A modified technique for high-resolution staining of myelin

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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.

Introduction

Several disciplines frequently require the visualization of myelin in post-mortem brain tissue. One example is neuropathology and the study of diseases that cause the degeneration of myelin, such as multiple sclerosis. Neuroscientists examining the function or organization of brain regions often find that variations in myelination can be used to distinguish areas anatomically.

Various techniques have been developed to visualize myelinated axons in the central nervous system, depending on the nature of the study. In wet, unstained sections, myelination can be seen and photographed using a light microscope with regular illumination (Richter and Warner, 1974) or with darkfield illumination (Horton and Hocking, 1997). Basic staining techniques such as the Weil stain (Weil, 1928, Berube et al., 1965), the Pal-Weigert Method (Weigert, 1884, Weigert, 1885, Clark and Ward, 1934), Luxol Fast Blue (Klüver and Barrera, 1953), and immunocytochemical localization of myelin basic protein (Horton and Hocking, 1997) can be used when high resolution of fibers is not required. When a detailed stain including very small myelinated fibers is required, however, many investigators use the silver staining technique first developed by Gallyas (1979), which is based on the binding of colloidal silver to myelin for viewing by light microscopy.

The Gallyas stain for myelin has proven capricious, as it gives variable results depending on the post-mortem handling of the tissue, the ambient temperature during physical development, the quality of reagents used, and the proficiency of the experimenter performing the technique. Because of these limitations, several techniques have been developed for staining myelin both rapidly and with ease. Such benefits have come at the expense of high-contrast and detailed staining of fine fascicles. Yet, the Gallyas method remains the standard used by many neuroanatomists and physiologists due to the quality of data it can produce, and its wide application for staining either frozen or embedded sections of variable thickness (5–100 μm).

Two issues with the silver stain for myelin are the difficulty in controlling the level and speed of staining, and the unpredictability of background levels from section to section. A chemical method to attenuate silver staining has been used to increase contrast and for general de-staining. In cytological preparations, Meywald et al. (1995) found that the use of a silver reducing agent, potassium ferricyanide, after the staining procedure was the best method for obtaining a high signal-to-noise ratio, in addition to allowing for complete de-staining, if necessary. The use of this “bleaching” step after development serves two purposes for the myelin staining procedure we describe; one is to de-stain tissue which has been developed too far, the other is to increase contrast in the staining pattern between myelin and background. Indeed, others have found this reducing agent helpful in obtaining the best contrast in silver staining for myelin in cerebral cortex (Jain et al., 1998).

We have developed a modification of the Gallyas silver stain, which offers higher reproducibility and greater control over development, while being less sensitive to error and contamination. Two critical steps are added to the standard Gallyas method: (1) the introduction of a fixation step, using 10% formalin, between impregnation and development helps to soften folds in tissue that may have been introduced in the lipid solvent step, opening the tissue to more even staining during physical development, and providing a safe stopping point mid-stain, making possible the short-term storage of half-processed sections. (2) The use of a ferricyanide bleaching step after developing increases the signal-to-noise and enables the tissue to be bleached out completely and re-developed if the bleaching is excessive or if staining is not at the desired level.

Section snippets

Materials and methods

Seven common marmosets (Callithrix jacchus), at the end of electrophysiological studies, were sedated with ketamine (IM) and euthanized with an IP lethal dose of pentobarbital sodium/phenytoin sodium (Euthasol Euthanasia Solution, Virbac) and perfused transcardially with heparinized phosphate buffer (pH ∼7.0) followed by 4% paraformaldehyde (EM Grade, Ted Pella #18501) in 0.1 M phosphate buffer (pH ∼7.0). The brains were immediately removed and cryoprotected in 20% sucrose (in 0.1 M phosphate

Results

Fig. 1 shows a series of rostral-to-caudal sections in one marmoset to illustrate the consistency of staining and quality of signal-to-noise using the new modification. The spacing between sections is not regular and is intended to show highlights of key areas such as the globus pallidus, lateral and medial geniculate, hippocampus, and areas of cerebral cortex (the primary sensory and motor areas are clearly distinguished from adjacent secondary areas by darker staining).

Several

Discussion

The modification presented in this paper has several advantages over the original Gallyas stain and the previously-used modified Gallyas (as in Jain et al., 1998). Two major benefits come from the addition of the fixation step, half-way through the staining procedure. One is that folding introduced during the pyridine-to-water steps can be softened and opened, allowing better uniform reagent penetration during development. There is no longer a need to keep sections flat through this process;

Acknowledgements

We gratefully acknowledge Karen Miller for her advice and assistance in developing this modification and Jennifer Liu for help with the tissue processing. We also thank Edward Bartlett for helpful comments on this manuscript. This research was supported by NIH grants DC003180 and DC005808 (X.W.), and EY013354 (S.H.).

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