Glossary of staining methods, reagents, immunostaining, terminology and eponyms

BIOLOGICAL STAIN COMMISSION
Glossary of Staining Methods, Reagents, Immunostaining, Terminology and Eponyms
Version 2.0. Compiled for the Biological Stain Commission by Richard W. Dapson, Richard W.
Horobin and John A. Kiernan.
Copyright © March 2021 Biological Stain Commission, Inc.
The headwords are in black bold, with cross references to other items in the glossary
in underlined bold blue italic. Click on an underlined term to move to its place; it will appear at
the top of your window or screen.
Ab. An informal abbreviation for antibody.
Absorption. (1) Neutralization of specific antibodies in an antiserum, by adding an
excess of the appropriate antigen; a negative control
for immunostaining. (2) Removal of unreacted fluorochrome from a solution
containing fluorescently labeled proteins, by treatment with activated
charcoal. (3) Treatment of a labeled antiserum with powdered acetone-fixed tissue,
such as liver or kidney, to remove unwanted labeled proteins that can bind to tissues
by non-immune mechanisms. (The tissue powder must not contain the antigen to
which the antiserum was raised.) See also Affinity-purified. (4) The wavelength of
light from a microscope’s lamp that is absorbed by a dye or pigment, or that
Microtechnique now includes sub-disciplines whose practitioners may not be familiar with related fields.
For example, a researcher or technologist familiar with immunohistochemistry may not understand the
rationale and terminology of staining with dyes, or of enzyme activity histochemistry. Likewise, a user of
fluorescent probes may have little knowledge of fluorescent labels, and vice versa. This glossary is for
the curious, such as those who have used a procedure and wonder, for example, who Mallory or
Papanicolaou was, or what an instruction to differentiate means. Many terms in the glossary provide
insight into the mechanisms of techniques. Similar words with different meanings are explained (e.g.,
albumen/albumin, affinity/avidity. selectivity/sensitivity). The glossary summarizes the properties of
many dyes and other reagents used to color or impart fluorescence to components of cells and tissues,
and the actions of fixatives and other specimen preparative techniques. Production of the glossary is an
ongoing task; the authors expand and update it from time to time. Version 2.0 contains approximately
150% as many items as the previous version, which was posted in January 2019.
If you find any mistakes, or want to suggest new items to include, please let us know. To do so, Click
here. Check the Questions and constructive criticisms item, and type your message in the space
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stimulates a fluorochrome to emit light of a different color.
Acetal. A compound formed by condensation of one molecule of an aldehyde with
two molecules of an alcohol to give the structure Rʹ–O–CH(R)–O–Rʹ. (R and Rʹ are
alkyl or aryl groups).
Acetic acid. A simple carboxylic acid, CH3COOH, present in vinegar (about 5%). The pure
substance is called glacial acetic acid because it freezes at 16.7°C to a transparent solid that looks
like glass or ice. It is a component of various pH buffers, fixative mixtures (in which it causes
swelling and precipitates nuclear chromatin) and staining solutions (to acidify dyes to pH 2–4).
Acetylcholinesterase (AChE). Enzyme that catalyzes hydrolysis of acetylcholine (Ach)
at synapses where Ach is the neurotransmitter; also present in
erythrocytes. Histochemical methods for AChE activity make use
of acetylthiocholine, a synthetic substrate.
Acetylthiocholine (AThCh). Its iodide, CH3C(O)S(CH2)2N+(CH3)3 I− is used in enzyme activity
histochemistry for localization of acetylcholinesterase (AChE). Hydrolysis of AThCh in the
presence of a complexed metal (usually Cu; sometimes Ag for EM methods) generates an
insoluble metal-thiocholine salt, which is then converted to a darkly colored insoluble salt such
as CuS, Cu2Fe(CN)6 or Ag2S. Butyrylthiocholine (BuThCh) is a similar substrate,
for butyrylcholinesterase.
Acid dye. A dye in which the colored component is an anion, e.g. Congo
red and eosin Y. Such dyes are not acids, the term derives from 19th century textile
dyeing, when they are used to color silk and wool from acid dyebaths.
Acid dyeing. The uptake of the anion of an acid dye into those cell and tissue structures
where biopolymers that are cationic predominate, typically proteins. These are cationic if acid
dyeing is carried out at low pH, when amines are present as –NH3
+ and carboxylic acids as non-
ionized free acids (i.e. –COOH). The affinity of an acid dye for such proteins is only partly due
to coulombic forces between dye anions and tissue cations, and substantially due to the various
short range dye–biopolymer van der Waals forces. However the coulombic forces do control
the selectivity of the staining.
Acid-fast stain. A stain for bacteria possessing a waxy cell wall (acid-fast
bacteria, Mycobacteria). Basic fuchsine dissolved in an alcoholic phenol solution (carbol
fuchsine) penetrates all bacterial cell walls but is retained during an acidic differentiation step
only in organisms with waxy cell walls, notably Mycobacterium tuberculosis. Several variants
are popular: Fite's, Kinyoun's and Ziehl-Neelsen's methods, differing in reagent concentration,
times, temperature and type of acid used in differentiation.
Acid fuchsin. See Acid fuchsine.

Acid fuchsine. An acid dye of moderate size, of the aminotriarylmethane class, made by
sulfonation of basic fuchsine. Acid fuchsine is used in mixtures with other acid dyes that have
bigger or smaller colored anions. A solution in saturated aqueous picric acid (smaller) is Van
Gieson’s stain, which colors collagen fibers red and cytoplasm yellow. Acid fuchsine is also
used in some trichrome methods, including Mallory’s stain and some variants of Masson’s
trichrome. The dye is very soluble in water and slightly so in ethanol. Synonyms: CI 42685,
Acid violet 19, acid fuchsin, acid magenta. Commercial lots are available certified by
the Biological Stain Commission.
Acid hematein stain. A staining method developed by J.R. Baker in 1946, later shown to be
specific for lecithins and sphingomyelins (choline-containing phospholipids). In the original
method specimens were fixed in a solution containing formaldehyde and CaCl2 and then
immersed in a potassium dichromate (K2Cr2O7) solution for 4 days at 60°C. Frozen sections of
the chromated tissue were stained for 2h with a freshly prepared solution of hematein and
differentiated in an alkaline solution of potassium ferricyanide, also for 2h. In recent
modifications the treatment with K2Cr2O7 is applied to the sections rather than the whole block
of tissue, shortening the time of chromation to 4h. Dark blue staining occurs where hematein
forms a metal coordination complex with chromium bound by choline-containing lipids.
Mitochondria and myelin are shown well. The tissue background is a rather unpleasing shade of
light yellow, due to staining with hematein, an acid dye, alone.
Acidophilic. Literally, acid [dye] loving. Usually proteins, which take up acid dyes.
Examples of acidophilic tissue components are collagen fibres, nuclear histones and
erythrocytes.
Acid phosphatase histochemistry. See Gomori’s acid phosphatase method.
Acridine orange. A basic dye in the acridine family, with small cations, used as
a fluorochrome. It can bind to nucleic acids, with different fluorescent emission
colors for DNA and RNA. Soluble in water; less soluble in ethanol. Synonyms:
CI 46005, Basic orange 14.
Acriflavine. A yellow basic dye of the acridine class with a small, planar conjugated
system. It is fluorescent (blue excitation, green emission) and is used in a wide variety
of staining and histochemical procedures. The dye is used also as an antiseptic and as
a systemic treatment for a variety of infections in animals. Acriflavine is soluble in
water, less so in alcohol. The dye is made by N-methylation of proflavine, a closely
similar dye, usually present (10–30%) as a contaminant. Synonyms: ACF, CI 46000,
trypaflavine.
Active site. The region of an enzyme molecule that binds the substrate and facilitates the
reaction catalyzed by the enzyme.
Addition reaction. See adduct. Contrast with condensation reaction.

Adduct. (1) Compound formed by combination of two molecules without the loss of
any atoms. Often used when the exact structure of the addition compound is
uncertain. (2) In fixation, the addition of a fixative molecule to its substrate. The
adduct retains chemical reactivity that could lead to crosslinking. Contrast
with condensation reaction.
Adjuvant. An additive that enhances the immune response to an antigen. See
also Freund’s adjuvant.
Affinity. (1) Often used to describe an attractive force between two molecules or the strength of
that force. See also avidity. (2) In a physicochemical context relevant to staining, affinity
is the degree to which a reagent (e.g. dye, enzyme substrate, labeled
antibody or silver ion) transfers from the staining solution into the tissue. The greater
the transfer, the higher the affinity. Contributions to affinity are factors that favor this:
for exmple, coulombic forces and other reagent-tissue attractions, but also positive
entropy changes such as occur during hydrophobic bonding. Synonym: substantivity.
Affinity-purified. This phrase describes a reagent made from an antiserum by
capturing antibodies to a specific antigen, using beads of an insoluble polymer with
the antigen covalently bound to their surfaces. After washing away unwanted
components of the serum, the antibodies are released from the beads by acidification,
which weakens antigen-antibody bonds. Suitably neutralized solutions of affinity-
purified antibodies are polyclonal antibodies; they have many uses
as primary and secondary reagents in immunostaining.
Agar. A mixture of polysaccharides from red seaweeds. It is composed of agarose, a long,
unbranched neutral galactan, and agaropectin, a mixture of other galactans that bear sulfate and
pyruvate ester groups. Agar dissolves in near-boiling water and, on cooling to about 40°C, sets to
a transparent gel that is used in media for culturing bacteria. The gel is firm at temperatures up to
85°C. Tiny specimens can be embedded in agar to make larger blocks that are more easily seen
and handled for processing into paraffin. Also used to cover frozen sections prior to staining, to
prevent losses of cell components such as enzymes into reagent solutions. Agar
is basophilic because of the sulfate ester groups of agaropectin. Synonym: agar-agar.
Agarose. A neutral polysaccharide that constitutes two-thirds of agar and is the component that
forms gels. Each agarose molecule consists of unbranched chains of some
800 D- and L-galactose units. Used as a matrix for chromatographic and electrophoretic
separations and as an intermediate embedding medium in the production of tissue microarrays.
Unlike agar, agarose is not basophilic.
Aggregation of dyes. Dye molecules bind to many things, including other dye
molecules. Resulting dye aggregates can contain two molecules (e.g. methylene blue)

