|Embryology - 9 Jul 2020 Expand to Translate|
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|A personal message from Dr Mark Hill (May 2020)|
|contributors to the site. The good news is Embryology will remain online and I will continue my association with UNSW Australia. I look forward to updating and including the many exciting new discoveries in Embryology!|
- 1 Introduction
- 2 Acid Phosphatase
- 3 Acridine Orange
- 4 Alcian Blue
- 5 Alizarine Blue
- 6 Alizarin Red
- 7 Azocarmine
- 8 Best's Carmine
- 9 Cason's Trichrome
- 10 Chromosome Banding
- 11 Congo Red
- 12 Erythrosin Orange
- 13 Golgi Method
- 14 Gomori's Trichrome
- 15 Gram Stain
- 16 Haematoxylin and Eosin
- 17 Heidenhain's Azan
- 18 Leishman's Stain
- 19 Luxol Fast Blue
- 20 Mallory Trichrome Stain
- 21 Masson’s Trichrome Stain
- 22 Movat's stain
- 23 Mucicarmine
- 24 Nissl
- 25 Orange G
- 26 Osmium Tetroxide
- 27 Papanicolaou stain
- 28 Periodic acid-Schiff
- 29 Phloxine B
- 30 PhosphoTungstic Acid Hematoxylin (PTAH)
- 31 Picro-Mallory
- 32 Regaud's Hematoxylin
- 33 Romanowsky Stain
- 34 Schmorl's Stain
- 35 Silver Stain
- 36 Sirius Red
- 37 Spalteholz Method
- 38 Sudan Black
- 39 Tartrazine
- 40 Toluidine Blue
- 41 Verde Luz-Orange G-acid Fuchsin
- 42 Verhoeff-Van Gieson
- 43 Weigert's Elastic Tissue
- 44 Wright
- 45 Whipf's Polychrome
- 46 Fixative
- 47 Mounting Media
- 48 Embedding
- 49 References
- 50 External Links
- 51 Glossary Links
This page gives a general overview of some histological stains used to identify structures in cells and tissues. This stains information should also be considered in relation to Histology Fixatives. To see related histology images use the Category:Histology link.
Medicine Foundations students do not need to know stain information in this detail.
|Histology Stains - Common Stains and Their Reactions|
|Haematoxylin||mucins - light blue|
|Eosin||colloid - pinkmuscle - red|
|Van Gieson||muscle: yellow/browncartilage - pink|
|Verhoeff's Elastin||elastic fibres - black|
|Silver Impregnation||reticular fibres - black|
|Nuclear Fast Red|
|Gomori's Trichrome||keratin - redmuscle - purple/red|
|Heidenhain's Azan||muscle - red|
|Osmium Tetroxide||myelin, lipids - black|
|Alcian Blue||mucins, - blue|
|Periodic acid-Schiff (PAS)||mucins, glycogen, glycocalyx - magenta|
|Phosphotungstic Acid-Hematoxylin (PTAH)||muscle bands - blue|
|Masson's Trichrome||cartilage, mucins - blue or green; muscle - red|
|Luxol Fast Blue||myelin - blue|
|Aldehyde Fuchsin||elastic fibres, mast cells - deep purple|
|Gallocyanin||nucleic acids, Nissl granules - dark blue|
|Romanowsky (e.g. Leishman's)||acidophils - red, basophils - blue, azurophilic - purple|
|Aldehyde Pararosanilin||elastic fibres - purple|
|Common Histology Stains|
These templates put a link to this page and the appropriate stain text.
Use the text in the curly brackets to insert the text in the curved brackets with a link to the Histology Stains page.
- Stains lysosomes due to their acid phosphatase content.
- Enzymatic activity of acid phosphatase (lysosomal enzyme) releases a precipitate of lead phosphate from solution. This is then converted to lead sulphide black deposit.
- Stains nucleic acid and detects cells undergoing apoptosis versus necrosis/necroptosis.
- enables measurement of RNA and DNA as proxies for cell viability.
