Efficient navigation zen

Tissue sections were analyzed by fluorescence microscopy (Axiovert 200 M, Zeiss, Germany) or Confocal microscopy (Olympus). Image analysis software Zen (Zeiss) or Fluoview 2.0 (Olympus) was used to quantitate fluorescence intensity (fluorescent pixels). Additionally, a manual cell count of fluorescent positive cells in a field of view (FOV) using the cell count module in Zen (Zeiss). Cells overlapping the border of FOV were also included. For each image, five such FOVs were counted and data represented as percent positive. Masson’s trichrome and Picrosirius red staining were done using standard procedure.

For immunofluorescence microscopy of fixed cells, microfluidic devices were removed and neurons fixed for 2–4 h with PBS containing 4% paraformaldehyde and 4% sucrose, then processed for immunocytochemistry as previously described67 (link). Permeabilization was performed using 0.1% saponin, 0.2% gelatin and 1% BSA in PBS. Imaging was carried out on a Zeiss LSM 710 confocal microscope and analysed with Zen (Zeiss) and ImageJ software. Mouse hippocampal neurons cultured on glass coverslips were treated in the same manner, except that 50 ng ml⁻¹ of CTB-Af555 was added to the buffer for 5 min before fixation with 4% paraformaldehyde and processing for immunocytochemistry as previously described66 (link). Imaging was carried out on a Zeiss LSM 510 inverted confocal microscope and analysed with Zen (Zeiss) and ImageJ software. All images were compiled using Illustrator CS 5.1 (Adobe). For analysis of immunofluorescence images from confocal microscopy, 3–5 different neuronal soma were selected from different positions in each field (394 μm × 394 μm), and 8–9 fields from three independent cultures were used per analysis. The morphology of the soma was derived from masks using the β-Tub III channel, and the fluorescence intensity of the masked regions was measured using Image J plugins.

GLO1 (+/+) and GLO1 (−/−) iPS cells were cultured as described above for 24 h after passage. Before fixation, nocodazole was added to the culture medium to destabilize unstable MTs. After 15 min of nocodazole treatment (1 μg/ml), the cells were fixed and permeabilized, and the MTs were stained with anti-α tubulin (DM1A) antibody (T6199, dilution 1/10,000; Sigma-Aldrich). The fluorescence intensity of the MTs was measured using ZEISS Efficient Navigation (Carl Zeiss).

For awake imaging, mice were habituated to the treadmill under head-fixed conditions for 40 min per day over 2 weeks. Ca²⁺ imaging was performed using a two-photon microscope (Zeiss LSM 7 MP, Carl Zeiss, Jena, Germany) equipped with a water immersion objective lens (Apochromat 20, NA = 1.0, Carl Zeiss). Two-photon excitation at 900 nm for GCaMP6s imaging was carried out using a mode-locked Ti:sapphire laser system (Chameleon, Coherent, USA). Data were acquired using ZEN software (Zeiss Efficient Navigation, Carl Zeiss) at 4.4 Hz for imaging of S1 and 32 Hz for imaging of the cerebellar Bergmann glia.

Calcium imaging was performed with a two-photon microscope (Zeiss LSM 7 MP, Carl Zeiss, Jena, Germany) equipped with a water immersion objective (Apochromat 20×, NA = 1.0, Carl Zeiss). Two-photon excitation for GCaMP6s imaging (900 nm) was provided by a mode-locked Ti: sapphire laser system (Chameleon, Coherent). Imaging was acquired using ZEN software (Zeiss Efficient Navigation, Carl Zeiss). All the experiments were conducted under anesthesia with isoflurane (1%) and the body temperatures of mice were maintained between 36 and 38°C using a heating pad (IL-H-80, Live Cell Instrument). For layer 2/3 neurons calcium imaging, time-lapse imaging (512 × 300 pixels, 0.4 μm/pixel, two line steps, 0.229 s per frame) was performed with imaging depth of 180–220 μm from the surface.

Dissection, fixation and immunostaining were performed as described by [68 (link)]. The following antibodies were used: mouse anti-Prospero (MR1A-c, Developmental Studies Hybridoma Bank (DSHB)) at 1:200; mouse anti-Arm (N2 7A1-s, DSHB) at 1:50; rabbit anti-Caspase3 (Cell Signaling, #9661) at 1:300; rabbit anti-DH31 (gift from Jan Veenstra [40 (link)]) and Michael Nitabach [31 (link)]) at 1:500. The secondary antibodies used were anti-mouse Alexa647, anti-rabbit Alexa488, anti-rabbit Alexa546 (Invitrogen). All secondary antibodies were used at 1:1000. Phalloidin-Alexa555 (Molecular Probes, A34055) were used at 1:500 2h at room temperature or 1:2000 overnight at 4°C. Guts were mounted in Fluoroshield-DAPI medium (Sigma) and observed with a Zeiss Axioplan Z1 with Apotome 2 microscope. Pictures in Fig 4M and 4N were acquired using a Zeiss LSM 880 confocal equipped with a Fast AiryScan. Images were analyzed using ZEN (Zeiss) and Photoshop software. Image acquisition was performed at the Microscopy platform of the Institut Sophia Agrobiotech (INRA 1355-UNS-CNRS 7254-Sophia Antipolis).