Axiophot

Imaging of embryos was performed using a Nikon SMZ1500 fluorescent stereomicroscope with a Nikon DS-5 digital camera or using a Zeiss Axiophot epifluorescent microscope with a Nikon DXM1200 camera. Images of HUVEC cultures were captured on a Nikon Eclipse TS100 microscope fitted with a DS-Fi1c camera with NIS-Elements D software and images of retinal explants were captured using a Nikon SMZ1500 microscope and DXM1200 camera with ACT-1 software. Data were analysed using Adobe Photoshop and Image J. Quantification of embryonic intersomitic vessel outgrowth and nerve outgrowth between treated and control conditions was measured using Image J. To take into account differences in embryo length results are displayed as a ratio between nerve or vessel length to body or somite length respectively. Statistical analyses were conducted using Prism 5.0 (GraphPad Software, La Jolla, CA) or SPSS 20.0 (SPSS Inc., Chicago, USA). Statistical significance was assessed using two-tailed unpaired Student’s t-test, Mann-Whitney U-test, One-way ANOVA or Kruskal-Wallis test followed by Tukey’s or Dunn’s test respectively. Error bars represent standard error of the mean.

Imaging of VNNs labeled for cFos following either centrifugation or sGVS was performed using a Zeiss Axioplan or Nikon Axiophot microscope with 4x-20x objectives in brightfield mode. Image capture software was Slidebook (Intelligent Imaging Innovations, Denver, CO). Counts of cFos-labeled VNNs were performed at 20x using an eye piece reticle with a 10 × 10 grid. Nissl-stained sections and a stereotaxic mouse brain atlas (29 ) were consulted when assigning cells to one VNN or another. Every stained section along the rostro-caudal axis of the VN complex was analyzed. We counted VNNs with strong nuclear cFos labeling in every section that contained the medial vestibular (MeV) or spinal vestibular (SpV) nucleus. cFos-labeled neurons in left and right sides of the brain were counted; counts for each side were averaged.
Imaging of VNNs labeled for cFos and Tubb3 was performed using an Olympus FV-1000 confocal microscope with a 60x oil objective.

Images were taken using a Nikon Eclipse E800, a Zeiss Axiophot or a Nikon AZ100M microscope and adjusted in Photoshop CS5. Confocal images were recorded on a Leica SP5 confocal microscope; confocal stacks were processed with the Leica software and adjusted in Photoshop.

Observation of live embryos and larvae was carried out as in Saavedra et al. (2014) (link) using a Leica inverted SP5 confocal microscope. Larval cuticles were prepared following Wieschaus and Nusslein-Volhard (1986) , L3 larvae were sliced longitudinally before mounting and examined under a Zeiss Axiophot equipped with a Nikon D-300 camera.