- Stains mucopolysaccharides or glycosaminoglycans
- cationic dye (positively charged molecule) for the demonstration of glycosaminoglycans.
- binds anionic (negative) sites on the polysaccharide.
- can be combined with H&E and VG staining methods.
Alizarine Brilliant Blue R
- Useful for identifying bone or other hight calcium structures.
- Stains insoluble calcium cations
- bright red stain
- Other metals such as barium, aluminium, mercury and magnesium (dark red)
- Stains nuclei a deep red and cytoplasm a pale red.
- Stains glycogen granules red.
- Periodic acid-Schiff is more commonly used and also colours glycogen red.
Stain developed by Cason (1950) for muscle and collagen Aniline blue (methyl blue) colours collagen fibres.
The term refers to the light and dark pattern, seen after staining with a dye, of individual chromosomes identified in metaphase. It is only in meiosis and mitosis during metaphase that chromosomes can be easily identified, during the normal cell life (interphase) the chromosomes are unravelled and distributed within the nucleus in chromosome territories. A band is that part of a chromosome which is clearly distinguishable from nearby regions by appearing darker or brighter with one or more banding techniques.
Depending on the type of stain used a number of different banding patterns can be seen:
Chromosome banding pattern seen by treating with trypsin and then staining with the dye Giemsa stain
Chromosome banding pattern seen as a of reverse giemsa chromosome banding, producing bands complementary to G-bands often used to determine whether there are large sequence deletions. Can also be fluorescent using the dye acridine orange.
Chromosome banding pattern seen by treating with a fluorochrome or the fluorescent dye quinacrin.
Chromosome banding pattern seen for centromeric or constitutive heterochromatin, the centromere appears as a stained band compared to other regions.
Metaphase is a cell division term referring to the third mitotic stage, mitotic spindle kinetochore microtubules align chromosomes in one midpoint plane. Metaphase ends when sister kinetochores separate. Originally based on light microscopy of living cells and electron microscopy of fixed and stained cells. A light microscope analysis called a "metaphase spread" was originally used to detect chromosomal abnormalities in cells.
- Congo red is water soluble, yielding a red colloidal solution; though its solubility is better in organic solvents, such as ethanol.
- Previously used as a dye, but its properties (toxicity, colour change) means that it is no longer used for this purpose.
- Has been used to detect amyloid in muscle and nerve fresh frozen sections.
- Erythrosin B orange solution
- dye for cytoplasm and connective
- Dominici method for bone marrow biopsies.
- Empirical Formula (Hill Notation) C20H8I4O5
- Molecular Weight 835.89
- CAS Number 15905-32-5
- (Golgi stain) A selective silver stain technique developed by Camillo Golgi (1843–1926) in 1873.
- This historic technique allowed Santiago Ramón y Cajal (1852–1934) to interpret the structure of the central nervous system.
- There are also a range of other silver staining techniques (see silver staining reticular fibres).
- Developed in 1950 by an American histologist George Gömöri (1904–1957).
A bacterial staining procedure using crystal violet and pink safranin counterstain that generally divides bacteria into either gram-positive or gram-negative and useful for considering associated pharmacology. The procedure was named after Hans Christian Gram (1853 - 1938).
- Purple crystal violet stain is trapped by layer of peptidoglycan.
- peptidoglycan forms outer layer of the cell.
- Outer membrane prevents stain from reaching peptidoglycan layer in the periplasm.
- outer membrane is composed of four major components: lipopolysaccharide, phospholipids, beta-barrel proteins, and lipoproteins.
- outer membrane then permeabilized.
- Pink safranin counterstain is trapped by peptidoglycan layer.
- Links: Histology Stains | Abnormal Development - Bacterial Infection | Medical Microbiology - Gram stain procedure
Haematoxylin and Eosin
One of the most common staining techniques in pathology and histology.
- Acronym "H and E" stain. (H&E, HE)
- UK - Haematoxylin, USA - Hematoxylin
(Stain - Haematoxylin Eosin) Note - if you have clicked the stain name link where it appears on a page or image on this site it will bring you here, use your web browser back button to return to your original page.
