Lsm 700 module

Images were acquired using a Zeiss Axio Oserver Z1 inverted microscope equipped with a Laser Scanning Microscope (LSM) 700 module (Zeiss, Oberkochen, Germany) for confocal images, and an AxioCam MRm Rev.3 monochromatic digital camera (Zeiss) for bright-field pictures, and operated with the Zen 2010 software (Zeiss). IHC and IF images were acquired using 10× or 40× objectives, respectively. For IF, fluorochrome signals were collected serially, with barrier filters manually set. Unless indicated otherwise, all images correspond to a single z-stack of 2-μm focal plane orthogonal projections. Identical confocal imaging settings were used within experiments.

Immunohistochemistry was performed as previously described25 (link). Briefly, animals were anaesthetized and transcardially perfused with 4% paraformaldehyde (PFA). Brains were dissected, post-fixed in 4% PFA overnight and both brain hemispheres were sectioned 30 μm thick on a freezing microtome. Free-floating or mounted sections were blocked with 3% normal donkey serum and 0.3% Triton-X. Sections were then incubated in primary followed by secondary antibodies diluted in blocking solution, 48 h at 4 °C and 2 h at room temperature (RT), respectively. In-between antibody incubation, sections were washed with Tris-buffered saline. Samples were imaged with a confocal laser-scanning microscope LSM 700 module from Zeiss and AIR from Nikon equipped with 4 laser lines (405, 488, 561 and 633nm) under 10 × , 20 × and 40 × objective lenses. Serial Z-stack images were obtained and collapsed to obtain a maximum intensity projection of lines and/or orthogonal crosshairs were displayed to indicate co-localization. A detailed list of antibodies is available in Supplementary Table 2.