All animals were euthanized by immersion in a 168mg/L tricaine-S solution (MP Biomedicals). Adult skeletons (Fig. 1 ): specimens were fixed for 3–7 days in 10% Normal Buffered Formalin (NBF). Skeletal staining was conducted according to39 (link). Larvae skeletons: 15dpf larvae, 1 month-old/1.4cm (TrL) and 2 month-old/1.7cm (TrL) juveniles were fixed for 3 days in 4% paraformaldehyde. Specimens stained with alizarin red and alcian blue as described with 60 mM MgCl240 (link). Live bone staining by calcein green and alizarin red was conducted as described in Kimmel et al., 2010 with modifications41 (link). Fish were stained for 24 hours in calcein green, returned to tanks for 14 days, and then chased with alizarin red for 24 hours. Fish were then returned to tanks for 24 hours before fixation overnight in 10% NBF at 4C and stored in 100% ethanol in the dark at 4°C until trypsin digestion as previously described41 (link). Preparations were phenotyped on either a Leica M165FC dissecting microscope or a Zeiss Axioskop 2 FS Plus compound microscope. The dissecting microscope was equipped with a Planapo 1.6X M-series objective, a Prior L200 fluorescent light source and a Leica DC7000T camera controlled by LAS X software. The compound microscope was equipped with a Qimaging Micropublisher 6 camera controlled by Ocular software.
M165fc
Manufactured by Zeiss
The M165FC is a stereo microscope from Zeiss. It provides high-resolution magnification and imaging capabilities for various laboratory applications.
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Lab products found in correlation
2 protocols using m165fc
1
Skeletal Staining and Live Bone Imaging
2
Quantifying Axonal and Glial Reconnections
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Re-established axonal connections and glial connections (“bridges”) were scored at the time point of interest in fixed immunolabelled samples (for axonal and glial bridges) and live transgenic animals (for axonal bridges). Larvae were directly visually evaluated using a fluorescent stereomicroscope (Leica M165 FC) or confocal imaging (Zeiss LSM 710, 880). Larvae were scored as described [3 (link)] with the observer blinded to the experimental condition. Briefly, a larva was scored as having a bridged lesion site when continuity of the axonal labeling between the rostral and caudal part of the spinal cord was observed. The same criterion was used for the assessment of the glial bridges. Continuity of labeling was defined as at least one fascicle being continuous between rostral and caudal spinal cord ends, irrespective of the fascicle thickness. Larvae in which the lesion site was obscured by melanocytes or the notochord was inadvertently injured were excluded from the analysis.
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