Dark-field microscopy visualization of unstained axonal pathways using oil of wintergreen

Despite enormous progress in the development of new morphological techniques, there is still not a simple technique for visualization of the fiber architecture in the mammalian brain. To develop such a technique, thick (400-600 microm) sections of the rat, mice, calf or postmortal human brain were fixed in paraformaldehyde, dehydrated in a series of ethanol and finally immersed in methyl salicylate. The major principle of this newly developed method was to make the neural tissue transparent, and then utilize the ability of neuronal fibers to deflect and deviate light directed from the side to render them visible. Dark-field illumination was used to create illuminating rays of light arriving at an angle exceeding the collecting angle of the objective lens, thus causing only the axonal pathways to be visible as a bright silver silhouette against a dark background. As a result, a three-dimensional structure of the whole white matter of the brain slice became clearly viewable. This technique worked equally well for mammalian brain frontal, sagittal and horizontal sections, as well as for the spinal cord sections. The method was appropriate for verification of axonal fiber courses in brain slice preparations used in electrophysiological experiments, including special applications, such as visualization of axonal bundles within neural transplants. Due to its simplicity, the technique can be successfully used even in an amateur laboratory having basic microscopy equipment and reagents.