Hematoxylin and thionin techniques for staining myelin and cells: variations and critical steps

The action of the delipidization step and the type of differentiation fluid was assessed for two stains, Harris' hematoxylin and thionin, in frozen sections of rat brains. For thionin, all procedural variations used produced a blue or purple cell staining. Myelin stained red when sections were delipidized and differentiated in acetic acid, acid-formalin, or acid-alcohol. Myelin remained white when the delipidization step was omitted or when differentiation was performed in alcohol. Myelin staining was achieved with Harris' hematoxylin when sections were delipidized and were differentiated in either acid-formalin or borax-ferricyanide. The former produced red fibers against a red-purple or light purple cell background with some cell staining. The latter produced purple fibers contrasting sharply with a buff background and minimal cell staining. Cells were further accentuated in the hematoxylin techniques by a thionin counterstain.