- UK - Haematoxylin, USA - Hematoxylin
- Stains nuclei blue to dark-blue.
- Stains the matrix of hyaline cartilage, myxomatous, and mucoid material pale blue.
- Stains myelin weakly but is not noticeable if combined with eosin stain.
- combined with Orange G (H & Or. G.) instead of eosin, specifically stains the granules of acidophilic cells of the adenohypophysis (anterior pituitary).
- Stains cytoplasm pink to red; red blood cells are also bright red.
- Common counterstain to hematoxylin.
- Stain intensity varies with the formula as well as the fixative.
- Eosin - (Greek, eos = dawn, rose-coloured) an acidic dye staining the basic cytoplasmic proteins pink.
- Eosinophil - (Greek, + philein = to love) a type of blood cell with distinct cytoplasmic granules which stain pink with eosin.
- Eosinophilic - having an affinity for eosin dye.
- Heidenhain (1915) introduced azocarmine G in place of the acid fuchsine in Mallory's trichrome method.
- Staining is azocarmine G followed by a solution containing PTA, orange G and aniline blue.
- Controlled the destaining to show different colours.
- cell nuclei - dark red
- collagen - blue
- cytoplasm - variety of colours
- Named after Rudolph Heidenhain (1834-1897) a German histologist and physiologist.
Heidenhain's Iron Haematoxylin
- an iron alum hematoxylin stain used for staining muscle striations and mitotic structures blue-black.
- Named after Rudolph Heidenhain (1834-1897) a German histologist and physiologist.
- William Boog Leishman (1865 – 1926) was a Scottish pathologist.
- Similar to other Romanowsky stains - Giemsa stain, Jenner's stain, and Wright's stain
- Used to identify leucocytes, malaria parasites, and trypanosomas.
- Methanol mixture of "polychromed" methylene blue (demethylated into various azures) and eosin.
- dried precipitate known as "methylene azure" is sold as Leishman's Stain
- Methanol also acts as fixative.
- variations include Wright's stain (America) and Giemsa and May-Grünwald stains in Germany and Europe.
- Links: Practical Lymphoid Tissues
Luxol Fast Blue
- Myelin and phospholipids blue to green.
- copper phthalocyanine dyes.
- Counterstain with Cresyl violet (Nissl stain) to show neuron.
- used as solutions in alcohol or other moderately polar liquids.
- Links: Practical - Neural Histology
Mallory Trichrome Stain
(Mallory triple stain, Mallory aniline blue stain)
- Stains connective tissue.
- Mallory's trichrome method uses acid fuchsine followed by a solution containing PTA, orange G and aniline blue, provides dark red nuclei, orange erythrocytes, and blue collagen fibres, cartilage matrix and mucus.
Masson’s Trichrome Stain
Claude L. Pierre Masson (1880–1959)
- Stains nuclei deep blue, skeletal and smooth muscles red, collagen and mucin blue.
- Stains brain and spinal cord parenchymal tissue dusky pink to red.
- Used to evaluate fibrosis
- Striations in skeletal muscles also shows up much better in Masson’s trichrome than in hematoxylin and eosin stain.
- Although called a trichrome, four dyes (hematoxylin, Biebrich scarlet, acid fuchsin, and analine blue) are utilized.
- black - (iron hematoxylin) stain nuclei
- light green - mesenchyme green
- orange - (azofuchsin) fibrin and parenchyma
- Connective tissue stain including cardiovascular tissue.
- Pentachrome (five colour) stain:
- Black - nuclei; elastic fibres
- Yellow- collagen fibres; reticular fibres
- Blue - ground substance; mucin
- Bright red - fibrin
- Red - muscle
- Named after Henry Zoltan Movat who originally developed in 1955 and later modified by H. K. Russell in 1972.
(Mucin Stain, Al. carm.) Staining of acid mucopolysaccharides in tissue sections. Dye contains a large 2:1 dye-aluminum cationic complex, which is red. the mucicarmine solution is applied to sections after staining nuclei blue with a hemalum. the red dye-metal complex is attracted to anionic sites in the tissue (mucus, cartilage matrix, etc.) but it does not displace the nuclear stain. A yellow anionic dye such as picric acid or metanil yellow may optionally follow mucicarmine, as a cytoplasmic counterstain.
