The Staining of the Myelin Sheath by Luxol Dye Techniques

The basic mechanisms by which luxol fast blue stains myelin sheaths was studied by experiments directed to elucidating the chemistry of the dye, the nature of the substrate, and the mode of binding of the dye to the substrate. The diphenylguanidine salts of a series of acid dyes were synthesized. All functioned as myelin stains demonstrating that the chemical basis of the staining reaction is the fact that luxol fast blue is the diarylguanidine salt of an acid dye and is not related to the specific structure of the dye. The nature of the substrate was investigated by chemical analysis of isolated myelin and tissue sections. It was determined that myelin contains a high proportion of non-polar amino acids and fatty acid residues which are not extractable by lipid solvents. It is suggested that these structures are concentrated in hydrophobic sites in the myelin substrate and that the dye gains access to these by virtue of its water insolubility. The mode of binding was studied by chemical analysis of the diphenylguanidine salt of congo red in its reaction with tissue sections. It was determined that when the dye is bound to the substrate the diphenylguanidine is released. Since with all luxol dyes the shade of the myelin sheath changes following the application of the basic counterstain, it is further proposed that these dyes have a double function in this staining reaction acting as both dyes and mordants.