Preservation and staining of myelinated nerve fibers

Six procedures are given for preservation of myelinated nerve fibers for light or electron microscopic studies. These procedures fall into two main categories: those with and those without aldehyde fixation. Essentially different effects are attained by application of tannic acid, saline, microwave or conventional heating, or a decreased temperature. All procedures end in osmication. Three main aspects of myelinated fiber morphology are taken into account when judging the quality of their preservation: axon, myelin sheath, and axon/myelin coherence. Each aspect can be preserved excellently, but always in combination with a less superior quality of the other two aspects. Superior myelin quality is attained using microwave irradiation, either with aldehydes to which tannic acid is added or without aldehyde fixatives. Superior axon quality is attained with aldehydes and (conventional) heating. Axon/myelin coherence is best preserved by decreasing the temperature during the rinse with saline. Another two procedures provide good, though less superior, preservation of both axon and axon/myelin coherence. Next, the fixed tissue is embedded in plastic blocks from which semithin and ultrathin plastic sections are cut for light and electron microscopy, respectively. In addition to the standard procedure for toluidine blue staining on semithin sections, two microwave-supported procedures are described, which can be used as alternatives if the staining result is unsatisfactory. Furthermore, a toluidine blue staining procedure is described for glycol methacrylate (GMA)-embedded material, which can be used if larger sections are needed.