Hematoxylin and eosin stain or haematoxylin and eosin stain (H&E stain or HE stain) is one

of the principal stains in histology. It is the most widely used stain in medical diagnosis and is
often the gold standard; for example when a pathologist looks at a biopsy of a suspected cancer,
the histological section is likely to be stained with H&E and termed "H&E section", "H+E
section", or "HE section". A combination of hematoxylin and eosin, it produces blues, violets,
and reds.

Contents

1 Principle

2 Overview

3 See also

4 References

5 External links
o 5.1 Protocol

Principle
The staining method involves application of hemalum, a complex formed from aluminium ions
and hematein (an oxidation product of hematoxylin). Hemalum colors nuclei of cells (and a few
other objects, such as keratohyalin granules and calcified material) blue. The nuclear staining is
followed by counterstaining with an aqueous or alcoholic solution of eosin Y, which colors
eosinophilic structures in various shades of red, pink and orange.
The staining of nuclei by hemalum 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 thionine or
toluidine blue. 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 coordinate 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.
The eosinophilic structures are generally composed of intracellular or extracellular protein. The
Lewy bodies and Mallory bodies are examples of eosinophilic structures. Most of the cytoplasm
is eosinophilic. Red blood cells are stained intensely red.
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, e.g. melanin.
Some structures do not stain well. Basal laminae need to be stained by PAS stain or some silver
stains, if they have to be well visible. Reticular fibers also require silver stain. Hydrophobic
structures also tend to remain clear; these are usually rich in fats, e.g. adipocytes, myelin around
neuron axons, and Golgi apparatus membranes.

Overview

Hematoxylin is a dark blue or violet stain that is basic/positive. It binds to basophilic

substances (such DNA/RNA - which are acidic and negatively charged).

o DNA/RNA in the nucleus, and RNA in ribosomes in the rough endoplasmic

reticulum are both acidic because the phosphate backbones of nucleic acids are
negatively charged. The negatively charged backbones form salts with basic dyes
containing positive charges. Therefore, dyes like hematoxylin will bind to DNA
and RNA and stain them violet.
Eosin is a red or pink stain that is Acidic / Negative. It binds to acidophilic substances
such as positively charged amino acid side chains (e.g. lysine, arginine).
o Most proteins in the cytoplasm are basic because they are positively charged due
to the arginine and lysine amino acid residues. These form salts with acid dyes
containing negative charges, like eosin. Therefore, eosin binds to these amino
acids/proteins and stains them pink. This includes cytoplasmic filaments in
muscle cells, intracellular membranes, and extracellular fibers.
So, in optical microscopy we can observe :[1]
Nuclei in blue/purple

Basophils in purplish red

Cytoplasm in red

Muscles in dark red

Erythrocytes in cherry red

Collagen in pale pink

Mitochondria in pale pink

See also

Papanicolaou stain, other popular staining technique

Cytopathology

Eosin

Acid-fast

References
1.
1.

http://www.histalim.com/accueil/activities/our-services/histology/hematoxylineosin-2/
Godwin Avwioro (2011). Histochemical Uses Of Haematoxylin - A Review. JPCS 1:2434. PDF
Baker JR (1962) Experiments on the action of mordants. 2. Aluminium-haematein. Quart.
J. Microsc. Sci. 103: 493-517.
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.
Marshall PN, Horobin RW (1973) The mechanism of action of "mordant" des - a study
using preformed metal complexes. Histochemie 35: 361-371.
Puchtler H, Meloan SN, Waldrop FS (1986) Application of current chemical concepts to
metal-haematein and -brazilein stains. Histochemistry 85: 353-364.

External links
Wikimedia Commons has media related to H&E stain.

SIGMA-ALDRICH H&E Informational Primer[dead link]

Protocol

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

Hematoxylin & Eosin (H&E) Staining Protocol

Rosen Lab, Department of Molecular and Cellular Biology, Baylor College of Medicine)
Step by step protocol[dead link]

e
Microbial and histological stains

Prussian blue

Sudan stain
o Sudan II
o Sudan III
o Sudan IV
o Oil Red O
o Sudan Black B

Periodic acid-Schiff stain

Amyloid

Congo red

Bacteria

Gram staining
o Methyl violet/Gentian violet

Iron/hemosiderin

Lipids

Carbohydrates

o Safranin

ZiehlNeelsen stain/acid-fast
o Carbol fuchsin/Fuchsine
o Methylene blue

Auramine-rhodamine stain
o Auramine O
o Rhodamine B

trichrome stain: Masson's trichrome stain/Lillie's trichrome

o Light Green SF yellowish
o Biebrich scarlet
o Phosphomolybdic acid
o Fast Green FCF

Van Gieson's stain

H&E stain
o Haematoxylin
o Eosin Y

Silver stain
o Grocott's methenamine silver stain
o WarthinStarry stain

Methyl blue

Wright's stain

Giemsa stain

Gmri trichrome stain

Neutral red

Janus Green B

Alcian blue stain

Movat's stain

Acidophilic

Basophilic

Chromophobic

Connective tissue

Other

Tissue
stainability
Categories:
Staining