Myelin is a critical element of the central and peripheral nervous systems of all higher vertebrates. Any disturbance
in the integrity of the myelin sheath interferes with the axon's ability to conduct action potentials. Thus,
the study of myelin structure and biochemistry is critically important. Accurate and even staining of myelin is
often difficult because of its lipid-rich nature and multiple tight membrane wraps, hindering penetration of
immunoprobes. Here we show a method of visualizing myelin that is fast, inexpensive and reliable using the
cross-linking fixative glutaraldehyde that produces strong, broad-spectrum auto-fluorescence in fixed tissue.
Traditionally, effort is generally aimed at eliminating this auto-fluorescence. However,we showthat this intrinsic
signal, which is very photostable and particularly strong in glutaraldehyde-fixed myelin, can be exploited to visualize
this structure to produce very detailed images of myelin morphology. We imaged fixed rodent tissues
from the central and peripheral nervous systems using spectral confocal microscopy to acquire high-resolution
3-dimensional images spanning the visual range of wavelengths (400–750 nm). Mathematical post-processing
allows accurate and unequivocal separation of broadband auto-fluorescence from exogenous fluorescent probes
such as DAPI and fluorescently-tagged secondary antibodies. Weadditionally show the feasibility of immunohistochemistrywith antigen retrieval,which allows co-localization of proteins of interest togetherwith detailedmyelin
morphology. The lysolecithin model of de- and remyelination is shown as an example of a practical
application of this technique, which can be routinely applied when high-resolution microscopy of central or peripheral myelinated tracts is required.