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Hematoxylin and eosin stain ( or haematoxylin and eosin stain or hematoxylin-eosin stain; often abbreviated as H&E stain or HE stain) is one of the principal tissue stains used in
histology Histology, also known as microscopic anatomy or microanatomy, is the branch of biology which studies the microscopic anatomy of biological tissue (biology), tissues. Histology is the microscopic counterpart to gross anatomy, which looks at large ...

histology
. It is the most widely used stain in
medical diagnosis Medical diagnosis (abbreviated Dx, Dx, or Ds) is the process of determining which disease or condition explains a person's symptoms and medical sign, signs. It is most often referred to as diagnosis with the medicine, medical context being implic ...
and is often the
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. For example, when a
pathologist Pathology is the study of the causesCauses, or causality, is the relationship between one event and another. It may also refer to: * Causes (band), an indie band based in the Netherlands * Causes (company), an online company See also * Ca ...

pathologist
looks at a
biopsy A biopsy is a medical test A medical test is a medical procedureA medical procedure is a course of action intended to achieve a result in the delivery of healthcare. A medical procedure with the intention of determining, measuring, or diagno ...

biopsy
of a suspected
cancer Cancer is a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. These contrast with benign tumor A benign tumor is a mass of cells Cell most often refers to: * Cell (biolo ...

cancer
, the
histological section Histology, also known as microscopic anatomy or microanatomy, is the branch of biology Biology is the natural science that studies life and living organisms, including their anatomy, physical structure, Biochemistry, chemical processes, Mol ...
is likely to be stained with H&E. H&E is the combination of two histological stains:
hematoxylin Haematoxylin or hematoxylin (), also called natural black 1 or C.I. 75290, is a compound extracted from heartwood Wood is a porous and fibrous structural tissue found in the stems and roots of trees and other woody plant A woody pla ...

hematoxylin
and
eosin Eosin is the name of several fluorescent light. Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a longer ...

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

nuclei
a purplish blue, and eosin stains the
extracellular matrix In biology Biology is the natural science that studies life and living organisms, including their anatomy, physical structure, Biochemistry, chemical processes, Molecular biology, molecular interactions, Physiology, physiological mechanisms, ...
and
cytoplasm In cell biology Cell biology (also cellular biology or cytology) is a branch of biology Biology is the natural science that studies life and living organisms, including their anatomy, physical structure, Biochemistry, chemical processes ...
pink, with other structures taking on different shades, hues, and combinations of these colors. Hence a pathologist can easily differentiate between the nuclear and cytoplasmic parts of a cell, and additionally, the overall patterns of coloration from the stain show the general layout and distribution of cells and provides a general overview of a tissue sample's structure. Thus, pattern recognition, both by expert humans themselves and by software that aids those experts (in
digital pathologyDigital pathology is a sub-field of pathology that focuses on data management based on information generated from Digitization, digitized specimen slides. Through the use of computer-based technology, digital pathology utilizes virtual microscopy. Gl ...
), provides histologic information. This stain combination was first introduced in 1876 by A. Wissowzky.


Uses

The H&E staining procedure is the principal stain in histology in part because it can be done quickly, is not expensive, and stains tissues in such a way that a considerable amount of
microscopic anatomy Histology, also known as microscopic anatomy or microanatomy, is the branch of biology Biology is the natural science that studies life and living organisms, including their anatomy, physical structure, Biochemistry, chemical processes, Mol ...
is revealed, and can be used to diagnose a wide range of
histopathologic Histopathology (compound of three Greek words: ''histos'' "tissue", πάθος ''pathos'' "suffering", and -λογία '' -logia'' "study of") refers to the microscopic The microscopic scale (from , ''mikrós'', "small" and σκοπέω, ''skop ...
conditions. The results from H&E staining are not overly dependent on the chemical used to fix the tissue or slight inconsistencies in laboratory protocol, and these factors contribute to its routine use in histology. H&E staining does not always provide enough contrast to differentiate all tissues, cellular structures, or the distribution of chemical substances, and in these cases more specific stains and methods are used.