or a hundred (e.g. Congo red). Aggregation may occur either in solution or in stained
tissues, see metachromasia. Aggregation is favored in dyes with large planar conju-
gated systems, facilitating dye–dye van der Waals forces. When aqueous staining
solutions are used, a hydrophobic character of the dye also favors aggregation,
facilitating dye-dye hydrophobic bonding.
Albumen. The principal protein of egg white. The letter e distinguishes it from the
term albumin.
Albumin. A protein that is soluble in water and precipitated by high concentrations
of salts (e.g. saturation with ammonium sulfate). Examples are serum albumin and
egg albumen. Compare with globulin.
Alcian blue. Any of several basic dyes with a large phthalocyanine chromophore bearing
pendent solubilizing cationic side-chains. Acidified aqueous solutions of alcian blue stain anionic
carbohydrate macromolecules but not nucleic acids (DNA and RNA). Solubility in water or
ethanol varies from batch to batch on account of additives, notably boric acid, included to
increase stability of the dye powder. Synonyms: CI 74240, Ingrain blue 1, alcec blue, alcian blue,
alcian blue pyridine variant. The Biological Stain Commission recognizes and certifies two
categories of alcian blue. Alcian blue 8G is converted to a permanently insoluble pigment by a
mildly alkaline rinse after staining; the side-chains probably are not the same as in the original
dye with this name. Alcian blue variant dye lots have similar staining specificity but do not
form an insoluble precipitate with an alkaline rinse and hence can be extracted from sections by
acids.
Alcian blue for acid mucopolysaccharides. Due to basic dyeing, this dye stains sulfate and
carboxyl groups at pH 2.5, but only sulfate groups at pH 1.0 because carboxylic acids are not
ionized at that low pH. The large size of alcian blue results in failure to stain DNA or RNA
because of steric hindrance. The large conjugated system and very hydrophilic character of
alcian blue prevent extraction by dehydration alcohols. Pretreating sections with hyaluronidase
removes connective tissue mucins containing hyaluronic acid, chondroitin-4- and 6-sulfate,
leaving epithelial mucins available for staining with alcian blue.
Alcian blue plus PAS. Detects both acidic and neutral mucopolysaccharides. Neutral mucins
stain magenta, sulfated mucins stain blue, and those containing carboxyl groups only may stain
purple. See alcian blue for acid mucopolysaccharides and periodic acid Schiff for mechanisms.
Alcoholic formalin. Usually 10% unbuffered aqueous formalin in 70% ethanol (i.e., 1 volume
of 40% w/v aqueous formaldehyde diluted with 9 volumes of 70% v/v aqueous ethanol),
although it can be buffered to neutrality. This fixative penetrates more quickly than aqueous
formalin, especially in fatty tissues.
Aldehyde fuchsine. See Gomori's aldehyde fuchsine.

Aliphatic hydrocarbon. Any of several organic molecules of low toxicity consisting only of
carbon and hydrogen atoms. Low molecular weight hydrocarbons, which are used as solvents
and clearing agents, may be unbranched (e.g. hexane, octane, decane) or branched (e.g. iso-
decane and other isoparaffinic compounds). Mixtures of higher molecular weight hydrocarbons
are present in paraffin wax which is used as an embedding medium. Synonym: alkane. Contrast
with aromatic hydrocarbon.
Alizarin red S. A small anthraquinone acid dye that can chelate metal ions. Its principal
application is histochemical localization of calcified deposits (carbonate or phosphate)
in tissues. It can also be used for vital staining of calcium, as in developing bones and
teeth, and, often in conjunction with alcian blue, for staining bone and cartilage in
cleared preparations of whole small animals. Alizarin red S is soluble in water;
slightly soluble in ethanol. Synonyms: CI 58005; CI Mordant red 3. Commercial lots
are available certified by the Biological Stain Commission.
Alizarin red S for calcium. This stain demonstrates calcium by chelation of the calcium ions in
the mineral deposits with the alizarin red S.
Alpha-helix. A molecular shape in three-dimensional space characterized as having a right-
handed coil structure that can be stretched out to a straight linear form (beta-strand). The alpha-
helix is characteristic of DNA and certain proteins.
Aluminon. The triammonium salt of an anionic hydroxytriarylmethane dye, aurin tricarboxylic
acid (CI 43810, Mordant violet 39). It forms colored complexes with various metals including
aluminum (blue), for which it has been used as a histochemical reagent. Soluble in water, less so
in alcohol. Synonym: aurin tricarboxylic acid.
Ammine. A metal coordination complex with ammonia, such as the silver diammine
ion Ag(NH3)2
+, which is present in many solutions used in silver
staining methods. Do not confuse ammines with amines, which are organic
compounds in which one or more hydrocarbon radicals replace hydrogen atoms of
ammonia.
Amphipathic. Synonym for amphiphilic.
Amphiphilic. Having both hydrophilic and hydrophobic domains in the same molecule, as in
soaps and other detergents. Synonym: amphipathic. Although dyes such as metanil
yellow and orange II are amphipathic, this has no impact on their staining applications.
However, the amphipathic properties of dyes are exploited in vital staining to provide selectivity
for certain cell membranes. For instance, the strongly amphipathic fluorochrome di-8-
ANEPPS is used to stain the plasmalemma, whereas the weakly amphipathic cationic
fluorochrome rhodamine B hexyl ester is used as a probe for the endoplasmic reticulum in
living cells.

Amphoteric. Describes a compound which can act as an acid (and donate protons) or a base
(and accept protons) depending on the pH of the local environment. Amphoteric dyes exist in
multiple ionic forms, e.g. neutral red has both cationic and non-ionic species,
whereas rhodamine B has cationic and zwitterionic species. pH indicators are amphoteric dyes
whose different ionic species are of markedly different colors; examples include indicators used
to check the pH of solutions (e.g., indigocarmine) and those used for vital staining to assess the
pH of cellular compartments (e.g., Carboxy-SNARF 1).
Amplification. The generation of several molecules of a visible substance at the site
of detection of each single molecule of interest in a specimen. For example, one
antibody molecule might carry several fluorescent labeling molecules. Enzymatic
labels can use indefinite amounts of substrate to generate insoluble, colored products.
A physical developer can deposit black metallic silver on submicroscopic colloidal
gold particles, increasing their diameters hundreds of times.
Amplification of staining. Using a secondary staining step to increase the coloration
intensity of an initial stain, without changing the initial pattern of selectivity. In this
way, physical development and gold toning can amplify a silver stain; and
a DAB stain derived from a peroxidase labeled antibody is amplified using a
silver stain. Amplification occurs in all techniques of enzyme histochemistry.
Amylases. A group of enzymes, present in malt, pancreas and saliva, that catalyze the hydrolysis
of starch and glycogen, yielding maltose, a soluble disaccharide. Synonyms: diastase, ptyalin.
With sections of animal tissues, either a mixture (such as human saliva) or purified α-amylase is
often used to remove glycogen prior to staining with the PAS method. Saliva is less reliable
because its amylase content varies among people.
Amyloid. A group of unrelated peptides or proteins that share several characteristics. Each one is
derived from a naturally occurring, soluble molecule of identical primary structure that becomes
misfolded and insoluble. Each then self-replicates and self-aggregates into larger (sometimes
massive) molecules. Amyloids are usually pathological, arising either from unknown etiology
(e.g., Alzheimer's disease, Parkinson's disease, Type II diabetes) or from genetic disorder,
although a few are actually functional. Some pathologic variants are transmissible (prions).
All amyloids are detectable with high pH Congo red solutions containing salt and alcohol, using
a combination of light, polarizing and fluorescence microscopy. Thioflavine T is another dye
used to detect amyloid, but its mode of action, selectivity and sensitivity are unknown.
Amylose. A polysaccharide made of glucose units. It is one of the two components of starch,
and has the same chemical configuration as glycogen.
Aniline blue. A mixture of triphenylmethane acid dyes, two of which are methyl blue and water
blue, with sulfophenylamino side-chains. Commercial samples of the dye contain sirofluor, a
by-product of manufacture that is a useful fluorochrome. The blue components of aniline blue
are not fluorescent. This large acid dye is used in mixtures with other acid dyes that have smaller

colored anions. A solution in saturated aqueous picric acid is similar to Van Gieson’s stain. In
the trichrome family of staining methods, aniline blue is often the component that colors
collagen fibers. It is soluble in water and much less soluble in ethanol. Synonyms: CI 42780,
Acid blue 93 (methyl blue), CI 42755, Acid blue 22 (water blue), aniline blue WS, cotton blue,
ink blue, soluble blue. Commercial lots are available certified by the Biological Stain
Commission.
Aniline dye. Old fashioned, but still strangely popular, term for any synthetic dye.
This derives from the mid-19th century when aniline was perhaps the most frequent
precursor of synthetic dyes.
Anion, Anionic. A negatively charged atom or molecule, for instance an ionic species
such as an acid dye or a chloride ion. Anions are attracted to the positive electrode
(anode) in electrophoresis.
Antibodies (singular: antibody, also Ab). Immunoglobulin proteins produced by an animal in
response to exposure to or administration of an antigen. Antibody molecules bind specifically to
parts of antigen molecules known as epitopes. See also monoclonal
antibody, polyclonal. In immunohistochemistry, labeled antibodies are used to identify the
locations of antigens in cells and tissues.
Antigen. Any material that stimulates an animal’s immune system to produce antibodies.
Macromolecules of infecting bacteria, fungi and viruses are not the only antigens. Almost any
protein, or even a much simpler compound, can be administered to an animal with an adjuvant to
evoke an immune response. See also epitope, immunoglobulin and marker.
Antigen retrieval (AR). The restoration of the native conformation of epitopes that have been
directly altered by a fixative, or that were masked (hidden) by crosslinks or hydrophobic
inversions. Procedures to achieve AR include citraconic acid treatment, enzyme
retrieval, glyoxal-specific antigen retrieval, and heat-induced epitope retrieval (HIER).
Antiserum (plural: antisera). Serum containing antibodies to an antigen. Commonly
only the globulin fraction is used. An antiserum is polyclonal and also contains
antibodies to antigens other than the one with which the animal was immunized.
Diluted solutions containing affinity-purified antibodies are nevertheless often called
antisera.
Apoptosis. A series of biochemical reactions leading to cell death that, unlike necrosis, does not
result in an inflammatory response. DNA is cleaved into fragments. The nucleus
undergoes pyknosis, then karyorhexis. The remains of the cell are inconspicuously taken up by
nearby living cells, not by extravasated phagocytes. Histochemical methods for apoptotic cells
include ISEL, TUNEL and immunostaining for cleaved caspase-3.
Aprotic solvent. A liquid polar organic compound that lacks a hydrogen atom that can be

released as a proton. Aprotic solvent molecules cluster around (solvate) cations, but
leave anions relatively unimpeded, so that the latter will be more reactive than when dissolved in
an ordinary (protic) polar solvent. Examples of aprotic solvents are dimethylsulfoxide and N,N-
dimethylformamide.
Aprotinin. A basic polypeptide that inhibits some proteolytic enzymes. When
conjugated with fluorescein isothiocyanate (FITC), it can be used as a fluorescent
stain for mucosubstances rich in uronic or sialic acids. Synonym: Trasylol.
Argentaffin. A substance (e.g. melanin) able to directly reduce Ag+ ions to produce a
deposit of metallic silver (Ag0) without the need for an added reducing agent.
Argentaffin also describes cells containing such substances, e.g. many of the
enteroendocrine (enterochromaffin) cell-types in the gastrointestinal tract. The
term argentaffin reaction thus describes a type of silver staining.
Argentaffin reaction. A silver stain wherein the tissue component reduces ionic silver
to elemental metallic silver without the need of an external reducing agent like
formaldehyde or hydroquinone. See Fontana-Masson argentaffin reaction.
Argyrophil. A tissue element giving rise to deposits of metallic silver (Ag0) following
uptake of Ag+ into the specimen, with the process driven by an added reducing agent.
The term argyrophil reaction thus describes a type of silver staining.
Argyrophil reaction. A silver stain wherein silver ions are first adsorbed onto tissue
elements and subsequently reduced by an external reagent such as formaldehyde or
hydroquinone. See Bielschowskys silver, Bodian protargol stain, Gomori's method
for reticular fibers, Grimelius argyrophil stain.
Aromatic hydrocarbon. In a histotechnical context: any of several organic
compounds, containing only carbon and hydrogen atoms, with ring structures
composed of alternating single- and double-bonded carbon atoms, e.g. benzene,
toluene, xylene. Aromatic hydrocarbons are associated with high levels of toxicity.
Contrast with aliphatic hydrocarbons.
Artifact. (1) Entity generated by a human being, in which sense all stained specimens
are artifacts. (2) In histotechnical usage, something not naturally present, resulting
from the preparative procedure; e.g. technical errors or lack of understanding of
specimen, specimen preparation, or the staining procedure. Synonym: arte
Auramine O. A diphenylmethane basic dye with small cations, used chiefly as
a fluorochrome in sensitive staining methods for detecting leprosy and tubercle bacilli and other
microorganisms. A fluorescent Schiff-type reagent is formed by reaction with sulfur dioxide.

