Figure 4. Confocal microscopy image of phrenic motoneuron and afferent fiber labeling in the C4 spinal cord following CT-β phrenic nerve application.

In this example, CT-β labelled phrenic afferents were visualized using a secondary antibody with green fluorescence, and a neuron specific marker (NeuN) was visualized with a red fluorescent secondary antibody. The punctate CT-β labeling of phrenic afferent projections can be observed in the dorsal horn (C) and intermediate grey region (E). Panels D and F were created to better highlight these projections, and show monochrome images derived from the original images using Image-J software. The monochrome images clearly show the afferent fiber projection (highlighted by the circles in D and F). The higher magnification shown in panels G and H illustrates the distinct morphological characteristics of CT-β positive terminal synaptic boutons. Scale bars: 200μm (A), 100μm (B-F), and 10 μm (G-H).