immunostaining of Human Dopaminergic Neurons

Differentiated human dopaminergic neurons were fixed by using 4% paraformaldehyde for 15 min and permeabilised by Triton X-100 at 0.04% as described before^{28 (link)}. Primary antibodies against human aSYN (Santa Cruz C20; Santa Cruz Biotechnology, Dallas, USA), β3-tubulin (Invitrogen, Karlsruhe, Germany) at a concentration of 1:200 were incubated overnight at 4 °C, followed by secondary anti-rabbit, anti-mouse antibodies coupled to fluorophore Dyelight 488, and Dyelight 649 (Invitrogen) at a concentration of 1:500. Images were obtained by using a CCD camera (DFC 360, FX, Leica, Wetzlar, Germany) connected to an epifluorescent microscope (DMI 6000 B, Leica). For co-localisation studies, cells were co-stained with MitoTracker Deep Red (200 ng/ml) for mitochondria and DAPI (4′,6-diamidino-2-phenylindole, 1 µg/ml) for the nucleus. Quantification and analysis of neuronal network was performed by using the Image J software.