Auramine O is moderately soluble in water, more soluble in ethanol, and slightly soluble in
xylene. Synonyms: CI 41000, Basic yellow 2. Commercial lots are available certified by
Aurin tricarboxylic acid. See aluminon.
Autofluorescence. Fluorescence seen in cells or tissues that have not been exposed to
a fluorochrome. Examples include elastin, lipofuscin and many components of plant tissues,
including chlorophyll. Autofluorescence of proteins is increased
by fixation in formaldehyde or glutaraldehyde, and can then make an unwanted background
in immunofluorescence techniques.
Autoradiography. The production of autoradiographs, which, for biological applications, are
records in a photographic emulsion that has been in contact with cells or tissue sections that
contain a radioactive probe, such as [3H]-thymidine or [3H]-uridine (incorporated into DNA or
RNA respectively), a radioactive amino acid (incorporated into proteins), or a radioactive
compound taken up from the environment. Synonym: radioautography.
Avidin. A basic glycoprotein (MW 70,000) of egg-white that can bind, with
high affinity and avidity, the heterocyclic ring system of 1 to 4 biotin molecules.
Fluorescent or enzymatic labels can be covalently attached to biotin, allowing
amplified detection of biotinylated secondary antibodies. In
another immunohistochemical method, a mixture of avidin with a biotinylated
labeling enzyme (avidin-biotin complex, ABC) binds multiple avidin and label
molecules to each biotinylated secondary antibody, greatly increasing the sensitivity
of detection. Natural avidin can bind to sites other than biotin-labeled antibodies, a
property attributed to its carbohydrate component that can result in false-positive or
“background” artifacts in immunostaining. Preferred reagents are deglycosylated
avidin and streptavidin, which also have 4 biotin-binding sites per molecule of
protein.
Avidity. The strength of binding that involves multiple weak forces (such as hydrophobic
bonding and van der Waals forces). The term is often used in relation to attachment
of antibodies to their antigens, or of complementary polynucleotides. The similar
term, affinity refers to the attractive force bringing molecules together. Affinity is the strength of
the attractive force between molecules whereas avidity is the strength of that binding. The terms
are not differentiated according to the types of molecules involved (antibody/antigen,
dye/substrate, drug/receptor, etc).
AZAN stain. See Heidenhain's AZAN stain.
Azocarmine B. An acid dye of moderate size in the aminoazine group, used chiefly in
the AZAN staining procedure, a trichrome method. This dye differs from azocarmine G by
having an additional sulfonate group, hence its greater water solubility. The dye is soluble in
water, and slightly soluble in ethanol. Synonyms: CI 50090, Acid red 103. Commercial lots are

available certified by the Biological Stain Commission.
Azocarmine G. An acid dye of moderate size in the aminoazine group, used chiefly in
the AZAN staining procedure, a trichrome method. The dye is soluble in water (less so
than azocarmine B), and slightly soluble in ethanol. Synonyms: CI 50085, Acid red 101.
Commercial lots are available certified by the Biological Stain Commission.
Azo coupling reaction. This is a chemical reaction between an aromatic diazonium salt (Ar–
N2
+) and a phenol (Ar–OH) or aromatic amine (Ar–NH2) to yield an azo dye, containing the
linking azo group Ar–N=N–Ar(OH or NH2). Note: Ar represents an aromatic component such as
phenyl, naphthyl or azine. Such scaffolds may carry a variety of additional substituents, e.g. –
S03
- in some azo acid dyes, =N(CH3)2
+ in some basic dyes, or –CH3 in some Sudan dyes.
Azo dye. Dye containing one (monoazo, e.g. orange G), two (bisazo or disazo,
e.g. Congo red) or more (polyazo, e.g. sirius red F3B) azo group(s), namely −N=N−.
Azure A. A small thiazine basic dye, one of the products of polychroming methylene blue. It is
used in the compounding of some of the Romanowsky blood stains. Azure A is soluble in water
and ethanol. Synonym: CI 52005. Commercial lots always contain other thiazine dyes derived
from methylene blue. Lots suitable for inclusion in blood stains are available certified by
Azure B. A small thiazine basic dye, one of the products of polychroming methylene blue. It is
present in all the Romanowsky blood stains and is indeed necessary for their correct
performance. Azure B chloride is soluble in water and ethanol. The perchlorate, tetrafluoroborate
and thiocyanate salts, which have the highest purity, are less soluble and require addition of
an aprotic solvent to achieve concentrations high enough for use in blood stains. Synonym: CI
52010. Commercial lots of azure B chloride usually also contain other thiazine dyes derived
Azure C. A small thiazine basic dye, one of the products of polychroming methylene blue. It is
used in the compounding of some of the Romanowsky blood stains. Azure C is soluble in water
and ethanol. Synonyms: CI 52002. Commercial lots always contain other thiazine dyes derived
Baker. Dr John Randal Baker (1900–1984), was an English zoologist and anthropologist at the
University of Oxford. His books Principles of Biological Microtechnique (1958) and Cytological
Technique (five editions, 1933–1966), are classics in the field of
mechanisms of fixation and staining. Techniques developed by Baker include the acid–
hematein method, mucicarmine, and methods for fixing and staining mitochondria and the
Golgi apparatus.
Banding patterns. See chromosome banding patterns.

Barbital. A barbiturate whose sodium salt is used, with HCl, as a pH buffer (pH range 7.0–9.6)
in histochemistry. It is a controlled substance, formerly used as a sedative. Its use is limited and
it has largely been replaced by TRIS and other buffers. Synonym of sodium salt: veronal.
Base pairs. The stair-step portion of double-stranded DNA that holds the double helix together
via hydrogen bonds. There are two base pairs: adenine and thymine, and guanine and cytosine.
Basic dye. A dye with a cationic colored component, e.g. alcian blue, hemalum,
or neutral red. Such basic dyes are not bases; the term derives from textile dyeing
usage, in which they were originally used to color silk and wool fibres from alkaline
(“basic”) dyebaths.
Basic dyeing. The uptake of the cation of a basic dye into those cell and tissue structures
where biopolymers which are anionic predominate. These biopolymers are most commonly
DNA and RNA (with phosphate anions), or glycosaminoglycans and polysaccharides (with
sulfate anions and, at higher pH, carboxylate anions). The affinity of a basic dye for such
biopolymers is only partly due to coulombic forces between dye cations and tissue anions, and is
substantially due the various short range dye–biopolymer van der Waals forces. However, the
coulombic forces do control the selectivity of the staining.
Basic fuchsin. See basic fuchsine.
Basic fuchsine. A rather small basic dye, in the aminotriarylmethane class, which
stains basophilic structures. Major uses of basic fuchsine are for detection of acid-fast bacteria
(with the Ziehl-Neelsen stain), and for preparation of Schiff’s reagent. Traditionally, basic
fuchsine was a mixture containing four similar dyes: pararosaniline, rosaniline, magenta II and
new fuchsine. Most of the basic fuchsine currently manufactured is the acetate or chloride salt
of pararosaniline, but at least one major American vendor still supplies basic fuchsine composed
of mixed dyes. The dyes comprising basic fuchsine are moderately soluble in water (heating
needed) and ethanol. Synonyms: basic fuchsin, magenta, CI 42500, Basic red 9 (pararosaniline),
CI 42510, Basic violet 14 (rosaniline), CI 42520, Basic violet 2 (new fuchsine). Note: there
disagreement between dictionaries, handbooks, vendors and end-users on the spelling of this dye
name, with Google Ngram indicating that books currently favor fuchsine
over fuchsin. Commercial lots are available certified by the Biological Stain Commission.
Basic fuchsine, special for flagella. A name applied by the Biological Stain Commission to
batches of the dye that work well in staining bacterial flagella. It may be either a 3:1 mixture of
the acetate and chloride salts of pararosaniline or another mixture of basic fuchsine components
that works equally well in Leifson’s flagella stain. Soluble, albeit slowly, in water and
ethanol. Commercial lots are available certified by the Biological Stain Commission.
Basophil. A type of leukocyte with large basophilic cytoplasmic granules.
Basophilia, basophilic. (1) Literally, basic dye lover. Basophilic tissue components,

with affinity for basic dyes, include cartilage matrix, nuclear chromatin, and mast cell
granules. (2) In hematology, basophilia means an abnormally large number of basophil
leukocytes in the blood, traditionally determined by counting cell types in a Romanowsky
stained blood film.
BCECF. A small fluorescent (503 nm excitation, 528 nm emission at pH 9)
polycarboxylated fluorescein derivative. The free acid and monoanionic forms are lipophilic and
so membrane permeant, more highly ionized species are hydrophilic and membrane impermeant.
The probe is usually applied to cells as its membrane permeant acetoxymethyl (AM) ester which
is converted to the active species, BCECF, by cellular esterases. BCECF is widely used as a
fluorescent pH probe of living cells. It is also used non-microscopically as a
physiological pH indicator. The free acid is soluble in neutral and alkaline aqueous solution, the
AM ester is soluble in DMSO. Synonyms: 2’,7’-Bis-(2-carboxyethyl)-5-(and
6)carboxyfluorescein; the ester is usually termed BCECF AM.
Best’s carmine. Traditional selective stain for glycogen, comprising a natural dye,
namely carminic acid, dissolved in alkaline, salty aqueous-methanol. Intriguingly, all these
components of Dr Best's stain are required for effective staining; and the technique provides an
example of dye-tissue hydrogen bonding.
Beta pleated sheet. A molecular shape of parts of certain proteins in three-dimensional space,
characterized as having an accordion-like folding. This results from strands of amino acids being
joined by hydrogen bonds between peptide linkages in the plane of the sheet. The side chains of
the amino acids are perpendicular to that plane, with hydrophobic side chains on one surface and
hydrophilic ones on the other. This configuration occurs in parts of most proteins and is
especially prominent in amyloid proteins.
Biebrich scarlet. An acid dye in the disazo class, with colored anions of moderate size, used in
some trichrome staining methods. It is soluble in water and slightly soluble in ethanol. Useful in
some experimental work because the color does not change over a wide pH range. Synonyms:
CI 26905, acid red 66, ponceau B.
Bielschowsky. Max Bielschowsky (1869–1940) was a German neuropathologist. He learnt
histological staining techniques from Weigert at the Senckenberg Pathology Institute, Frankfurt-
am-Main; but was later purged from German academia in the anti-Semitic pogrom following
1933, eventually emigrating, via Spain, to the UK. The eponymous silver stain bearing his name
was an improvement of a procedure developed by Ramon y Cajal.
Bielschowsky's silver. Perhaps the original argyrophil reaction to demonstrate axons, dendrites,
neurofibrils; as well as the neurofibrillary tangles and senile plaques of Alzheimer's disease.
Sections are treated with silver nitrate, after which bound silver Ag+ is reduced with
formaldehyde to precipitate metallic silver Ag0, and finally gold toned to heighten contrast.
This silver staining procedure was highly capricious and so has been modified numerous times
by many authors.
Bile pigments. Principally bilirubin. A colored product of the metabolism of the heme

