Electron microscopic imaging of multiple markers in glutaraldehyde fixed CNS tissue of Macaca fascicularis: maximizing information from a single experimental animal

Abstract

Sensory and motor pathways in the central nervous system (CNS) of macaque monkeys were visualized by anterograde or retrograde axonal transport of wheatgerm agglutinin-horseradish peroxidase (WGA-HRP) reacted with the chromagen tetramethylbenzidine (TMB), or by the use of anterograde degeneration after specific ablation lesions. To maximize information from each animal we combined the results of the anterograde and retrograde axonal transport with several pre- and post-embedding markers at both the light and electron microscopic levels while maintaining good preservation of tissue. Pre-embedding techniques included those for cytochrome oxidase activity and the calcium-binding proteins calbindin D-28k and parvalbumin. Post-embedding techniques included immunocytochemistry for gamma-aminobutyric acid (GABA) or other amino acid neurotransmitters. We believe that the methods described here provide superior tissue preservation, thus permitting a more detailed analysis of tissue prepared after experiments concerned with neural circuitry.