Zen 2013

Images were acquired on a Zeiss LSM 880 laser-scanning microscope at ×20, ×40, or ×63 primary magnification. The emission spectrum for each dye was limited as follows: Alexa488/Cy2 (505–530 nm), Cy3 (560–610 nm), and Alexa647/Cy5 (650–720 nm). Biocytin-filled neurons were 3D‐reconstructed from z-stack images acquired at ×40 primary magnification using the ZEN2013 software package (Zeiss). For the analysis of phospho^{40 (link)}-TH and TH immunofluorescence, montages of confocal micrographs in single optical sections spanning the entire PeVN were acquired at ×20 or ×10 primary magnification. Three serial sections/animal were selected for quantification of fluorescently labeled neurons. The PeVN was divided into three levels: rostral, bregma, approx. −0.2 mm; mid, bregma −0.6 mm (at the suprachiasmatic nucleus); and caudal, bregma −0.9 mm (at the retrochiasmatic nucleus). All images were acquired with the same laser power, pinhole, master, and digital gain settings on an LSM 880 microscope. Quantitative analysis of the immunofluorescence intensity was performed in Fiji ImageJ for the individual color channels. To determine diurnal changes in neuromedin-S expression, labeled neurons were inspected in the SCN. Two sections/animal were selected for quantification of the fluorescence intensity using the ZEN2013 software.

Cells were fixed with 4% paraformaldehyde (PFA) in PBS for 15 min at RT, washed 3 times with PBS, and pre-treated with phosphate buffered saline (PBS) containing 1% BSA/0.5% Triton X-100. Incubation with primary antibodies was carried out overnight at 4 °C and with secondary antibodies for 1–2 h at RT. Cells on coverslips were mounted with Aqua-Poly/Mount (Polysciences Inc., Warrington, UK). Images were taken with a 20× (0.8, Apochromat), a 63× (1.40 oil, Plan-Apochromat) or a 100× (1.3 oil, Plan-Neofluar) objective as stacks of multiple optical sections at a Zeiss Axio Imager Z1 equipped with an ApoTome or with a Zeiss LSM 710 (Zeiss, Oberkochen, Germany). Projections were calculated with the Zen 2013 software (Zeiss) and 3D reconstructions were generated using Imaris (Bitplane, Zurich, Switzerland). For quantification of subcellular aSyn levels, signal intensities of aSyn stainings were determined with the Zen 2013 software for the nuclear and extra-nuclear regions (including the soma and neurites).