component of hemoglobin, normally excreted by the liver into the intestine. With excessive
destruction of erythrocytes, inadequate liver function or obstruction of the duct leading to the
gut, bilirubin accumulates in the blood and in tissues, causing abnormal yellow coloration
(jaundice). The exact sites of the pigmentation can be identified histochemically with Fouchet’s
stain and by several other methods.
Biogenic amines. Small molecules that act as hormones, neurotransmitters or mediators of
immune responses, including dopamine (dihydroxyphenylalanine), adrenaline (epinephrine),
noradrenaline (norepinephrine), serotonin (5-hydroxytryptamine, 5HT) and histamine. These
small molecules rapidly diffuse away from their cells, so their distribution is usually studied
by immunohistochemical detection of enzymes involved in their synthesis. Two exceptions are
amines in the chromaffin cells of the adrenal medulla (adrenaline, noradrenaline) and the
enterochromaffin cells of the small intestine’s epithelium (5HT). These retain enough biogenic
amines to be histochemically stainable in paraffin sections of appropriately fixed specimens.
Biological Stain Commission (BSC). A not-for-profit corporation that ensures the
quality of dyes through independent assays and other tests, promotes cooperation and
dialogue among manufacturers, vendors and users of dyes in all fields of biological
and biomedical sciences, educates users of biological stains about sources of reliable
dyes and how they might best be used, and publishes information about innovations
and improvements in biological staining and histochemistry. Dye batches that pass the
BSC’s tests are certified stains. The BSC also publishes a journal, Biotechnic &
Histochemistry, a book, Conn’s Biological Stains, and conducts correspondence and
annual meetings, maintaining dialogue among scientists, manufacturers and vendors
concerned with biological stains. For more information about the BSC, go
to http://biostain.com.
Biomolecules and biopolymers Compounds characteristic of living organism. Those
of high molecular weight include nucleic acids, glycosaminoglycans,
polysaccharides, proteins etc.Lipids are biomolecules that are not polymers.
Biopsy. A small sample of tissue for diagnostic examination, taken by excision, needle or
scraping, without the need for surgery.
Biotin. An easily attached organic label that can be detected by virtue of its specific
affinity for avidin or streptavidin, large proteins that can themselves be labeled with
either fluorochromes or enzymes.
Birefringence, birefringent. An optical property of a substance that causes rotation of a beam
of polarized light passing through it. A polarizing microscope is needed to visualize
birefringence; birefringent objects appear bright against a dark background. Some substances in
tissues are naturally birefringent (e.g. cellulose microfibrils in plant cell walls, collagen fibers,
the A bands of striated muscle fibers, and many crystals). Birefringence can be greatly enhanced

by staining with certain dyes, including Congo red for amyloid and sirius red F3B for collagen.
Bisbenzimide. A polymethine basic dye in the benzimidazole class, notable for
its fluorescence (UV excitation, blue emission) and for its strong affinity for DNA. The
dye cations bind to the minor groove of the DNA helix. It is widely used as a fluorescent stain
for DNA in living cells (vital staining) and as a counterstain for immunofluorescence and in
situ hybridization. Bisbenzimide is soluble in water and dimethylformamide, but not in
phosphate buffers. Synonym: Hoechst 33258.
Bismarck brown Y. A basic dye of the disazo class, with cations of moderate size that
stains basophilic structures. It has been used to stain mucus, amyloid, plant cell walls and
bacteria, and it is a component of some Papanicolaou stain variants that are used for cancer
diagnosis. It is soluble in water and ethanol. Synonyms: CI 21000, Basic brown 1, vesuvine.
Commercial lots are available certified by the Biological Stain Commission.
Blockade, blocking reaction. Conversion of tissue sites able to bind or generate stain
into non-binding, non-generating forms. Examples include: attachment of unlabeled
antibodies to antigenic tissue sites to block subsequent binding of labeled
immunoglobulins; enzyme inhibition following exposure to glutaraldehyde; and
esterification of tissue acids to reduce basophilia.
Blocking. In immunohistochemistry, treatment of sections with a soluble protein that binds
nonspecifically to the tissue. This suppresses unwanted nonspecific binding of antibodies.
Blocking agents include BSA, casein and diluted, unlabeled, non-immune serum from the
species in which the secondary antibody was raised. If a tissue contains endogenous biotin, this
must be blocked if a biotin-labeled secondary antibody is to be used. Sections are treated with a
solution avidin and then with biotin to occupy vacant binding sites of avidin. For this purpose,
diluted egg-white and skimmed milk are convenient and cheap sources of avidin and biotin,
respectively.
Bluing agent or reagent. A mildly alkaline solution following a hemalum stain, used
to shift the reddish violet color to blue-violet or blue. Hard (alkaline) tap water, a
dilute lithium carbonate solution or Scott's tap water substitute are the agents most
commonly used.
Bodian. David Bodian (1910-1992) was an American medical scientist. In 1936,
while a student of comparative neuroanatomy in Chicago, he published a silver
staining method for axons and nerve endings using protargol, a technique with which
his name is associated. Bodian later became professor of anatomy at Johns Hopkins
University in Baltimore, where he conducted important research into the pathogenesis
of poliomyelitis. See also: Bodian's protargol stain and protargol.
Bodian's protargol stain. Demonstrates nerve fibers in paraffin sections. Certain silver
proteinate solutions, called protargol-S, and certified by the Biological Stain Commission, are

used to impregnate axons. Neurofilaments within the axons bind the silver Ag+, which is then
reduced with hydroquinone in an argyrophil reaction. The section is then gold toned to enhance
contrast.
BODIPY dyes. A large class of fluorescent probes containing the boron-dipyrromethene
scaffold, widely used for vital staining. The BODIPY scaffold sometimes carries substituents,
which result in specific localization within living cells; e.g. BODIPY 493/503 is widely used as
a specific stain for lipid droplets. Other dyes, such as BODIPY TRX SE, carry reactive
substituents that enable the fluorescent labeling of macromolecules such as
peptides, phospholipids, polynucleotides, proteins and other biochemicals. This labeling is used
both to generate vital stains and to facilitate in vivo tracing. Unfortunately many biomedical
authors report their applications of “BODIPY” dyes without specifying which of the of many
compounds were used.
Bouin’s fluid. A rationally contrived fixative mixture of three ingredients that can stabilize an
immersed animal tissue in different ways. Formaldehyde penetrates quickly and modifies
proteins. Acetic acid enters cells and their nuclei, precipitating chromatin. Picric acid coagulates
proteins and also opposes the tissue swelling caused by acetic acid.
Bound water. Water molecules hydrogen-bonded to specimen molecules, effectively insulating
them spatially and electronically from one another. Contrast with free water.
Bovine serum albumin (BSA). Albumin from the serum of cattle. Tissue sections are often pre-
treated with diluted BSA prior to applying antibodies in immunohistochemistry. This pre-
treatment is often called blocking. BSA molecules occupy sites in the tissue that can
nonspecifically bind proteins, thereby limiting the attachment of the antibodies to their
specific antigens.
Bradford protein assay. A simple, rapid spectrophotometric assay for proteins. An acidified
solution of coomassie brilliant blue G250 is added to a solution containing protein. In the
absence of protein, the dye solution is the reddish color of the fully protonated anion. With
binding to protein, by electrostatic forces and hydrophobic bonding, both the dye’s anionic sites
are neutralized by cationic sites in the protein, and the bound dye has the blue color of the
unprotonated compound. This blue color is measured with a spectrophotometer as the
absorbance at 595 nm. The assay is named for Marion M. Bradford of the University of Georgia,
who published the method in 1976.
Brilliant blue (G, R). Synonyms for coomassie brilliant blues.
Brilliant cresyl blue. A basic dye in the oxazine class with small cations, used for vital staining.
Two major applications are the detection of reticulocytes in blood, and the assessment of the
suitability of oocytes of various species for in vitro fertilization purposes. The dye is soluble in
water and ethanol. Synonyms: CI 51010, brilliant cresyl blue ALD. Commercial lots are
available certified by the Biological Stain Commission. Note: one major vendor currently sells
brilliant cresyl blue lots which are stated to be a “mixture of toluidine blue and water blue”.

Brilliant green. A basic dye in the triphenylmethane class with cations of moderate size that
stains basophilic structures, and has been used for coloration of bacteria and of fungal hyphae.
Its principal use, however, is to inhibit growth of coliform bacteria in cultures for detection
of Salmonella infection. Brilliant green is soluble in water, and more soluble in ethanol.
Synonyms: CI 42040, Basic green 1, aniline green, diamond green G, emerald green, fast green
J, malachite green G, solid green JJO. Commercial lots are available certified by the Biological
Stain Commission.
Brilliant indocyanine 6B. A synonym for coomassie brilliant blue R250.
Bromodeoxyuridine (BrDU). An analog of thymidine. When cells divide in the presence of
BrDU, they incorporate it into their DNA. This can be detected
by immunohistochemistry or autoradiography and serves as a marker for rapidly dividing cells,
as in animal embryos or in cancer.
Brown-Brenn Gram stain. A Gram stain variant for smears, that uses basic fuchsine as the
stain for Gram negative bacteria and picric acid as a background stain.
Brown-Hopps Gram stain. A Gram stain variant similar to the Brown-Brenn procedure that
was modified for tissue sections instead of smears.
Buffer. A solution that maintains a constant pH after addition of small amounts of
acid or base. It is typically made of a salt and a weak acid, which are in equilibrium in the
solution. If additional hydrogen ions (more acid) are added, they combine with the base of the
salt. If more alkali (OH-) is added, it neutralizes the weak acid. In either case, the pH does not
change. Specific buffer solutions maintain pH over limited ranges, typically about 2
pH units.
Buffy coat. The thin pale greyish yellow layer, consisting of leukocytes, that settles on top of the
erythrocytes and below the plasma when anticoagulated whole blood is centrifuged.
Butyrylcholinesterase (BuChE). An group of enzymes similar to acetylcholinesterase, formed
principally in the liver and needed for the metabolism of some drugs. It is also present in nervous
tissue, and can confuse the interpretation of sections stained histochemically for
acetylcholinesterase activity because both enzymes catalyze the hydrolysis of AThCh.
Butyrylthiocholine is a selective substrate for BuChE. The two enzymes can also be
distinguished by using selective inhibitors in the incubation medium. Synonyms: nonspecific
cholinesterase, serum cholinesterase.
Cajal. Santiago Felipe Ramon y Cajal (1852–1934) was a Spanish physician and anatomist, best
known for providing microscopic evidence in support of the neuron theory but also for
investigation of the retina and central visual pathways, and the degenerative and regenerative
changes that follow injury to the central and peripheral nervous systems. For this he received the
Nobel Prize in Physiology or Medicine, together with Golgi, in 1906. The name Cajal is
associated with a variety of cell-types in the nervous and digestive systems; with several silver

