Luxol fast blue stain, abbreviated LFB stain or simply LFB, is a commonly used

stain A stain is a discoloration that can be clearly distinguished from the surface, material, or medium it is found upon. They are caused by the chemical or physical interaction of two dissimilar materials. Accidental staining may make materials app ...

to observe

myelin Myelin Sheath ( ) is a lipid-rich material that in most vertebrates surrounds the axons of neurons to insulate them and increase the rate at which electrical impulses (called action potentials) pass along the axon. The myelinated axon can be lik ...

under

light microscopy Microscopy is the technical field of using microscopes to view subjects too small to be seen with the naked eye (objects that are not within the resolution range of the normal eye). There are three well-known branches of microscopy: optical, el ...

, first developed by

Heinrich Klüver Heinrich Klüver (; May 25, 1897 – February 8, 1979) was a German-American biological psychologist and philosopher born in Holstein. After having served in the Imperial German Army during World War I, he studied at both the University of Ham ...

and

Elizabeth Barrera Elizabeth or Elisabeth may refer to: People * Elizabeth (given name), a female given name (including people with that name) * Empress Elisabeth (disambiguation), lists various empresses named ''Elisabeth'' or ''Elizabeth'' * Princess Elizabeth ( ...

in 1953. ''Luxol fast blue'' refers to one of a group of three chemically and histologically similar dyes. LFB is commonly used to detect

demyelination A demyelinating disease refers to any disease affecting the nervous system where the myelin sheath surrounding neurons is damaged. This damage disrupts the transmission of signals through the affected nerves, resulting in a decrease in their con ...

in the

central nervous system The central nervous system (CNS) is the part of the nervous system consisting primarily of the brain, spinal cord and retina. The CNS is so named because the brain integrates the received information and coordinates and influences the activity o ...

(CNS), but cannot well discern myelination in the

peripheral nervous system The peripheral nervous system (PNS) is one of two components that make up the nervous system of Bilateria, bilateral animals, with the other part being the central nervous system (CNS). The PNS consists of nerves and ganglia, which lie outside t ...

History

Luxol fast blue dyes were produced by

DuPont Dupont, DuPont, Du Pont, duPont, or du Pont may refer to: People * Dupont (surname) Dupont, also spelled as DuPont, duPont, Du Pont, or du Pont is a French surname meaning "of the bridge", historically indicating that the holder of the surname re ...

since at least 1944. ''Luxol'' refers to the original trade name used first by DuPont, and later, the Rohm & Haas division of

Dow Chemical The Dow Chemical Company is an American multinational corporation headquartered in Midland, Michigan, United States. The company was among the three largest chemical producers in the world in 2021. It is the operating subsidiary of Dow Inc., ...

. Du Pont produced three blue dyes sold under the ''Luxol'' trade name, in addition to various other "fast" dyes. The first method of using a luxol fast blue was described by Klüver and Barrera in 1953.

Types and Chemical Structure

There are three types of luxol fast blue: luxol fast blue MBS, luxol fast blue ARN, and luxol fast blue G. LFB MBS is the original and most widely used luxol stain, and was the stain used by Klüver and Barrera. Researchers have since developed similar stain protocols using luxol fast blue ARN. LFB MBS is the bis ,3-di(2-tolyl)guanidinium

salt In common usage, salt is a mineral composed primarily of sodium chloride (NaCl). When used in food, especially in granulated form, it is more formally called table salt. In the form of a natural crystalline mineral, salt is also known as r ...

of a

copper phthalocyanine Copper phthalocyanine (CuPc), also called phthalocyanine blue, phthalo blue and many other names, is a bright, crystalline, synthetic blue pigment from the group of dyes based on phthalocyanines. Its brilliant blue is frequently used in paints ...

-disulfonic acid. The chemical formula for the MBS dye is C32H14CuN8Na2O6S2; the acid, known as luxol fast blue MBS free acid, has the chemical formula 32H16CuN8O6S22-. LFB MBS is a

phthalocyanine Phthalocyanine () is a large, aromatic, macrocyclic, organic compound with the formula and is of theoretical or specialized interest in chemical dyes and photoelectricity. It is composed of four isoindole units linked by a ring of nitrogen ato ...

dye. LFB ARN and LFB G, by contrast, are diarylguanidine salts of sulphonated

azo dyes Azo dyes are organic compounds bearing the functional group R−N=N−R′, in which R and R′ are usually aryl and substituted aryl groups. They are a commercially important family of azo compounds, i.e. compounds containing the C−N=N−C li ...

. LFB ARN is better known as anazolene sodium, with the chemical formula C26H16N3Na3O10S3. LFB G has the formula C57H46N10O13S4.

Mechanism of action

Luxol fast blue is used primarily to stain the myelin sheaths of

neurons A neuron (American English), neurone (British English), or nerve cell, is an membrane potential#Cell excitability, excitable cell (biology), cell that fires electric signals called action potentials across a neural network (biology), neural net ...

. Luxol fast blues undergoes an acid-base reaction to bind to the bases of

phospholipids Phospholipids are a class of lipids whose molecule has a hydrophilic "head" containing a phosphate group and two hydrophobic "tails" derived from fatty acids, joined by an alcohol residue (usually a glycerol molecule). Marine phospholipids typi ...

; while the exact bases involved are unknown, previous research has shown strong affinities towards the phospholipids phosphotidyl choline, phosphotidyl ethanolamine, phosphotidyl serine, and

sphingomyelin Sphingomyelin (SPH, ) is a type of sphingolipid found in animal cell membranes, especially in the membranous myelin sheath that surrounds some nerve cell axons. It usually consists of phosphocholine and ceramide, or a phosphoethanolamine hea ...