Method of application

There are many ways to prepare the hematoxylin solutions (formulation) used in the H&E procedure, in addition, there are many laboratory protocols for producing H&E stained slides, some of which may be specific to a certain laboratory. Although there is no standard procedure, the results by convention are reasonably consistent in that cell nuclei are stained blue and the
cytoplasm In cell biology Cell biology (also cellular biology or cytology) is a branch of biology Biology is the natural science that studies life and living organisms, including their anatomy, physical structure, Biochemistry, chemical processes ...
and
extracellular matrix In biology Biology is the natural science that studies life and living organisms, including their anatomy, physical structure, Biochemistry, chemical processes, Molecular biology, molecular interactions, Physiology, physiological mechanisms, ...
are stained pink. Histology laboratories may also adjust the amount or type of staining for a particular pathologist. After tissues have been collected (often as
biopsies A biopsy is a medical test A medical test is a medical procedure performed to detect, diagnose, or monitor diseases, disease processes, susceptibility, or to determine a course of treatment. Medical tests such as, physical and visual exams, ...
) and fixed, they are typically dehydrated and embedded in melted
paraffin wax Paraffin wax (or petroleum wax) is a soft colorless solid derived from petroleum Petroleum, also known as crude oil and oil, is a naturally occurring, yellowish-black liquid A liquid is a nearly incompressible fluid In physics, ...

paraffin wax
, the resulting block is mounted on a
microtome A microtome (from the Greek ''mikros'', meaning "small", and ''temnein'', meaning "to cut") is a cutting tool In the context of machining truck of the US Army with machinists working on automotive parts Machining is a process in which a materi ...
and cut into thin slices. The slices are affixed to microscope slides at which point the wax is removed with a solvent and the tissue slices attached to the slides are rehydrated and are ready for staining. Alternatively, H&E stain is the most used stain in
Mohs surgery Mohs surgery, developed in 1938 by a general surgeon In modern medicine, a surgeon is a physician who performs surgery. There are also surgeons in podiatry, dentistry, orthodontics, and veterinary medicine. History The first person to documen ...
in which tissues are typically frozen, cut on a
cryostat A cryostat (from ''cryo'' meaning cold and ''stat'' meaning stable) is a device used to maintain low cryogenic A medium-sized dewar is being filled with liquid nitrogen by a larger cryogenic storage tank In physics Physics is the na ...

cryostat
(a microtome that cuts frozen tissue), fixed in alcohol, and then stained. The H&E staining method involves application of
haematoxylin Haematoxylin or hematoxylin (), also called natural black 1 or C.I. 75290, is a compound Compound may refer to: Architecture and built environments * Compound (enclosure), a cluster of buildings having a shared purpose, usually inside a fen ...

haematoxylin
mixed with a metallic salt, or
mordant A mordant or dye fixative is a substance used to set (i.e. bind) dye A dye is a color Color (American English) or colour (Commonwealth English) is the visual perception, visual perceptual Physical property, property correspondin ...

mordant
, often followed by a rinse in a weak acid solution to remove excess staining (''differentiation''), followed by ''bluing'' in mildly
alkaline In chemistry, an alkali (; from ar, القلوي ''al-qaly'' "ashes of the saltwort") is a base (chemistry), basic, ionic compound, ionic salt (chemistry), salt of an alkali metal or an alkaline earth metal. An alkali can also be defined as ...

alkaline
water. After the application of haematoxylin, the tissue is
counterstain A counterstain is a stain A stain is a discoloration that can be clearly distinguished from the surface, material, or medium it is found upon. They are caused by the chemical or physical interaction of two dissimilar materials. Accidental stai ...
ed with eosin (most commonly
eosin Y Eosin Y also called C.I. 45380 or C.I. Acid Red 87, is the form of eosin Eosin is the name of several fluorescent light. Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation ...

eosin Y
).


