Axioscop microscope

Immunohistochemistry detection of VGLUT1-3 on human brain sections was performed as previously described with minor modifications. Paraffin embedded sections (5 μm) of the cerebellum were obtained from formalin-fixed brains as already described (Torres-Platas et al., 2014 (link)). VGLUT1-3 immunostaining was performed with the anti-VGLUT1 antiserum (1:500 dilution) as described previously (Kashani et al., 2008 (link)) and then processed with biotinylated goat anti-rabbit IgG (1:500 dilution), streptavidine (1:1000 dilution) and 3′3-diaminobenzidine reagents (Jackson Immunoresearch, Brulington Ontario, Canada). Images were obtained with a Zeiss Axioscop microscope.

Fixed 129SvPas/C57Bl6-Thy1-eYFP brains were cut into 40-µm slices using a cryo-microtome (Microm, H560S); slices were mounted on Superfrost plus slides (Menzel-Gläser). Surface quantifications were performed on coronal slices. Epifluorescence of sections was digitized using an Axioscop microscope (Zeiss) equipped with a 5x dry objective and an EMCCD Quantum camera controlled by Metamorph software (Roper Scientific). ROIs were manually drawn and surface areas were calculated using ImageJ software. Since the cerebral volume for WT and KO mice was not the same^{21 (link)}, the area of the corticospinal tract was quantified on coronal brain sections selected based on morphometric landmarks. Thus, slice B corresponded to the end of the dentate gyrus (Bregma: −1.34 mm); slice C corresponded to the start of the habenular commissure (Bregma: −2.30mm); Slice D corresponded to the end of cerebral peduncle lateralization and the start of the rubrospinal tract (Bregma: −4.04 mm); and slice E corresponded to the 10^th slice before the appearance of pyramidal tract decussation (Bregma: −7.56 mm). Epifluorescence for the pyramidal tract was further analyzed on sagittal sections using a confocal microscope (LSM 710, Zeiss), or in whole-mount brainstems using a stereo-microscope (Olympus SZX12).

Cells grown during 5–7 days in shaken liquid BG11₀ NH₄⁺ medium or heterocyst preparations were observed and photographed with a Zeiss (Oberkochen, Germany) Axioscop microscope equipped with a Zeiss ICc1 digital camera. GFP fluorescence was analyzed by confocal microscopy. Samples from cultures of Anabaena sp. strain CSMI21 or the wild-type PCC 7120 as a control grown in bubbled cultures with BG11 or BG11₀ medium were visualized using a Leica HCX PLAN-APO 63X 1.4 NA oil immersion objective attached to a Leica TCS SP2 confocal laser-scanning microscope. GFP was excited using 488-nm irradiation from an argon ion laser. Fluorescence emission was monitored by collection across windows of 500–520 nm (GFP imaging) and 630–700 nm (cyanobacterial autofluorescence). Under the conditions used, optical section thickness was about 0.4 μm. GFP fluorescence intensity was analyzed using ImageJ 1.43 m software (http://imagej.nih.gov/ij/).