Fig. 3

Visualizing DSCAM expression in cleared Xenopus brain tissues. a, b Whole tissue clearing followed by immunostaining was used to characterize DSCAM expression in intact Xenopus laevis tadpoles. (a) Low-magnification confocal imaging (tiled scan) of a tadpole head shows DSCAM immunoreactivity (red), and 3A10 antibody co-immunostaining (green). Optic nerve fibers (solid white arrowhead) and RGC axon terminals within the tectal neuropil (empty white arrowheads) are visualized by the 3A10 immunostaining. In addition to the optic nerve, the 3A10 antibody stains axonal fibers in sensory and motor cranial nerves. b Individual confocal plane from a z-stack of a brain (imaged ventrally) shows differential distribution of DSCAM and 3A10 immunoreactivity along the optic nerve as it enters the optic chiasm (arrow). DSCAM immunoreactivity also localizes to cell bodies (cb) and neuropil (np). c Individual confocal planes from horizontal z-stacks further illustrate co-localization of DSCAM and 3A10 immunoreactivity in the midbrain neuropil (from dorsal-left to ventral-right). Stronger DSCAM immunoreactivity is observed on the dorsal-most portion of the tectum, where axon terminals extensively branch (solid white and yellow arrowheads). The 3A10 antibody staining also reveals RGC axon fibers as they enter the midbrain (empty arrowheads). d Higher magnification confocal images of dissected brains illustrate strong punctate DSCAM immunoreactivity in the area of the neuropil where axon terminals localize, as identified by the 3A10 immunostaining (arrowheads; yellow lines indicate location of x–z and y–z orthogonal planes, thickness of sample imaged was 85 µm). e Magnified, one-micron z-section of the cleared brain in d reveals coincident punctate DSCAM immunostaining (red) in 3A10-immunolabeled growth cones (green). f The fluorescence intensity of DSCAM (top graph) and 3A10 (bottom graph) immunostaining was measured in whole brain tissues using a 10 μm circular ROI tool and analyzed using MetaMorph, with ten measurements obtained across ten regions each at the level of the lateral neuropil, axon terminals, and axon tract. Measurements were taken from both brain hemispheres equally (n = 4 tadpoles). Data is normalized to the mean fluorescence intensity per channel in the lateral neuropil. Error bars indicate S.E.M. **** p ≤ 0.0001. Scale bars: 100 μm for a, b and c; 25 μm for d; 10 μm for e