. Together, these phosphoglycerides make up 27.6% of the dry weight of isolated myelin. The various luxol fast blues are histologically similar, with only minor variations in affinity towards certain phospholipids.

Procedure

In the staining procedure, tissue sections are stained with a solution consisting of one of the luxol fast blues and ethanol (sometimes, glacial acetic acid is added). There are two main LFB staining protocols: conventional LFB staining and the MCOLL protocol, and are primarily performed on paraffin sections. A typical conventional LFB staining is performed as follows: # Dewax sections in

xylene In organic chemistry, xylene or xylol (; IUPAC name: dimethylbenzene) are any of three organic compounds with the formula . They are derived from the substitution of two hydrogen atoms with methyl groups in a benzene ring; which hydrogens are su ...

. # Hydrate sections several times, alternating pure

ethanol Ethanol (also called ethyl alcohol, grain alcohol, drinking alcohol, or simply alcohol) is an organic compound with the chemical formula . It is an Alcohol (chemistry), alcohol, with its formula also written as , or EtOH, where Et is the ps ...

and 95% alcohol. # Stain sections for at least 16-24 hours at around 56°C. # Rinse (or immerse) in 95% alcohol at least once, then rinse once in distilled water. # Differentiate in 0.05%

lithium carbonate Lithium carbonate is an inorganic compound, the lithium salt of carbonic acid with the chemical formula, formula . This white Salt (chemistry), salt is widely used in processing metal oxides. It is on the WHO Model List of Essential Medicines, Wor ...

. # Continue differentiation in changes of 70% alcohol until grey and white matter can be distinguished. # Wash in distilled water. # (if necessary, continue differentiation with several changes of 70% alcohol). # Stop differenitation in distilled water. # Rinse or immerse in multiple changes of 95% alcohol. # (if necessary, rinse or immerse in multiple changes of pure ethanol). # Transfer to xylene. # Mount slides. In the MCOLL protocol, the following steps are added after differentiation is stopped, and before the transfer to xylene and mounting: # Rinse in distilled water. # Stain sections in picro-sirius red solution at room temperature for 30 minutes. # Rinse in multiple changes of distilled water. # Counterstain with Harris'

hematoxylin Haematoxylin American and British English spelling differences#ae and oe, or hematoxylin (), also called natural black 1 or Colour Index International, C.I. 75290, is a chemical compound, compound extracted from wood#Heartwood and sapwood, heart ...

for 3 minutes. # Rinse in tap water for 3-5 minutes. # Dehydrate in successively increasing concentrations of ethanol from 70% (or 95%) to 99%. In pure LFB stains, myelin fibers appear blue, with areas of the highest concentration of myelin appearing darker. The blue stain appears on a white background. Typically, cresyl violet is used as

counterstain A counterstain is a stain with colour contrasting to the principal stain, making the stained structure easily visible using a microscope. Examples include the malachite green counterstain to the fuchsine stain in the Gimenez staining techniqu ...

. Cresyl violet binds to Nissl substance, which is concentrated around a neural cell's

nucleus Nucleus (: nuclei) is a Latin word for the seed inside a fruit. It most often refers to: *Atomic nucleus, the very dense central region of an atom *Cell nucleus, a central organelle of a eukaryotic cell, containing most of the cell's DNA Nucleu ...

; such a counterstain allows differentiation between myelenated axons, cell bodies, and unmyelenated axons or

glial cells Glia, also called glial cells (gliocytes) or neuroglia, are non-neuronal cells in the central nervous system (the brain and the spinal cord) and in the peripheral nervous system that do not produce electrical impulses. The neuroglia make up ...

. In such a stain,

fibers appear blue,

neuropil Neuropil (or "neuropile") is any area in the nervous system composed of mostly unmyelinated axons, dendrites and glial cell processes that forms a synaptically dense region containing a relatively low number of cell bodies. The most prevalent ...

appears pink (or faint purple), and neuron cell bodies appear purple.

Other combination methods

Though the typical counterstain for LFB staining is cresyl violet, LFB protocols are frequently combined with other common staining methods. The combination of LFB with counterstain or other staining methods provides the most useful and reliable method for the demonstration of pathological processes in the CNS. After cresyl violet, LFB is most often combined with

H&E stain Hematoxylin and eosin stain ( or haematoxylin and eosin stain or hematoxylin–eosin stain; often abbreviated as H&E stain or HE stain) is one of the principal tissue stains used in histology. It is the most widely used stain in medical diag ...

(

and

eosin Eosin is the name of several fluorescent acidic compounds which bind to and from salts with basic, or eosinophilic, compounds like proteins containing basic amino acid residues such as histidine, arginine and lysine, and stains them dark red ...

), which is abbreviated ''H-E-LFB'', ''H&E-LFB''. Other common staining methods include the

periodic acid-Schiff Periodicity or periodic may refer to: Mathematics * Bott periodicity theorem, addresses Bott periodicity: a modulo-8 recurrence relation in the homotopy groups of classical groups * Periodic function, a function whose output contains values tha ...

Oil Red O Oil Red O (Solvent Red 27, Sudan Red 5B, C.I. 26125, C26H24N4O) is a lysochrome (fat-soluble dye) diazo dye used for staining of neutral triglycerides and lipids on frozen sections and some lipoproteins on paraffin sections. It has the appearance ...

, phosphotungstic acid, and

Holmes silver nitrate Holmes may refer to: People and fictional characters * Holmes (surname), a list of people and fictional characters ** Sherlock Holmes, a fictional detective * Holmes (given name), a list of people * Gordon Holmes, a penname used by Louis Trac ...

method.

References