Figure 2.

Key aspects of initial CN V differentiation can be resolved with a new rapid ex vivo direct fluorophore-conjugated biocytin injection/tracing method combined with high-resolution laser confocal microscopic imaging in mid-gestation mouse embryos. (A) Injection strategies to resolve initial topographic segregation (left), precise versus exuberant axon growth (middle) and single axon trajectories (right) in E11.5 embryos. (B) Summary of post-injection incubation/survival (1.5 h at 37° in Leibovitz L-15 medium; left) with histological processing limited to whole embryo clearing after fixation (middle) prior to confocal imaging of the whole embryo (right). A complete sagittal optical section from a 3D image set of a whole embryo that received a single fluorochrome-conjugated biocytin injection into distal Ba1B (md) is shown. (C) Z-stacks of images of single labeled axons can be analyzed to assess the trajectory and branching of the axon in 3D (left). These images are then further digitally analyzed to track individual axons through the optical sections of the Z-stack (middle) and then display each axon in 3D space to assess CN V organization with single axon resolution (right).