staining methods, a method for astrocytes; and with a combination of carmine, picric
acid and indigocarmine (Cajal's trichrome) that provides red nuclei, yellow erythrocytes, green
muscle and blue collagen fibers.
Cajal's gold sublimate. This demonstrates neuroglia, particularly astrocytes. Tissues are fixed in
a mixture of ammonium bromide and formaldehyde, with release of hydrogen ions to lower the
pH to 1.5. Sections are stained in an aqueous solution of chloroauric acid ("gold chloride") and
mercuric chloride ("sublimate") to impregnate the cells. Sodium thiosulfate halts the reaction.
Astrocytes are dark purple or black; other cells acquire lighter shades of purple. See also Cajal.
Calcein. A hydroxyxanthene fluorescent probe used to detect calcium and other metals, as
a marker for cytoplasmic continuity between embryonic cells, and as a marker for apoptosis in
living cells and organisms. Both a hydrophilic form and a hydrophobic form are available; the
latter membrane permeable compound can be converted to the former by intracellular enzymes.
Synonym: fluorexon.
Calcofluor white M2R. The disodium salt of a hydrophilic organic compound with
large anions consisting of disulfonated stilbene with attached triazinyl and phenyl rings and
hydroxyethyl groups, making a large conjugated system. It is fluorescent (near UV absorption,
blue-white emission) and is used as a stain for cellulose cell walls, fungi, chitin, as
a counterstain for in situ hybridization with plant tissues, and to evaluate viability of plant and
animal cells. Synonyms include calcofluor white and tinopal, with various other associated
letters; CI 40622, Fluorescent brightener 28.
Callose. A plant cell-wall polysaccharide composed of β-1,3-glucosyl units. It occurs normally
in the sieve plates that separate cells of the phloem and is added to cell walls at sites of
injury. Aniline blue and sirofluor are useful fluorescent stains for callose.
Canonical forms. Different structures of an organic compound in
which resonance occurs. In drawing canonical structures, only bonds and sites of
electric charge may be varied; the positions of the atoms may not be changed.
Carbol-fuchsine. A solution of basic fuchsine (about 0.5%), ethanol (about 10%) and
phenol ("carbolic acid", about 5%) in water. It is used in the acid-fast stain for
mycobacteria.
Carbowax 1540. A water-soluble wax-like polyethylene glycol that melts at 43–46°C. See
also DTT-Carbowax,
Carboxyfluorescein. An anionic xanthene dye closely related to fluorescein, containing an
additional carboxyl group. It has been used as a fluorescent probe in plants and animals, for
intracellular pH, gap junctions between cells, and leakage from liposomes.
Carboxy SNARF-1. An anionic xanthene dye closely related
to fluorescein and carboxyfluorescein. It is used principally to measure

intracellular pH.
Carmine. An aluminum complex of carminic acid, of variable composition, manufactured
from cochineal. This dye-metal coordination complex is used in staining solutions devised to
provide red coloration of cell nuclei, chromosomes, glycosaminoglycans or glycogen. Solutions
containing carmine or carminic acid plus aluminum salts are termed carmalum. Synonyms: CI
75470, Natural red 4. Commercial lots are available certified by the Biological Stain
Commission.
Carminic acid. A red anthraquinone natural dye derived from cochineal, which can form metal
coordination complexes. Some of these, especially with aluminum or iron, are used as biological
stains, in particular for chromosomes. Synonyms: CI 75470, Natural red 4.
Casein. A protein from milk, used (often as skim milk) to suppress non-specific attachment
of antibodies in immunohistochemistry (see also bovine serum albumin. Skim milk also
contains biotin and can block free binding sites of avidin or streptavidin (see also blocking).
Catalase. A peroxidase enzyme that destroys hydrogen peroxide. It is used to inhibit
endogenous peroxidase in enzyme histochemistry and immunohistochemistry.
Catecholamines. A family of biogenic amines derived from tyrosine, including dopamine,
epinephrine (adrenaline), and norepinephrine (levarterenol, noradrenaline).
Cation, Cationic. A positively charged atom or molecule, an ionic species such as
a basic dye, or a sodium ion. Such species are attracted to the negative electrode
(cathode) in electrolysis or electrophoresis.
Cationized ferritin. Ferritin that has been treated with N,N-dimethyl-1,3- propanediamine to
confer a positive charge. It is used to show, by EM, the negatively charged surfaces of bacteria
and other cells.
C-banding. See chromosome banding patterns.
Celestine blue. An oxazine basic dye with small colored cations that are also able to form larger
cationic dye-metal complexes. The complex formed with ferric salts has staining properties
similar to those of hemalum. Celestine blue is soluble in water and ethanol. Synonyms: CI
51050, Mordant blue 14, celestin blue, celestine blue B, coelestin blue, coreine 2R, gallo sky
blue B.
Cell death. See apoptosis and necrosis.
Centromere. The dense area of a chromosome during cell division where spindle fibers attach
and the chromatids are joined in an X shape. See chromosome banding patterns.

Ceramide. A type of lipid; fatty acid amides of sphingosine containing no phosphorus.
Cerebrosides. Glycolipids in which ceramide is glycosylated galactose or glucose. They are
abundant in the central nervous system, and in Gaucher’s and Krabbe’s diseases they accumulate
in phagocytic cells. The deposits can be detected in frozen sections with the PAS technique.
Certified stain. Currently the Biological Stain Commission (BSC) offers testing and
certification for over 60 stain powders. The tests and required standards are published,
and are revised from time to time as quality improves and applications change. For a
batch of dye that passes the tests, the BSC issues a certificate to the vendor. The
certificate identifies the vendor’s batch number, the BSC’s identification code for the
batch, and the uses for which the dye is certified. Small labels, which are difficult to
fake, also carry the BSC’s identification code; they are made available for affixing to
bottles of BSC-certified stains. The BSC laboratory keeps samples of all certified (and
rejected) dye batches, and records of sales of certified dyes from submitting vendors
to other vendors. Parts of the same batch often have different vendors’ numbers, but
the BSC’s identification code never changes. BSC certificates are valid for 10 years
for all stains except alcian blue, for which the time is 5 years. There are vendors
claiming to sell “certified stains” who have not had their products independently
tested by the BSC. The web page http://biologicalstaincommission.org/vendors-
list/ shows manufacturers and vendors who have recently submitted samples that
received BSC testing and certification.
Chelating agent. Some are mordant dyes which react with metal ions giving rise to
stains. Examples include carminic acid, celestine
blue, gallocyanine, hematein and nuclear fast red. Some are used to generate
colored products from tissue constituents, e.g.: demonstration of calcium ions
by chelation with alizarin red S; or of nickel ions by chelation
with dimethylglyoxime. Another chelating agent, EDTA, is used for decalcification of
tissues.
Chelation. Formation of a metal coordination complex by the reaction of a metal ion
with a chelating agent. This involves a compound carrying two or more metal binding
groups (such as CO2
-, OH or C=O), which are able to form a complete covalently
bonded ring that includes the metal ion. Such chelate rings often are chemically stable
structures.
Chemical dehydration. See dehydration.
Chemiluminescence. The emission of light but not heat during a chemical reaction. Some dyes
(e.g., lucifer yellow CH) are chemiluminescent when oxidized.

Chlorazol black E. An acid dye of the polyazo class, with large anions, which can stain all
components of tissues in black and shades of gray. Colored impurities impart other colors to
some materials including chitin (green) and glycogen (pink). It is used with animal (especially
insect) and plant tissues, and to detect parasitic protozoa in fecal smears. Synonyms: CI 30235,
Direct black 38, Erie black GXOO, pontamine black E. Commercial lots are
Cholesterol. A steroid component of cell membranes and atherosclerotic lesions,
demonstrable histochemically with the perchloric acid–naphthoquinone (PAN) reaction. It also
may be insolubilized with digitonin before processing and subsequently stained. The high
melting point of cholesterol (150°C) precludes its staining with the non-ionic Sudan dyes used
to demonstrate other hydrophobic lipids.
Cholesterol esters. Compounds in which the hydroxy group of cholesterol is esterified by
a fatty acid. They are more hydrophobic than cholesterol, have much lower melting points (20–
40°C), and can be stained with Sudan dyes.
Cholinesterases. Acetylcholinesterase and butyrylcholinesterase. Both are competitively
inhibited by eserine, which is included in the incubation medium to provide negative controls
in enzyme histochemistry.
Chromaffin reaction. A fixation method that also demonstrates adrenaline and noradrenaline in
the adrenal medulla. Potassium dichromate in the fixative oxidizes these monoamines to
brown quinones.
Chromatin. Material in the nucleus of a cell that is stained by cationic dyes and by
some dye–metal complexes such as carmine and hemalum (aluminum–hematein).
Chromatin comprises DNA and the nucleoprotein (histone) of the chromosomes.
Chromic acid. In a biological staining context, this term describes acidified dichromate
(Cr2O7
2¯) solutions. Chromic acid been used inaccurately as a synonym for the anhydrous form
(chromium trioxide, CrO3), which is used to make such solutions.
Chromogen. A substance that reacts to give a coloured product, such as DAB and
other compounds used in histochemical methods for localization
of peroxidase activity.
Chromophore. The part of a molecule that absorbs light in part of the visible range of
the spectrum (400-700 nm), thereby making the compound colored. Traditional
chromophores include the azo group and quinonoid ring systems, which can be seen
in the structural formulae of most dyes.
Chromosome banding patterns. Alternating dark and light bands on metaphase chromosomes,
seen after staining with suitable dyes (e.g., Romanowsky stains like Giemsa’s and Wright's or

with the fluorochrome quinacrine). Regions rich in guanidine-cytosine appear dark because
they stain strongly (C banding) with Romanowsky stains, and bright (Q banding) with
quinacrine; thymine-adenine rich regions appear light with Romanowsky stains (R or reverse
banding) and dark (not fluorescent) with quinacrine. Banding patterns are characteristic of
specific chromosomes, thus allowing for karyotyping.
Chromosome painting. See fluorescent in-situ hybridization (FISH).
Chromotrope 2R. A hydrophilic red acid dye of moderate size that can also
form metal coordination complexes. Very soluble in water; slightly soluble in
ethanol. Used as a cytoplasmic stain, especially in trichrome methods,
including Gomori's one-step trichrome technique. Synonyms: CI 16570, Acid red 29,
Mordant blue 80. Commercial batches have dye contents up to 75%.
Chromoxane cyanine R. This alternative name for eriochrome cyanine R was used in the 8th
and 9th editions of Conn's Biological Stains (1969, 1977) and the dye is sold with this name by
some vendors. The name eriochrome cyanine R was preferred in the 10th edition
of Conn's (2002) because it is the one used in publications about the chemistry, purity and
analytical applications of the dye.
Churukian. Charles J. Churukian (1929–2011) was a member of the Biological
Stain Commission and served as a histological technician in the Pathology Laboratory of the
University of Rochester Medical Center. He wrote several articles in the Journal of
Histotechnology and 11 editions of his Manual of Special Stains were locally published, 1977-
2008. The last edition, available online, includes a long list of recommended expiration times
for solutions of dyes and other stock solutions.
Churukian-Schenk method for argyrophil granules. See Grimelius argyrophil stain.
Citraconic acid or citraconic anhydride. The acid HOOC–CH2–CH(CH3)–COOH
predominates in dilute solutions. An antigen retrieval agent used at elevated temperature, that
removes the formaldehyde adduct from crosslinks. Solutions of "citraconic anhydride" are said
by some to be a universal antigen retrieval method, but may be widely effective simply because
of high temperature and mild alkalinity.
Clearing agent. A solvent used to remove the dehydrant and unbound lipids from a tissue
specimen during tissue processing, and to prepare the specimen for immersion into paraffin
wax; typically an aromatic hydrocarbon (xylene or toluene), an aliphatic hydrocarbon (various
proprietary mixtures), or d-limonene. Clearing agents are also used to remove wax
from sections prior to staining, and to remove the dehyrant from sections prior
to coverslipping with a non-aqueous mounting medium.
Cleland’s reagent. See dithiothreitol.

