Axiocam icc 5 camera

Following in situ hybridization, OPC cultures were imaged using ×40 and ×60 air objectives on an Axio Lab.A1 microscope with an Axiocam ICc5 camera and Axiocam software (Zeiss, Germany). Confocal fluorescent images of cultures and tissue sections were collected using an UltraView spinning disk confocal microscope with Volocity Software (Perkin Elmer, MA) with standard excitation and emission filters for DAPI (Hoechst 33342), FITC (Alexa Fluor‐488), TRITC (Alexa Fluor‐568) and far red (Alexa Fluor‐647). Hoechst 33342 was used to consistently define regions of interest within each tissue section. For low magnification images for cell number quantification, confocal stacks were collected at 2 μm Z‐intervals using a ×20 or ×40 objective and were stitched using Volocity Software. For high magnification images for quantification of assembled primary cilia, confocal stacks were collected at 0.5 μm Z‐intervals using a 100x oil objective and stitched using Volocity Software. Cell counts were performed manually from exported images, using ImageJ (NIH, Washington DC) and Adobe Photoshop CS6 by an experimenter blind to genotype, timepoint or treatment. Images were collected from ≥3 mice per genotype for in vivo experiments and ≥ 3 independent biological replicates per treatment condition for in vitro experiments.

Images of intact nonfixed sporophytes were obtained using a MZ16 stereo microscope, a DFC295 camera and LAS AF software (Leica Microsystems, Wetzlar, Germany). Images of sporogenous cells released from disrupted sporangia and mounted in 30% glycerol were obtained using a DMI4000B microscope with differential interference contrast optics, a DFC360FX camera and LAS AF software (Leica Microsystems). Images of GUS‐stained sporophytes mounted in 30% glycerol and Toluidine blue‐ or GUS‐stained sporophyte sections were obtained using an Axioscope A1 microscope, an AxioCam ICc 5 camera and Zen Blue software (Zeiss). Adobe photoshop CC was used to adjust intensity and contrast, remove background and trace structures in figures. Numerical distance data were generated by measurements in micrographs using the ImageJ Fiji platform (Schindelin et al., 2012 (link)). Microsoft excel was used to create bar charts, calculate means and SD, and to perform Student’s t‐tests.

Investigators were blinded for experimental conditions during image acquisition. In HE‐ and MAP2‐stained sections a ×2.5 magnification was used to visualize both hemispheres using a light microscope (Zeiss, Oberkochen, Germany) with an AxioCam ICc 5 camera (Zeiss). For immunofluorescent stainings, images of both hemispheres were acquired using a Cell Observer microscope with an AxioCam MRm camera (Zeiss, Oberkochen, Germany). For MBP/NF200 stainings ×2.5 images were taken to visualize the cortex. In addition, three adjacent ×40 images were taken at a fixed distance from the external capsule into the cortex (layer III/IV), superjacent to the ipsi‐ and contralateral hippocampal areas. For exact locations, we refer to van Tilborg et al. (2017). For both Iba1 and GFAP analyses, three ×40 images were acquired in the corpus callosum. Moreover, one ×40 image of the CA1 region of the hippocampus (dorsal from the dentate gyrus) was obtained for GFAP area analyses. For CC1/Olig2, Ki67/Olig2, and Caspase3/Olig2 stainings two ×20 micrographs were taken in the corpus callosum and the cortex, directly dorsal from the external capsule.