Whole-mount procedures for simultaneous visualization of nerves, neurons, cartilage and bone

• Published in: Brain research. Brain research protocols Vol. 4; no. 2; pp. 115 - 123
• Main Authors: Rosa-Molinar, E., Proskocil, B.J., Ettel, M., Fritzsch, B.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.07.1999
• Subjects: 3,3'-Diaminobenzidine; Alcian Blue; Alizarin red S; Animals; Anthraquinones; Anti-acetylated α-tubulin; Biotin - analogs & derivatives; Bone; Bone and Bones - ultrastructure; Cartilage; Cartilage - ultrastructure; Coloring Agents; Cyprinodontiformes - anatomy & histology; Dextran-biotin; Dextrans; Dorsal root ganglion; Female; Fluorescent Dyes; Immunohistochemistry; Male; Nerve Fibers - ultrastructure; Nerve Tissue Proteins - analysis; Neurons - ultrastructure; Pectoral fin afferent; Posterior lateral line nerve; Posterior lateral line ventral ramus; Retrograde transport; Septal branch of the ventral spinal nerve; Specimen Handling; Staining and Labeling - methods; Tissue Fixation; Tubulin - analysis; Whole-mount; Immunohistochemistry; Septal branch of the ventral spinal nerve; Dextran-biotin; Retrograde transport; Anti-acetylated α-tubulin; Posterior lateral line ventral ramus; Alizarin red S; Whole-mount; Posterior lateral line nerve; Cartilage; Pectoral fin afferent; Bone; Dorsal root ganglion; Alcian blue
• ISSN: 1385-299X
• DOI: 10.1016/S1385-299X(99)00007-0

Herein are described two techniques to selectively label and/or stain afferent and efferent fibers and their cell bodies and simultaneously stain bone and cartilage. One technique uses anti-acetylated α-tubulin immunohistochemistry to follow the course, peripheral branching, and origin of the ventral spinal nerve innervating the axial musculature and a second uses anterograde and retrograde transport of selectively applied 3 kDa biotin dextran amines to identify specific afferent and efferent projections and their cell bodies. Both procedures can be 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. Myelinated and unmyelinated nerve fibers are stained dark brown with the 3,3′ diaminobenzidine tetrahydrochloride (DAB) reaction product, whereas cartilage is stained blue (alcian blue) and bone is red (alizarin red S). The combination of these procedures provides a simultaneous three-dimensional understanding of the topography of afferent and efferent projections and their cell bodies and the axial and appendicular skeletal system, which is key to understanding the compartmental relationships of the spinal cord with respect to the axial and appendicular skeleton. Themes: Cell biology Topics: Staining, tracing, and imaging techniques