Cochineal. The dried, sometimes also powdered, bodies of gravid female insects (Dactylopius
coccus, also called Coccus cacti), which contain carminic acid. Synonyms: confusingly, carmine
and carminic acid are used as synonyms, especially in food and cosmetics.
Colchicine. A drug from Colchicum autumnale (autumn crocus). Applied to growing
plant roots or to cultured animal cells, it arrests mitosis in metaphase, allowing the
preparation of smears that display the chromosomes in a contracted state, suitable for
staining to show banding patterns and for karyotyping.
Collagen types. There are at least 30 different structural types of collagen, a few of which are of
histological importance. Type I is the most common, being found in virtually all animal
organs. Type II is in hyaline cartilage. Reticular fibers are a subtype of collagen that includes
immature Type I fibers and thin fibers (Type III) in skin, muscle, lung etc. Basement
membranes contain Type IV, which is not fibrillary and is associated with other proteins
and glycosaminoglycans). Trichrome and Van Gieson stains can show all types of collagen.
Reticular fibers and basement membranes are often demonstrated with silver
methods (see methenamine-silver procedures). With picro–sirius red staining, all types of
collagen are colored red, but only Types I and III are birefringent when examined with
a polarizing microscope.
Collagenases. Proteolytic enzymes (EC 3.4.24) of mammalian cells and bacteria that break
down collagen. They have been used occasionally as histochemical reagents to verify
that staining can be attributed to collagen. Most fixatives other than ethanol make collagen
inaccessible to collagenase. Bacterial collagenases are useful for releasing cells from animal
tissues for subsequent culturing or biochemical studies.
Colloid. A substance composed of either macromolecules or aggregates of smaller
molecules, dispersed in a liquid medium. Sizes of colloidal particles range from 1 to
500 nm. Individual suspended particles as small as 1 nm can be detected with visible
light, but the distinction between one or two particles (resolution) cannot be made
with a conventional light microscope if the size and separation are less than 200 nm.
Colloidal gold. A dispersion of tiny gold particles stabilized by
their affinity for macromolecules. Particle size is determined by conditions for
chemical reduction of the AuCl4
− ion. In aggregate, the smallest gold particles provide
a blue label and the largest particles show as red. Individual particles are clearly
shown by electron microscopy. Antibodies are macromolecules and gold can serve as
a label useful in immunostaining. Colloidal gold particles can also serve as catalytic
sites for amplification by physical development.
Colloidal iron. A method for demonstrating acid mucopolysaccharides.
(glycosaminoglycans and proteoglycans). Ferric chloride is converted to a colloidal suspension
of ferric oxide, whose positively charged particles bind to carboxylate and sulfonate anions in a
manner analogous to basic dyeing. Potassium ferrocyanide is then applied to form Prussian

blue pigment via the ferric-ferrocyanide reaction.
Colloid stabilizer. (1) Synonym for the protective colloids found in physical
developers. (2) Water soluble, high molecular weight compound (for example, agar or
polyvinyl alcohol added to some incubation solutions in enzyme histochemistry to
prevent losses of biopolymers from the cells and tissues.
Color (US), Colour(elsewhere). A quality of sensation of light induced in the eye
by electromagnetic radiation with wavelength in the range 350-800 nm, the color
being determined by the wavelength. Objects or materials that absorb in that complete
range are seen as black. Absorption only of light with lower wavelengths (blue-violet)
shows reflected or transmitted light with longer wavelengths (yellow, orange or red)
and vice versa. Green materials absorb light in the blue and red parts of the spectrum.
See also: dye and fluorescence.
Colour Index (CI). A database jointly maintained by the Society of Dyers and
Colourists (SDC, in the UK) and the American Society of Textile Chemists and
Colorists (ASTCC, in the USA). Information about some 13,000 different dyes and
pigments is available online as the Colour Index International. Each compound has its
unique CI number, which places it in a chemical category, and a unique generic name
that includes indications of the usual industrial method of application and the color.
The mode of synthesis is also given. Common names and trade names are also listed.
For example, CI 26125 or Solvent red 27 is the dye commonly known as oil red O.
The current version of the CI is available only to subscribers who can pay more than
$700 per year. The last printed edition (3rd, 1973, in several volumes) is in libraries.
The Colour Index Heritage Edition is a DVD publication that brings together almost
17500 pages of information published in the three editions of the Colour Index
between 1924 and 1999 prior to the publication of the 4th Edition online.Again, this is
expensive ($800), but may be available via an academic library. The CI database does
not contain much information about fluorescent compounds that are used only as
biological stains.
Common ion effect. A salt becomes less soluble when the concentration of one of its
ions in a solution greatly exceeds that of the other ion.
Concanavalin A (ConA). A lectin extracted from the seeds of Canavalia ensiformis, the jack
bean. Its specific affinity is for α-D-glucosyl and α-D-mannosyl groups, which occur
in glycosaminoglycans and glycoproteins. ConA can be labeled with a fluorochrome or with an
enzyme such as HRP, and was among the first lectins to be used in carbohydrate histochemistry.
Condensation reaction. In organic chemistry, combination of two molecules with
elimination of a small molecule such as water. Contrast with adduct.

Confocal microscopy. A type of fluorescence microscopy that produces a three-dimensional
image from successive planes of focus through the depth of a section, providing superior contrast
and higher resolution than can be obtained from a simple fluorescent image of a whole section.
Glare-free images can be obtained from specimens a hundred times thicker than the usual
sections or cell monolayers. There are various types of confocal microscope. With most, the
section is scanned by a laser with an excitatory wavelength. The filtered fluorescent emissions
from each focal plane are isolated at a pinhole and captured by a digital camera. The whole
process is controlled by a computer and software, which also combine the collected images.
Conformation. For a macromolecule, this is its overall shape. For a protein this stems from
its primary structure (i.e., the amino acid sequence) and the various intra- and inter-molecular
interactions that bend or fold the molecules into higher order 3-dimensional shapes. See
primary, secondary, tertiary and quaternary structure.
Congo red. An acid dye of the disazo class, with large anions. It has been used in
many histological staining methods, as a counterstain for blue stained nuclei, and in
solutions devised to impart more selective coloration to cellulose, collagen fibers,
elastic fibers and neurosecretion products. In recent decades the principal application
has been in specific staining methods for amyloid. Congo red is soluble in water, less
soluble in ethanol. Synonyms: CI 22120, Direct red 28, Congo, Cotton red B,
Kongoröt. Commercial lots are available certified by the Biological Stain
Commission.
Congo red for amyloid. Various formulations of the solution exist, but the most reliable
and selective is Puchtler's alkaline Congo red.
Conjugated bond number. Numerical structure parameter describing the extent of
conjugation within a stain or staining reagent by a count of the number of conjugated
bonds in a molecule. Usual abbreviation: CBN. Dyes vary widely regarding CBN
values. Small molecules such as methylene blue and picric acid both have values of
16, whereas large molecules such as alcian blue and sirius red F3B have values of 48
and 64, respectively.
Conjugated bonds, conjugated system. A chain of atoms linked by alternating
single and double covalent bonds in which spatial localization of all the bonding
electrons is not possible. For instance, the benzene carbon skeleton is conventionally
drawn as comprising three carbon–carbon single bonds plus three carbon–carbon
double bonds. Because the double bonds are conjugated (alternating), all six bonds are
identical, due to the delocalized π-electrons. Such delocalized electrons are mobile,
and electrical influences are readily propagated from one part a conjugated molecule
to another, enhancing dipoles and polarizability, and favoring van der Waals forces.

Conn. Harold J Conn (1886–1975). A senior figure in the Society of American
Microbiologists, based at the New York State Agricultural Laboratory in Geneva NY. He was
part of the group whose efforts, in the early 1920s, established the Biological Stain
Commission as a focus of efforts to standardize dyes used as biological stains.
Conn’s Biological Stains. A book documenting the properties and uses of dyes and other
colorants, including fluorochromes and pigments, used in the biological and medical sciences.
The first seven editions (1925-1961), entitled Biological Stains, were written by HJ Conn.
Conn's name was added to the titles of later editions: the eighth (1969) and ninth (1977) by RD
Lillie and the tenth (2002) edited by RW Horobin and JA Kiernan, with chapters by the editors
and 8 other authors.
Coomassie brilliant blue dyes (G250, R250). Two similar aminotriphenylmethane acid
dyes with large, hydrophilic anions. The one more commonly encountered is coomassie
brilliant blue R250, CI 42660, Acid blue 83, also known as brilliant blue R, brilliant
indocyanine 6B and kenacid blue R. A major application is in the FRAME cytotoxicity assay.
The dye is also used as a selective stain for proteins in sections of tissues and in cultured
cells. Coomassie brilliant blue G250 is CI 42655, Acid blue 90, also known as brilliant blue
G250. It is less soluble in water than coomassie brilliant blue R250, and is used in the Bradford
protein assay, which is based on a change in absorption maximum of an acidified solution from
470 nm to 595 nm for protein-bound dye. Both dyes are also used to stain separated proteins in
electrophoretic gels; R250 is the more sensitive for this purpose.
Coulombic forces. Electrical attractions and repulsions due to positively and
negatively charged species in tissues and stain molecules, such as –NH3
+ and –SO3¯.
Named from the coulomb, the SI unit for a quantity of electricity: 1.036×10−5 moles
of protons or 6.242×1018 electrons. Also called electrostatic forces.
Counterstain. A staining step carried out to provide a contrasting background to the staining of
some specific structure or component. Use of such a process is termed counterstaining.
Covalent bond. Link between two atoms resulting from the sharing of, usually, an
electron pair as in a C–C single σ (sigma) bond. Double bonds involve sharing two
pairs of electrons, for instance C=O and C=C. Although drawn as equal, one electron
pair form a σ (sigma) bond while the second pair constitute a π (pi) bond in which the
electrons are more loosely held between the two atoms than in the first pair.
Coverslip. A thin piece of glass (rarely plastic) that fits over a tissue section on a
microscope slide to prevent damage to the specimen. A coverslip is glued down
with mounting medium. Coverslipping describes the act of applying a coverslip to a
slide, either by hand or through the use of a coverslipping machine.
Cresyl violet. This name has been used for at least three basic dyes of the oxazine series with the
same ring structure but different attached amino and methyl groups. These dyes bind

