As noted previously, luxol fast blue (LFB) stains are used to identify myelin in nervous tissue. LFB is a sulfonated copper phthalocyanine dye and is the alcohol-soluble counterpart to alcian blue, which is water-soluble. In the LFB staining method, lipoproteins within the myelin are responsible for the staining reaction. The staining mechanism of the LFB method is that of an acid-base reaction. The lipoprotein base replaces the base of the LFB dye, resulting in a dark blue precipitate. The excess dye is removed with 95% ethanol. The tissue section is then differentiated in lithium carbonate and 70% ethanol until the gray matter and white matter are distinguished and contrast sharply with each other. The white matter will be blue to blue-green. LFB can be combined with other staining procedures to demonstrate other cellular components of the nervous system.
LFB is often combined with cresyl echt violet to demonstrate both myelin (by the LFB) and nissil (by the cresyl echt violet).
As mentioned in a previous section, cresyl echt violet is used to stain for neuronal cell bodies and processes. Nissl substance has a high ribonucleic acid (RNA) content, making it very basophilic. Cresyl echt violet is a basic aniline dye used to selectively stain Nissl by immersing tissue sections in the staining solution and then differentiating in alcohol until the background is colorless. Sections will appear unstained to the naked eye.