Metafluor software control

Fluorescent images of the RINm-5F cells were acquired with a Nipkow spinning disk confocal system (Yokogawa CSU-10; Yokogawa Electric Corporation, Tokyo, Japan) attached to a Diaphot 200 microscope (Nikon, Kanagawa, Japan) and equipped with a 60×1.40/NA oil immersion objective (Nikon). Fluorescence was excited at 488 nm with argon ion laser (Melles-Griot, Didam, The Netherlands) and detected at 520/35 nm using cooled EM-CCD camera (iXon+ DU888; Andor, Belfast, Northern Ireland, UK) under MetaFluor software control (Molecular Devices Corp., Downingtown, PA).
Images of the human caudate nucleus were acquired by an Olympus FV1000 confocal laser scanning microscope (Olympus) equipped with a 20×/0.95W XLUMPlanFI objective. Emission spectra for each dye were limited as follows: Hoechst (361–497 nm) and Cy5 (630–647 nm). For co-localization analysis of PDYN and Hoechst z-stacks of 24 images were acquired with a depth interval of 0.5 µm. The open source platform Image J (NIH, MD) was used for 3D projections and orthogonal co-localization analysis.