A modified technique for high-resolution staining of myelin

Abstract

This report describes a new modification of the Gallyas method for staining myelin in fixed brain tissue and compares results of multiple myelin-visualizing techniques in normal common marmoset (Callithrix jacchus), normal macaque monkey (Macaca mulatta), and a human with multiple sclerosis. The new modification involves immersion in 10% formalin following impregnation in ammoniacal silver nitrate, and the use of a low concentration of 4% paraformaldehyde in the developer. This improved technique is less sensitive to post-mortem tissue handling, temperature, and minor contaminants, allowing a more straightforward implementation in the laboratory setting. It permits simple user-controlled development of the reaction product to maximize contrast in the area of interest, resulting in high contrast staining not only of large axonal bundles, but also thin fascicles throughout tissue sections. Myelin staining in visual cortex of an Old World monkey and a New World monkey reveals similar patterns in the new myelin silver stain, the Gallyas stain, and myelin basic protein immunohistochemistry. The most heavily myelinated areas occupy the edges of blobs, but neither the most lightly stained nor the most darkly stained areas are always in our outside a blob. This indicates a more complex pattern between myelinated axons and blobs than previously suggested. While the new myelin silver stain, darkfield microscopy, the Luxol Fast Blue stain, the Gallyas stain, and myelin basic protein immunohistochemistry all permit visualization of myelin in the CNS, each technique has its own merits and pitfalls; careful evaluation of individual study requirements would best determine which methods are the most useful.