- Pink/Red - Mucin
- Red - Capsule of Cryptococcus
- Blue - Nuclei
- Yellow - Other Tissue Components
- Staining nucleic acids (ribosomes, RER, heterochromatin, nucleoli).
- methylene blue, toluidine blue or cresyl violet is used.
- A general cytoplasmic stain similar to eosin.
- Stains cytoplasm yellow or orange.
- Combined with Haematoxylin or Periodic acid-Schiff to specifically stain granules of acidophilic cells of the adenohypophysis (anterior pituitary).
- has a great affinity for lipids and is chemically bound to the fat and as such acts as a fixative.
- using osmium as a 'stain' makes use of the fact that osmium oxidises tissue fats and forms a black substance which is easily seen in the light microscope.
- adipose tissue, adipose cells, myelin sheaths.
- (Stain - Osmium)Note - if you have clicked the stain name link where it appears on a page or image on this site it will bring you here,
use your web browser back button to return to your original page.
(Papanicolaou's stain, Pap stain) This histology technique was originally described in a publication by George Nikolas Papanicolaou in 1942. A multichromatic (five dyes) staining histological technique has been used to stain many different human bodily fluids (CSF, semen, aspirations), used mainly in the "pap smear" histology. The technique has also been modified several ways (Bismarck brown Y deleted) from the original published technique.
- Haematoxylin - nuclear stain is used to stain cell nuclei.
- Orange G - stains keratin.
- Eosin Y - stains superficial epithelial squamous cells, nucleoli, cilia, and red blood cells.
- Light Green SF yellowish - stains the cytoplasm of non-keratinized squamous cells.
- Bismarck brown Y - stains nothing specifically, often omitted.
- (Stain - Papanicolaou)
Note - if you have clicked the stain name link where it appears on a page or image on this site it will bring you here,
use your web browser back button to return to your original page.
- Links: Menstrual Cycle - Histology
- Stains glycogen, mucin, fungus, basement membrane and other substances.
- Stain used to detect fungal organisms and cytoplasmic accumulation of glycogen.
- Stains glycogen red, used in place of Best's Carmine.
- Stains lysosomes granules red-purple, can be used in recognition of macrophages.
Periodic acid-Schiff / Orange G
- Basophil cells - magenta
- Acidophil cells - yellow
- Red blood cells - yellow
- Nuclei - blue/black
- Chromophobes - pale blue/grey
- Links: Pituitary Histology
- derivatives of eosin
- used in the hematoxylin phloxine saffron (HPS) stain
- stains paneth cell granules in Lendrum's phloxine-tartrazine method
- used to demonstrate alcoholic hyaline
- Formula - C20H2Br4Cl4Na2
PhosphoTungstic Acid Hematoxylin (PTAH)
- Stains nucleus and cytoplasm detail and connective tissue fibers.
- Stains collagen pink, fibrin blue, and striated muscle blue.
- Historic stain used to show CNS reactive astrocytes now used immunochemistry for glial fibrillary acidic protein (GFAP).
(Picro-Mallory trichrome stain)
- a modification of Mallory trichrome stain that involves the addition of picric acid.
- Regaud's modification of iron hematoxylin.
- Used to identify mitochondria by light microscopy.
- material fixed in potassium dichromate and formalin and subsequently mordanted in dichromate.
There are a series of different histological stains that can be classified as Romanowsky stains.
- Leishman's stain - commonly used version of Romanowsky stain for blood smears.
- Giemsa stain
- Jenner's stain
- Wright's stain
- Stains canaliculi and lamellae in compact bone sections.
- Stain has 2 colouring agents, ammoniacal thionin and aqueous saturated picric acid.
- thionin precipitates within the lacunae and canaliculi (dark brown)
- picric acid forms picrates in the bone matrix (brownish-yellow)
- Named after Christian Georg Schmorl (1861 - 1932) a German pathologist.
