Multicolor Labeling for Multidimensional Microscopy: Labels to Dye for

Abstract

We previously described two techniques to selectively label and/or stain myelinated and unmyelinated nerve fibers and simultaneously stain bone and cartilage. We used antiacetylated α-tubulin immunohistochemistry and anterograde and retrograde transport of selectively applied 3000 molecular weight (MW) biotin dextran amines (Molecular Probes, Inc. Eugene OR) to follow the course, peripheral branching, and origin of the ventral spinal nerve innervating the axial musculature. Those procedures were combined with an enzyme clearing and staining procedure for the simultaneous visualization of bone (alizarin red S) and cartilage (alcian blue) in whole-mount preparations. Use of the techniques enabled us to describe spatial and temporal changes of nerve fibers and the corresponding changes of the osseous elements during the transposition of the anal fin appendicular support of the Western Mosquitofish, Gambusia affinis affinis.We have extended these techniques by selective multicolor anterograde and retrograde labeling of spinal motor neuron cell bodies, their dendritic arbors, and their ventral motor nerves. We used low (3000) molecular weight (MW) fluorescent lysine-fixable dextrans (Texas Red® and fluorescein) and biotinylated lysine-fixable dextrans (biotin and fluorescein conjugated to biotin), Nissl stains, and fluorescent conjugates of cholera toxin subunit B (Molecular Probes, Inc, Eugene OR).