Chromate/Fluoro-Jade: A high resolution and contrast fluorescent staining method for localizing myelinated fibers in paraffin embedded brain tissue sections.

BACKGROUND

Options for fluorescent myelin stains are limited to immunofluorescent localization of myelin basic protein (MBP) and a proprietary lipid soluble dye, FluoroMyelin, for use with frozen sections and aqueous antifade mounting media. The present Chromate/Fluoro-Jade (Cr/F-J) stain was developed to enable the stable, high contrast and resolution fluorescent labeling of myelin in paraffin processed tissue sections.

NEW METHOD: Tissue sections were delipidized, rehydrated and transferred to a warm solution of potassium chromate. They were then stained in a Fluoro-Jade C solution in 45% ethanol, rinsed, solvent dehydrated and cover-slipped with DPX.

RESULTS

This stain exhibits the high contrast fluorescent labeling of both large myelinated tracts as well as fine individual myelinated axons. This staining pattern correlated closely with that seen with immunofluorescent labeling using antibodies against MBP. Furthermore, the stain binds to purified MBP in a concentration dependent manner. Omission of the chromium pretreatment abolished all staining as did the substitution of carboxyfluorescein for FJ-C.

COMPARISON WITH EXISTING

METHODS

Unlike immunofluorescent staining or the use of lipid soluble fluorochromes, advantages of the present method include speed, sensitivity, a lack of fading or diffusion, a disclosed chemical identity, compatibility with paraffin embedded tissue, aqueous or solvent based mounting media and various fluorescent counterstains.

CONCLUSION

The Cr/F-J staining procedure can be used for the high contrast and resolution fluorescent labeling of myelinated fibers in paraffin embedded brain tissue sections. It is suitable for multiple labeling studies and can be used to demonstrate both normal and pathological myelin.