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Author(s): Kiernan JA (Kiernan, John A.)
Title: Histochemistry of staining methods for normal and degenerating myelin in the central and peripheral nervous systems
Source: JOURNAL OF HISTOTECHNOLOGY 30 (2): 87-106
Date: 2007 JUN
Document Type: Journal : Review
DOI:
Language: English
Comment:
Address: Univ Western Ontario, Dept Anat & Cell Biol, London, ON N6A 5C1, Canada.
Reprint: Kiernan, JA, Univ Western Ontario, Dept Anat & Cell Biol, London, ON
N6A 5C1, Canada. E-mail: jkiernan@uwo.ca
Author Keywords: birefringence; DAB fluorescence; degeneration; eriochrome cyanine R;
immunohistochemistry; lipid histochemistry; luxol fast blue; marchi
reaction; myelin staining; osmium; palladium; pseudoplasmal reaction;
Sudan black B; tract tracing
KeyWords Plus: CHROMOXANE CYANINE-R; SUDAN BLACK-B; FAST BLUE ARN; OSMIUM-TETROXIDE;
SPINAL-CORD; HUMAN-BRAIN; WALLERIAN DEGENERATION; FIXATIVE SOLUTIONS;
FLUORESCENT STAIN; TIGHT JUNCTIONS
Abstract: This review Summarizes the function, structure, and chemistry of the myelin sheaths of axons; discusses the mechanisms of several staining techniques using inorganic compounds, dyes, and antibodies; and provides technical instructions for 13 staining methods for normal and degenerating myelin. Myelin has alternating hydrophobic and hydrophilic layers. The former contain the hydrocarbon tails of lipid molecules and hydrophobic segments of transmembrance proteins the latter contain the anionic heads of polar lipids and also basic proteins. Aqueous fixatives allow some myelin lipids to be retained in paraffin sections. Stains for normal myelin include histochemical reactions that detect choline-containing phospholipids (Baker's chromation-acid hematein) or double bonds (unsaturation) in the fatty acid components of lipids (osmium tetroxide or palladium chloride reduction; the pseudoplasmal reaction and DAB fluroscence, for oxidized double bonds). Traditional fat stains (Sudan dyes) stain normal myelin less intensely than fat or degenerated myelin. Dyes of the luxol fast blue group have colored anions balanced by hydrophobic organic cations. Applied from an organic solvent and differentiated in an aqueous alkali, these dyes are retained in myelin, which is more hydrophilic than fat but more hydrophobic than cytoplasm or collagen. Dye-metal complexes, notably those of ferric iron with hematein ("iron-hematoxylin") or eriochrome cyanine R (also called chromoxane cyanine R, solochrome cyanine R, and Mordant blue 3), are excellent myelin stains for paraffin sections. They probably attach to basic proteins and resist extraction from a relatively hydrophobic environment. Immunohistochemical methods can detect characteristic proteins and lipids of myelin, some of which are different in the central and peripheral nervous systems. When an axon is disconnected from its neuronal cell body, or if the cell body dies, both the axon and its myelin sheath degenerate. Myelin degenerates also in autoimmune and toxic diseases. The hydrophobic products of degeneration contain crystals of cholesterol esters, brightly visible in frozen sections viewed with crossed polars. Hydrophobic lipids of degenerated and phagocytosed myelin take up fat stains (Sudan dyes, Nile red) but they are not colored by the myelin stains used on paraffin sections. Degeneratnic, myelin fragments are selectively blackened in Marchi's reaction, which is with an aqueous solution containing osmium tetroxide and a hydrophilic oxidant (K2Cr2O7 or KClO3).
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