There are a variety of specialised silver staining techniques.
- Stains connective tissue collagen (type I) a grey/brown and the specialised reticular fibre collagen (type III) a dark black.
- Silver deposits can be formed by 3 different reactions: argentaffin, argyrophil, and Ion-exchange.
(Bielschowsky's silver staining)
- Max Bielschowsky (1869 – 1940) was a Polish neuropathologist born in Breslau.
- an improved method based upon Ramon y Cajal's historic method for selectively silver staining nerve fibres in neural tissue.
- Links: Santiago Ramón y Cajal
(Jone's Methenamine Silver)
- Stains the basement membrane of the glomerulus in the kidney.
- A routine stain on kidney biopsies.
- Periodic acid oxidizes the carbohydrate components of the basement membrane which produce aldehydes.
- Released aldehydes reduce the silver to a visible metallic silver (black).
- Links: Renal System Histology
- collagen stain.
- collagen is red on a pale yellow background.
- polarized light microscopy - larger collagen fibers are bright yellow or orange, and thinner ones (including reticular fibres) are green.
- Picro-sirius red method is an addition that prevents the loss of dye, that happens if the stained sections are washed in water.
The Spalteholz method (technique) is not a histological stain but a historic method for clearing macroscopic specimens. One drawback is that this clearing procedure, that includes peroxide, severely damages formalin-fixed tissue and inhibits further histological processing. This technique has been recently modified to allow further histological investigation. Named after Werner Spalteholz (1861-1940) a German anatomist.
- adipose tissue stain.
- stain colours fat (lipid) droplets black.
- several different Sudan dyes
- Sudan III and Sudan IV (Scarlet R.) stain fat droplets red (as does Oil-red-O).
- reacts against lipofuscin.
- lipofuscin related to many ageing processes.
- accumulate in senescent cells.
- can be combined with Phloxine and Haem
- tartrazine acts as a yellow counterstain for tissues stained red with phloxine
- pancreatic islets - display pancreatic beta cells
- Stains nucleus blue and cytoplasm light blue.
- Basic thiazine metachromatic dye with high affinity for acidic components, staining tissues rich in DNA and RNA.
- Connective tissue - (mucins and acid mucins) stain purple to red, background is stained blue.
- Mast cell granules - stain purple, due to the presence of heparin and histamine.
- Amyloid - stain blue, under polarized light they give a bright red birefringence.
- A synthetic dye in the thiazins family, see review
Verde Luz-Orange G-acid Fuchsin
- Verde Luz-orange G-acid fuchsin (VOF) stain.
- This stain can differentiate hard and soft connective tissues.
- Verhoeff-Van Gieson or elastic-Van Gieson (EVG) stain.
- This is a combination of Verhoeff’s elastic stain which is a hematoxylin stain containing ferric chloride and Wright’s iodine solution and Van Gieson stain which contains acid fuchsin, picric acid, and hematoxylin.
- Stains elastic fibers blue-black to black, collagen pale red, other tissue elements yellow, and nuclei blue to black.
- Named after Ira Thompson Van Gieson (1866-1913) an American neurologist and neuropathologist and Frederick Herman Verhoeff (1874–1968) an American ophthalmic surgeon and pathologist.
- Links: Colon Histology
Weigert's Elastic Tissue
- Weigert's elastic tissue stain.
- Elastic fibers purple or black.
- combination of basic fuchsin, resorcin, ferric chloride, water and alcohol.
Both Wright and Giemsa stains are types of Romanowsky stains used to stain peripheral blood and bone marrow smears.
- Wright's Blood Stain used on dried blood smears.
- Nuclei blue or purple.
- Granules - basophilic granules blue, acidophilic granules red, and neutrophilic granules reddish lilac.
- Red blood corpuscles orange.
- eosinates of polychromed methylene blue are dissolved in absolute methyl alcohol.
- methyl alcohol acts as the fixative.
A connective tissue staining technique.
Mounting medium refers to the solution in which the specimen after staining is embedded on the slide under a cover glass. It may be liquid, gum or resinous, soluble in water, alcohol or other solvents. Some require sealing from the external atmosphere, while some are self-sealing media. (See also review.)