to basophilic materials; applied from suitably acidified solutions, they strongly stain cell nuclei
and Nissl substance (rRNA in the cell bodies of neurons). Most dyes sold as cresyl violet are
soluble in water and in ethanol. Dyes sold as cresyl violet perchlorate are not soluble enough to
be used as biological stains. Synonyms: cresyl echt violet, cresyl fast violet, cresyl violet acetate.
Names of commercial products generally do not correspond to chemical entities. These dyes do
not have Colour Index numbers and names. Commercial lots designated as cresyl violet acetate
are available certified by the Biological Stain Commission.
Cresyl violet for Nissl substance. An acidified cresyl violet solution which demonstrates Nissl
substance (RNA) in the cytoplasm of neurons by basic dyeing. Cresyl violet is frequently used as
a counterstain after Luxol fast blue has been used to stain myelin in sections of brain or spinal
cord.
Crocein scarlet. An acid dye in the disazo class, with colored anions of moderate size, used in
the Movat pentachrome stain. It is soluble in water and ethanol. Synonyms: CI 27290, Acid red
73, woodstain scarlet.
Crosslink. A connection or bridge involving covalent bonds, between polymeric
chains or lower molecular weight molecules. (1) Generated by fixative agents, e.g.
crosslinking of proteins by formaldehyde or glutaraldehyde. Unsaturated lipids can
be crosslinked by osmium tetroxide. (2) Present in native proteins, most usually as
disulfide bridges within or between polypeptide chains. (3) Occurring in some plastic
embedding media after polymerization of a monomer.
Cryofixation. Rapid plunging of very small tissue specimens or monolayers of cells into
isopentane, liquid ethane or liquid propane cooled with liquid nitrogen. This results in temporary
stabilization (not fixation) of the specimen's macromolecules. Direct immersion into liquid
nitrogen is less effective because a layer of gaseous nitrogen forms around the immersed object
and delays freezing.
Cryogenic spray. A gas (usually freon or carbon dioxide) under pressure in a can, that, when
sprayed onto a specimen, causes freezing due to evaporative cooling. Used in cryotomy.
Cryoprotectant. A substance used to minimize damage due to formation of ice crystals when
tissues or cells are frozen. Sucrose (15-30% in water or a buffer) is commonly used for fixed
animal tissues intended for cryotomy. Dimethylsulfoxide or glycerol is the preferred additive for
cultures or suspensions of cells.
Cryosection. A frozen section produced on a cryostat. See also tissue section.
Cryostat. A microtome mounted in a freezing cabinet, used to produce frozen
sections (see cryotomy).
Cryotomy. The production of tissue sections from frozen specimens using a cryostat.

The advantage of frozen sections is that slides can be produced in minutes rather than
hours, often while the patient is still in the surgical suite. Typically used for biopsies.
See also Mohs surgery.
Crystal violet. A moderately large basic dye, of the aminotriarylmethane class, used as an
antiseptic and in a variety of staining methods for animal and plant tissues, notably the Gram
stain for bacteria. Crystal violet is soluble in water and much more soluble in ethanol.
Synonyms: CI 42555, Basic violet 3, gentian violet (in USA only; elsewhere this name refers
to methyl violet), hexamethyl pararosaniline, methyl violet 10B. Commercial lots are
Curcumin. A hydrophobic polymethine acid dye (CI 75300, Natural yellow 3) from the turmeric
plant (Curcuma longa) used as a pH indicator, changing color from yellow to red in the pH range
of 7.4–8.6, and then from violet to orange in the pH range of 10.2–11.8. The dye is poorly
soluble in water, freely so in organic solvents. It has also been used for staining tissue sections,
as a substitute for eosin. Curcumin is fluorescent (blue excitation, green emission), and has been
shown to enter the endoplasmic reticulum and lysosomes when used as a vital stain for cultured
cells.
Cyanine dyes. A group of dyes characterized by one or more C–(C=C–) (methine) groups
between two aromatic rings, one of which acts as an electron donor and the other as an electron
acceptor. Most are used as fluorescent probes. See also DiD, DiI, DiO and merocyanine 540.
Cyanoacrylate. An ester of cyanoacrylic acid, such as ethyl-2-cyanoacrylate,
CH2=C(CN)COOC2H5, the principal ingredient of adhesives with names like crazy-
glue and super-glue. Cyanoacrylates polymerize rapidly when exposed to traces of
moisture. The polymerized glue is insoluble in water but (fortunately) soluble in many
organic solvents. Used to stick specimens to the chuck of a vibrating microtome.
Cysteic acid method for cystine. Cystine (as in keratin or neurosecretory material in the
pituitary gland) is oxidized with performic acid or acidified potassium permanganate to cysteic
acid, which is then stained with alcian blue at pH<1.
Cytocentrifuge. Mechanical device used for preparing uniform layers of peripheral
blood cells and other cell suspensions. Synonym: spinner.
Cytochemistry. Strictly speaking, the body of techniques used to identify the chemical nature of
cells in smears, although it is often used as a synonym for histochemistry.
Cytology. The microscopic study of cells, typically in smears as opposed to sections (histology).
DAB. See Diaminobenzidine.
Dansyl chloride. A fluorescent compound (UV excitation, 372 nm; blue emission, 429 nm, in

chloroform) that forms covalent bonds with amines, especially lysine. It is used to label proteins,
especially antibodies for use in immunohistochemistry.
DAPI. 4’,6-diamidino-2-phenylindole dichloride. A cationic fluorochrome (344 nm excitation,
450 nm emission, in water) that provides a selective stain for DNA. It is much used as
a counterstain for nuclei in immunofluorescence preparations. DAPI can also serve as
a probe for cells connected by gap junctions.
Darrow red. A small basic dye of the oxazine series that binds to basophilic materials. Applied
from a suitably acidified solution, it stains both DNA and Nissl substance (rRNA in the cell
bodies of neurons). It was introduced in 1960 and is named for Mary A. Darrow, the technologist
in charge of the Biological Stain Commission's laboratory from 1926 to 1959. The dye dissolves
slowly in water (heating needed) and is poorly soluble in ethanol. Commercial lots are
DASPI. A fluorescent vital stain (blue excitation, 344 nm; yellow emission, 605nm, in water)
used as a probe for mitochondria in living cells. Synonyms: DASPMI,
dimethylaminostyrylmethylpyridinium chloride.
Debye forces (dipole-induced dipole forces). A type of van der Waals attraction in
which a polar group or molecule induces a dipole moment in the conjugated
system of an adjacent non-polar entity.
Decalcification. Removal of calcium salt deposits from tissue specimens prior
to microtomy. Reagents used include formic, hydrochloric and nitric acids, and
the chelating agent EDTA (ethylenediaminetetraacetic acid).
Deglycosylatedavidin. The protein avidin with its carbohydrate components enzymatically
removed, reducing the MW from 70,000 to 60,000. The biotin-binding properties are unchanged,
but the modified protein is much less prone to nonspecific binding to tissue components that do
not contain biotin-labeled antibodies. See also streptavidin.
Dehydrant, dehydrating agent. A solvent, such as methanol, ethanol, isopropanol or glycol
ether, that replaces water in a specimen by diffusion; or a ketal, that chemically reacts with
water. See also dehydration.
Dehydration. In histotechnology, the removal of water from a tissue sample or section by
passing it through a series of solvents of increasing hydrophobicity (e.g., 70%, 80%, 95% and
100% ethanol). The gradual removal of water minimizes shrinkage. The most commonly used
dehydrants are ethanol and isopropanol. An alternative method is chemical dehydration by
immersing specimens in acidified 2,2-dimethoxypropane (DMP). This liquid reacts with water
as it penetrates the tissue, producing methanol and acetone. Following dehydration by either
method a dehydrated specimen or section is usually moved into a clearing agent. See also over-
dehydration.

Dehydrogenases. A class of oxidoreductase enzymes that remove protons from a substrate.
These enzymes are typically demonstrated with tetrazolium salts, which are reduced to insoluble
colored formazans.
Delafield's hematoxylin. A regressive version of hemalum containing hematoxylin and
aluminium ammonium sulfate, as well as water and ethanol as solvents and glycerol as a
stabilizer to prevent over-oxidation. The solution is oxidized slowly by air and sunlight over a
period of months.
Delocalized π-electrons. Pi-electrons, which are shared by more than two atoms and
therefore cannot be said to form part of any individual covalent bond. π-electrons are
associated with double or triple bonds, and with resonant structures such as aromatic
rings.
Denaturant. Something which can cause denaturation of proteins. For instance heat,
organic compounds such as ethanol and urea, and chemically reactive compounds
such as dichromates or formaldehyde. Fixatives are typically denaturants.
Denaturation. Destruction of secondary (or higher level) organization
of biopolymers, typically proteins. In this latter case hydrophobic amino acid residues
become exposed on the molecular surface, leading to insolubility and aggregation.
Denaturation can be achieved by heating or by using a wide variety of
chemical denaturants. Denaturation usually reduces enzymic activity of proteins and
may diminish antigenicity, although some denaturant fixatives (e.g., glyoxal) actually
protect antigenicity. Curiously, heat (boiling buffered water)
can renature macromolecules to the point where lost antigenicity is restored
(see heat-induced epitope retrieval).
DEPC (diethyl pyrocarbonate). A reagent used to decontaminate glassware and water from
trace amounts of RNase. It combines with histidine, lysine, cysteine and tyrosine, thereby
inactivating the enzyme.
Dialysis. Passage of small, but not large, molecules through a membrane. Also
technique for concentrating solutions of proteins (or other macromolecular
substances) using tubing that is permeable only to small molecules.
Diaminobenzidine (DAB). A polyamino primary aromatic amine. The free base is
only slightly soluble in water; the tetrachloride dissolves easily. Oxidation of DAB,
which can be catalysed by the enzyme peroxidase, gives rise to an insoluble brown
polymeric pigment.
Di-8-ANEPPS. A zwitterionic probe that binds to the outer leaflet of the cell membrane. Used as
a vital stain that fluoresces (green excitation, red emission) in response to electrical changes in