Results

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

nuclei
of
cells Cell most often refers to: * Cell (biology), the functional basic unit of life Cell may also refer to: Closed spaces * Monastic cell, a small room, hut, or cave in which a monk or religious recluse lives * Prison cell, a room used to hold peopl ...
blue or dark-purple, along with a few other tissues, such as
keratohyalin Keratohyalin is a protein structure found in cytoplasmic granules of the keratinocyte Keratinocytes are the primary type of Cell (biology), cell found in the epidermis (skin), epidermis, the outermost layer of the skin. In humans, they constit ...
granules and
calcified Calcification is the accumulation of calcium salts in a Tissue (biology), body tissue. It normally occurs in the formation of bone, but calcium can be deposited abnormally in soft tissue,Miller, J. D. Cardiovascular calcification: Orbicular origi ...
material. Eosin stains the
cytoplasm In cell biology Cell biology (also cellular biology or cytology) is a branch of biology Biology is the natural science that studies life and living organisms, including their anatomy, physical structure, Biochemistry, chemical processes ...
and some other structures including
extracellular matrix In biology Biology is the natural science that studies life and living organisms, including their anatomy, physical structure, Biochemistry, chemical processes, Molecular biology, molecular interactions, Physiology, physiological mechanisms, ...
such as
collagen Collagen () is the main structural protein Proteins are large biomolecule , showing alpha helices, represented by ribbons. This poten was the first to have its suckture solved by X-ray crystallography by Max Perutz and Sir John Cowder ...

collagen
in up to five shades of pink. The
eosinophilic Eosinophilic (Greek suffix -phil- A philia is the love or Fixation (psychology), obsession with a particular thing or subject. The suffix -philia is used to specify the love or obsession with something more specific. It is antonymic to -phobia. ...
(substances that are stained by eosin) structures are generally composed of intracellular or extracellular
protein Proteins are large biomolecule , showing alpha helices, represented by ribbons. This poten was the first to have its suckture solved by X-ray crystallography by Max Perutz and Sir John Cowdery Kendrew in 1958, for which they received a No ...

protein
s. The Lewy bodies and Mallory bodies are examples of eosinophilic structures. Most of the
cytoplasm In cell biology Cell biology (also cellular biology or cytology) is a branch of biology Biology is the natural science that studies life and living organisms, including their anatomy, physical structure, Biochemistry, chemical processes ...
is eosinophilic and is rendered pink.
Red blood cell Red blood cells (RBCs), also referred to as red cells, red blood corpuscles (in humans or other animals not having nucleus in red blood cells), haematids, erythroid cells or erythrocytes (from Greek language, Greek ''erythros'' for "red" and ''k ...

Red blood cell
s are stained intensely red.


Mode of action

Although
hematein Hematein (US spelling) or haematein is an oxidized derivative of haematoxylin Haematoxylin or hematoxylin (), also called natural black 1 or Colour Index International, C.I. 75290, is a chemical compound, compound extracted from wood#Heartwood ...

hematein
, an
oxidized (mild reducing agent) are added to powdered potassium permanganate (strong oxidizing agent), a violent redox reaction accompanied by self-ignition starts. Redox (reduction–oxidation, pronunciation: or ) is a type of chemical reaction A ...

oxidized
form of hematoxylin, is the active colorant (when combined with a mordant), the stain is still referred to as ''hematoxylin''. Hematoxylin, when combined with a mordant (most commonly aluminum
alum An alum () is a type of chemical compound A chemical compound is a chemical substance composed of many identical molecules (or molecular entity, molecular entities) composed of atoms from more than one chemical element, element held together ...

alum
) is often considered to "resemble" a basic, positively charged, or
cationic An ion () is a particle In the Outline of physical science, physical sciences, a particle (or corpuscule in older texts) is a small wikt:local, localized physical body, object to which can be ascribed several physical property, physical or ...
stain. Eosin is an
anionic An ion () is an atom An atom is the smallest unit of ordinary matter In classical physics and general chemistry, matter is any substance that has mass and takes up space by having volume. All everyday objects that can be touched are ...
(negatively charged) and acidic stain. The staining of nuclei by hemalum (a combination of aluminum ions and hematein) is ordinarily due to binding of the dye-metal complex to DNA, but nuclear staining can be obtained after extraction of DNA from tissue sections. The mechanism is different from that of nuclear staining by basic (cationic) dyes such as or
toluidine blue Toluidine blue, also known as TBO or tolonium chloride (International Nonproprietary Name, INN) is a blue cationic (basic) dye used in histology and sometimes clinically. Test for lignin Toluidine blue solution is used in testing for lignin, a co ...
. Staining by basic dyes occurs only from solutions that are less acidic than hemalum, and it is prevented by prior chemical or enzymatic extraction of nucleic acids. There is evidence to indicate that co-ordinate bonds, similar to those that hold aluminium and hematein together, bind the hemalum complex to DNA and to carboxy groups of proteins in the nuclear
chromatin Chromatin is a complex of DNA Deoxyribonucleic acid (; DNA) is a molecule File:Pentacene on Ni(111) STM.jpg, A scanning tunneling microscopy image of pentacene molecules, which consist of linear chains of five carbon rings. A molecu ...
. The structures do not have to be acidic or basic to be called basophilic and eosinophilic; the terminology is based on the affinity of cellular components for the dyes. Other colors, e.g. yellow and brown, can be present in the sample; they are caused by intrinsic pigments such as
melanin Melanin (; from el, μέλας ''melas'', "black, dark") is a broad term for a group of natural pigments found in most organisms. Melanin is produced through a multistage chemical process known as melanogenesis, where the oxidation of the ami ...