Historically, Canada balsam (Gurr) was the most common mounting media.
Special mounting media have been developed for fluorescent dyes and microscopy.
Embedding (mountain) mediums are used in histology to enclose and support the specimen prior to sectioning and staining. Embedding also provides an easy long-term solution for long-term storing the tissue "blocks". Each embedding media has slightly differing properties, and needs to infiltrate the tissue, with paraffin and celloidin the two common embedding media.
Celloidin embedding - allows good morphological preservation though is difficult to remove, and may affect immunostaining.
Paraffin embedding - the most common historical technique and continues to be routinely used today in research and pathology laboratories, allows immunostaining to be performed, but preservation of fine cellular detail can vary.
- mixture of straight chain or n-alkanes with a carbon chain length of between 20 and 40.
- solid at room temperature (RT)
- melts at temperatures up to 65°C or 70°C
- can be supplied with melting points at different temperatures
- at melting point tends to be slightly viscous, decreases as the temperature is increased (use about 2°C above melting point)
- most common melting point for histological use 56°C–58°C (better infiltration of tissue at higher temperature)
Polyester wax embedding - has a low melting point (37°C), is organic solvent soluble, is water tolerant, and sections easily.
- Plemel JR, Caprariello AV, Keough MB, Henry TJ, Tsutsui S, Chu TH, Schenk GJ, Klaver R, Yong VW & Stys PK. (2017). Unique spectral signatures of the nucleic acid dye acridine orange can distinguish cell death by apoptosis and necroptosis. J. Cell Biol. , 216, 1163-1181. PMID: 28264914 DOI.
- Cason, (1950) Stain technology, vol.25, pp.225
- Kiernan, J.A., Carbohydrate Histochemistry Chapter 9, 75-92.
- Papanicolaou GN. (1942). A NEW PROCEDURE FOR STAINING VAGINAL SMEARS. Science , 95, 438-9. PMID: 17842594 DOI.
- Steinke H & Wolff W. (2001). A modified Spalteholz technique with preservation of the histology. Ann. Anat. , 183, 91-5. PMID: 11206989 DOI.
- Evangelou K & Gorgoulis VG. (2017). Sudan Black B, The Specific Histochemical Stain for Lipofuscin: A Novel Method to Detect Senescent Cells. Methods Mol. Biol. , 1534, 111-119. PMID: 27812872 DOI.
- Sridharan G & Shankar AA. (2012). Toluidine blue: A review of its chemistry and clinical utility. J Oral Maxillofac Pathol , 16, 251-5. PMID: 22923899 DOI.
- Ravikumar S, Surekha R, Thavarajah R. Mounting media: An overview. J NTR Univ Health Sci (serial online) 2014;3, Suppl S1:1-8. Available from: http://www.jdrntruhs.org/text.asp?2014/3/5/1/128479
- Davies F. (1940). Mounting media and the fading of histological preparations. J. Anat. , 74, 464-70. PMID: 17104830
- Bancroft JD and Stevens A. Theory and practice of histological techniques. 3rd ed. Churchill Livingstone, 1990.
- Freeman B, Glossary of histological and micro-anatomical terms Department of Anatomy, School of Medical Sciences, UNSW, revised 2000.
External Links Notice - The dynamic nature of the internet may mean that some of these listed links may no longer function. If the link no longer works search the web with the link text or name. Links to any external commercial sites are provided for information purposes only and should never be considered an endorsement. UNSW Embryology is provided as an educational resource with no clinical information or commercial affiliation.
- Mayo Medical Labs Mallory's Phosphotungstic Acid Hematoxylin (PTAH) Stain
- Sociedad Argentina de Citología Papanicolaou staining protocol
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Cite this page: Hill, M.A. (2020, July 9) Embryology Histology Stains. Retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/Histology_Stains
- © Dr Mark Hill 2020, UNSW Embryology ISBN: 978 0 7334 2609 4 - UNSW CRICOS Provider Code No. 00098G