membrane potential. Synonyms: dioctylaminonaphthylethylenepyridiniumpropyl
sulfonate; pyridinium, 4-[2-[6-(dioctylamino)-2-naphthalenyl]ethenyl]-1-(3-sulfopropyl)-, inner
salt.
Diastase. A mixture of amylases, usually extracted from pancreas or from malt. Also an obsolete
synonym for α-amylase.
Dichlorofluorescin diacetate. A colorless lipophilic compound formed by reduction of
2,7-dichlorofluorescein diacetate. It can enter cells, where the acetate groups are removed by the
action of cytoplasmic esterases. If reactive oxygen species (such as superoxide ions, singlet
oxygen or hydroxyl radicals) are being formed in the cell, the hydroxyxanthene structure is
restored by oxidation, generating 2,7-dichlorofluorescein, a hydrophilic anionic dye that is
also fluorescent. Synonyms: 2,7-dichlorodihydrofluorescin diacetate, H2DCFDA. Some authors,
misleadingly, describe this compound as a fluorescein.
DiD. One of a number of cationic cyanine dyes. Like DiI and DiO, it is very lipophilic and
serves as a fluorescent probe for membranes in living and fixed cells.
Differential stain. A solution of variously colored dyes with selective affinities for different
tissue components (e.g., Romanowsky stains, trichrome mixtures.
Differentiation. In histotechnical usage: controlled de-staining of a stained section.
Digitonin. An amphiphilic steroid glycoside extracted from seeds of Digitalis
purpurea (foxglove). It is soluble in ethanol and ethanol-water mixtures but almost insoluble in
chloroform, ether and water. Digitonin is used to solubilize lipids in an aqueous environment,
specifically to permeabilize cell membranes. With cholesterol, but not with cholesterol esters,
digitonin forms an adduct that is insoluble in acetone; it can be used to insolubilize cholesterol in
specimens prior to either histological processing or histochemical staining of frozen sections by
the perchloric acid-naphthoquinone method.
Digoxigenin. A steroid made by hydrolysis of the drug digoxin, from Digitalis lanata (white
foxglove). It can serve as a hapten, with high antigenicity. When conjugated in vivo to
deoxyuridine triphosphate it is incorporated into oligonucleotides for use in in situ
hybridization or in situ nick translation techniques. The bound digoxigenin is then
detected immunohistochemically, with an anti-digoxigenin antibody.
DiI. One of a number of Cationic cyanine dyes. Like DiD and DiO, it is very lipophilic and
Dimer. A compound formed by the union of two identical molecules.
2,2-Dimethoxypropane (DMP). A liquid ketal that, with acid catalysis, reacts with water; the
products are methanol and acetone. DMP is miscible with dehydrants and with clearing agents.
A specimen can be chemically dehydrated by immersion in a sufficient volume of acidified

DMP, and then processed for embedding in either paraffin or a plastic embedding medium. See
also dehydration.
Dimethylglyoxime. A chelating agent giving rise to insoluble, brightly colored metal
coordination complexes with several metal ions, including nickel.
Dimethyl sulfoxide (USA) or dimethyl sulphoxide (elsewhere). Often a single
word, dimethylsulfoxide. See DMSO.
DiO. One of a number of Cationic cyanine dyes. Like DiD and DiI, it is very lipophilic and
Dipole. The occurrence of a partial negative charge on the most electronegative atom
of a covalently linked pair, with the other atom carrying a partial positive charge. For
instance, in a nitro group (–NO2) the oxygen atoms are more electronegative than the
nitrogen atom, and thus the former carry partial negative charges. An entire molecule
may serve as a dipole, with one end carrying a partial positive charge and the other
end having a partial negative charge, as in many dyes.
Dipole-dipole interactions. See Keesom forces.
Dipole-induced dipole forces. See Debye forces.
Dipole moment. A dipole property: the product of the magnitude of the charge on
the electronegative atom, and the distance between the electronegative
and electropositive atoms; the unit of measure is the Debye.
Direct dyeing. A textile dyeing term, describing the coloration of cotton textiles using
large, planar, hydrophilic acid dyes, which “directly” bind to the fibers. In the Colour
Index these dyes fall into the CI Direct dye application class. Historically such
“direct” dyes were so named to distinguish them from colorants that only colored
cotton following pre-treatment with a metal salt or tannin.
Disazo (also bisazo). A word indicating that an azo dye has two azo groups (−N=N−)
in its structural formula.
Dispersion forces. See London forces.
Dithiothreitol. A reducing agent used to either break disulphide groups in proteins or to prevent
their formation, which can cause crosslinking of proteins. It is an ingredient of DTT-Carbowax,
a transport medium, and has also been used in conjunction with histochemical methods
that demonstrate cysteine and cystine in proteins. Synonyms: Cleland's reagent,

dithioerythritol, DTT.
DMSO. Abbreviation for dimethyl sulfoxide, (CH3)2SO. A polar solvent that is incapable
of hydrogen bonding (aprotic) but is effective in solubilizing polar and nonpolar substances, and
is miscible with water and most common solvents. It has been used as the solvent
for Romanowsky stains and as a cryoprotective agent. DMSO is colorless, odorless, of low
toxicity, and melts at 180C.
DNase (deoxyribonuclease). A family of enzymes that cleave DNA at phosphate ester linkages,
yielding small, soluble oligonucleotides. A DNase can be used to remove DNA from a tissue
section. A much milder treatment is used to provide positive controls in ISEL techniques for
detecting apoptosis.
DTT-Carbowax. A transport medium used to transport specimens containing unfixed cells
from a clinic to a laboratory for diagnostic cytology. It consists of 3% Carbowax 1540 in 60%
ethanol, a stable solution, to which DTT (2 g/100 ml) is added immediately before use. The DTT
serves to suppress crosslinking of glycoproteins, which would increase the viscosity of mucus in
the specimen.
Dye. An organic ion or molecule that can absorb visible light (and so is seen as
colored), that can attach to and impart color to other materials. Absorption of light is
due to the chemical bonds forming an extended conjugated system.
Dyeing. See staining.
EDTA. A reagent that binds metal ions through chelation in a soluble form. In histology it is
used to decalcify bone, and also in some antigen retrieval techniques. The disodium salt,
Na2EDTA, is the form usually used in histotechnology. Another common use of EDTA is
to prevent coagulation of samples of blood.
Synonyms: edathamil, ethylenediaminetetraacetic acid, (ethylenedinitrilo)tetraacetic
acid, sequestrene, versene.
Ehrlich. Paul Ehrlich (1854–1915) trained in four different medical schools, where he was
considered a mediocre student. Ehrlich’s major achievements in stain technology were made in
his early years, when he was the first investigator to appreciate the differences between acid
dyes and basic dyes for staining biological material. He also prepared the first neutral dye, albeit
it not a Romanowsky stain.
Elastin. The principal protein of mature elastic fibers in connective tissues and elastic
laminae of arteries; an amorphous, hydrophobic protein composed of abundantly
crosslinked polypeptide chains. Staining of elastin is by hydrophobic
interactions between dye and substrate, with the dye bonding to elastin through van
der Waals forces. Staining procedures include aldehyde-fuchsine, orcein, Verhoeff's
stain and Weigert's resorcin-fuchsine.

Electronegative, electronegativity. The tendency of an atom to attract an electron.
For instance, a chlorine atom is very electronegative, so Cl− ions are stable. However,
if the attractive tendency is very low the atom is termed electropositive, and such
atoms can lose electrons to form stable cations, e.g. the Na+ ion.
Electron microscope (EM). A microscope that uses a directed beam of electrons rather than
light (photons) to visualize the ultrastructure of specimens. Resolution can be up to 5,000 times
that of the best light microscope. A transmission EM is used for ultrathin (30-60 nm) sections of
plastic-embedded specimens. A scanning EM is for examining fine details of surfaces.
Electron microscopy (EM). The study of the ultrastructure of specimens, biological or
otherwise, using an electron microscope.
Electropositive, electropositivity. See electronegative.
Electrostatic forces. These attract electrons (negative) towards nuclei of atoms
(positive). Thus, cations are attracted to anions. Also called coulombic forces.
ELISA (Enzyme-linked immunosorbent assay). A variety of sensitive techniques that detect
tiny amounts of proteins or other antigens in serum, urine, tissue culture media etc. The
substance to be detected is immobilized (adsorbed) onto a prepared surface and then made
visible by a method similar to immunohistochemistry, using a secondary antibody labeled with
an enzyme (often HRP or alkaline phosphatase) to produce a colored product.
Embedding. Cells and tissues are immersed in a liquid, such as molten paraffin wax
or the monomer of a plastic embedding medium. After the liquid is solidified, by
freezing or polymerization respectively, the embedded specimen is interpenetrated
and supported by a solid matrix, the embedding medium.
Emission. The wavelength of light emitted by a fluorescent substance.
Enantiomers. Isomers with three-dimensional structures that are mirror images.
Enzyme histochemistry. The demonstration of enzymes in cells and tissues by
utilizing the catalytic activities of these biopolymers. Specimens are immersed
in enzyme substrates which are enzymatically converted – directly or indirectly – into
colored final reaction products, marking the enzyme’s site. In the case of indirect
conversion, intermediate reaction products are transformed into final reaction products
by reaction with visualization agents.
Enzyme label. These may be visualized using the simple, reliable methods of enzyme

histochemistry. Horseradish peroxidase (HRP) and alkaline phosphatase are the most
popular labels of this type. Usually the final reaction product is black, brown or blue,
and is viewed by ordinary bright-field microscopy.
Enzyme retrieval. A type of antigen retrieval utilizing a proteolytic enzyme (and sometimes
adjuncts like calcium to improve enzyme activity) that opens access to masked epitopes; it is not
effective against epitopes directly changed by fixation.
Enzyme substrate. The compound acted upon by a particular enzyme. See enzyme
histochemistry.
Eosin B. A moderately large, hydrophilic dibromodinitrofluorescein (i.e. xanthene) acid dye.
Sometimes used as the counterstain in Romanowsky stains, and originally specified as such for
the Leishman and Wright variants. The dye is soluble in water and ethanol. Synonyms: 45400,
Acid red 91. Commercial lots are available certified by the Biological Stain Commission.
Eosin Y. A moderately large tetrabromofluorescein (i.e. xanthene) acid dye. This dye is widely
used in histology, notably in the hematoxylin and eosin, Papanicolaou and Romanowsky stains.
Also used to stain various acidophilic structures such as eosinophil granules and Negri bodies;
and as a cytoplasmic counterstain in various procedures, such as silver stains. Eosin Y is soluble
in water, but poorly soluble in alcohol except under acidic conditions. Synonyms: CI 45380,
Acid Red 87, eosin. Commercial lots are available in a fairly pure form certified by
Epitope. The part of an antigen molecule to which an antibody molecule attaches.
Typically an epitope consists of 5 or 6 amino acids, either in sequence (a linear
epitope), or brought into proximity by the folding of a polypeptide chain into the
conformation of a protein molecule (a discontinuous epitope).
Epitope retrieval. See antigen retrieval.
Eriochrome cyanine R. An anionic hydroxytriarylmethane dye with a non-planar conjugated
system. It is freely soluble in water and alcohol and is a pH indicator: the red aqueous solution
changes to blue at pH 11-12 or to yellow at pH 1-2. The dye forms colored coordination
complexes with metals, including aluminum, chromium and iron. An acidic solution of the dye
and a ferric salt is useful as a blue stain either for nuclei (instead of the hemalum in
routine H&E for histopathology) or for myelin (instead of luxol fast blue in the Kluver and
Barrera and similar techniques). Synonyms: CI 43820, Mordant blue 3, chromoxane cyanine R,
solochrome cyanine R. This dye still has many other trade names and industrial uses. It can be an
inexpensive substitute for hematoxylin. The Biological Stain Commission tests
and certifies batches that meet its criteria for identification, dye content and staining
performance.
Erythrosin B. A large tetraiodofluorescein (i.e. xanthene) acid dye, whose major species at
neutral and alkaline pH is a lipophilic anion. Despite an extensive range of reported applications

Glossary of staining methods, reagents, immunostaining, terminology and eponyms

Glossary of staining methods, reagents, immunostaining, terminology and eponyms

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Glossary of staining methods, reagents, immunostaining, terminology and eponyms