melanin
.
Basal lamina The basal lamina is a layer of extracellular matrix In biology Biology is the natural science that studies life and living organisms, including their anatomy, physical structure, Biochemistry, chemical processes, Molecular biology, molecu ...

Basal lamina
e need to be stained by
PAS stain PAS or Pas may refer to: Companies and organizations * Malaysian Islamic Party (PAS), a Malaysian political party * Pakistan Academy of Sciences * Pakistan Administrative Service * Pan Am Southern, a freight railroad owned by Norfolk Southern and ...
or some
silver stainIn pathology Pathology is the study of the causes and effects of disease A disease is a particular abnormal condition that negatively affects the structure or function of all or part of an organism, and that is not due to any immediat ...

silver stain
s, if they have to be well visible.
Reticular fiber Reticular fibers, reticular fibres or reticulin is a type of fiber in connective tissue Connective tissue is one of the many basic types of animal Animals (also called Metazoa) are multicellular A multicellular organism is an org ...
s also require silver stain. Hydrophobic structures also tend to remain clear; these are usually rich in fats, e.g.
adipocyte Adipocytes, also known as lipocytes and fat cells, are the cells that primarily compose adipose tissue Adipose tissue, body fat, or simply fat is a loose connective tissue Connective tissue is one of the many basic types of animal An ...

adipocyte
s,
myelin Myelin is a lipid-rich (fatty) substance that surrounds nerve cell axons (the nervous system's "wires") to Insulator (electricity), insulate them and increase the rate at which electrical impulses (called action potentials) are passed along the a ...
around neuron
axon An axon (from Greek ἄξων ''áxōn'', axis), or nerve fiber (or nerve fibre: see spelling differences Despite the various English dialects Dialect The term dialect (from Latin , , from the Ancient Greek word , , "discourse", from ...
s, and
Golgi apparatus The Golgi apparatus (), also known as the Golgi complex, Golgi body, or simply the Golgi, is an organelle In cell biology Cell biology (also cellular biology or cytology) is a branch of biology Biology is the natural science that stu ...

Golgi apparatus
membranes.


Examples of H&E stained tissues


References


Further reading

* Kiernan JA (2008) Histological and Histochemical Methods: Theory and Practice. 4th ed. Bloxham, UK: Scion. * Lillie RD, Pizzolato P, Donaldson PT (1976) Nuclear stains with soluble metachrome mordant lake dyes. The effect of chemical endgroup blocking reactions and the artificial introduction of acid groups into tissues. Histochemistry 49: 23–35. * Llewellyn BD (2009) Nuclear staining with alum-hematoxylin. Biotech. Histochem. 84: 159–177. * Puchtler H, Meloan SN, Waldrop FS (1986) Application of current chemical concepts to metal-haematein and -brazilein stains. Histochemistry 85: 353–364.


External links


SIGMA-ALDRICH H&E Informational Primer


Protocol


Routine Mayer's Hematoxylin and Eosin Stain (H&E)



Rosen Lab, Department of Molecular and Cellular Biology, Baylor College of Medicine) Step by step protocol
{{DEFAULTSORT:HandE stain Microscopy Microbiology techniques Laboratory techniques Histopathology Histotechnology Staining dyes Staining