Glossary of Commonly Used Stains and Techniques
AAF (Acid Aniline Fuchsin)
The staining solution is a mixture of acid fuchsin and aniline. The technique requires tissue to be fixed in formalin and potassium-dichromate. Following staining, the tissue is differentiated in sodium carbonate or alcohol until most of the red dye is lost from the cytoplasm, but is still retained in the mitochondria. The tissue is counterstained with a blue or green dye. This technique was one of the early methods (1900), used to convincingly demonstrate mitochondria. Based on the use of this stain, Bensley convinced himself of the existence of mitochondria and subsequently, successfully isolated mitochondria from tissues by homogenization and differential centrifugation.
- Collagenous Fibers - blue
- Nuclei and Mitochondria - red
- Neuroglial fibers - red
- Myelin - yellow
- Ground Substances of Cartilage, Bone, Mucus, Amyloid - blue
A cationic dye which is commonly used to demonstrate negatively charged molecules. Glycosaminoglycans are negatively charged due to the presence of sulfate ester groups or carboxyl groups of uronic acids or both of these groups.
- Acid mucins/ mucosubstances = blue
- Nuclei (when counterstaining with Nuclear Fast Red) = reddish-pink
AF (Aldehyde Fuchsin)
This stain is a dye complex between aldehyde fuchsin in an acid medium with paraldehyde. The dye complex is apparently specific for sulfate groups in tissue; as such it will stain sulfated acid mucopolysaccharides as well as disulfide bonds which have been oxidized to SO4. An example of the latter would be the stain ability of the \xDF-cells of the pancreatic islets. An example of aldehyde fuchsin staining of acid mucopolysaccharides can be seen in mucous cells of salivary glands. Aldehyde fuchsin was first described as being specific for elastic. The nature of this staining reaction is not known, although it appears to be a physical complex between the dye molecule and the ground substance of elastin. The primary stain is counterstained with some contrasting color. We frequently use a mixture of acid fuchsin and ponceau which provides a pink/red coloration to the cytoplasm and then counterstain the collagen green with fast green. The latter is an aniline dye specific for collagen.
- Elastic Fibers – purple
- Mast Cells – purple
- Pancreatic βcells – purple
- Sulphated Mucins – purple
- Background – as the counterstain (green)
Ag (silver) Reactions
These techniques, of which there are many variants, involve the impregnation of tissue with silver and development with a developer such as formalin or commercial photographic developer. The techniques can be made specific for nerve fibers. They will also demonstrate certain cell types of the adrenal medulla and, with modification, can be used to stain reticulin. The Wilder silver method, used in your class sets for the reticulin stain, is a variation of the Bielschowsky method. The resultant impregnation appears different due to the fact that the sections have been toned with gold-chloride which changes the appearance of the silver deposited in the tissue.
- Structure of interest (depending on the stain, could be nerve fibers, reticular fibers, or certain cell types) - black, gray, or yellow
A mixture of methyl blue and water blue, this dye is used specifically to stain for collagen (as in the trichrome stain), connective tissue (as in Mallory's stain), or as a general contrast dye for differential staining.
This basophilic dye is used as a single stain for demonstrating DNA and RNA. It is especially useful in demonstrating Nissl bodies in neurons as reddish-violet discrete cytoplasmic granules. DNA is not as intensely stained.
H&E (Hematoxylin and Eosin)
The hematoxylin and eosin stain is routinely used for histology and pathology. The hematoxylin component of this staining sequence behaves as a basic dye and eosin is the classic acidic dye. Those tissue components with an affinity for a basic dye such as hematoxylin are referred to as basophilic (philia from the Greek to "to love"). Eosin will stain those tissue components that have an affinity for an acidic molecule and are thus eosinophilic or acidophilic. These terms refer to the proton donor or proton acceptor concept of tissue organic acids and bases. Hematoxylin is an extract of a bark of a peculiar tree grown in South America. In the presence of a suitable metallic salt, a complex is formed that changes color to a dark blue to black staining molecule. The typical hematoxylin will stain acidic tissue components such as DNA (chromatin) or RNA (RER). The staining complex is due to the affinity for the phosphate groups in the RNA or DNA. In the case of eosin, large concentrations of protein amino groups will be stained, thus areas rich in protein such as collagen, striated muscle, smooth muscle, or membrane rich systems will all have varying degrees of affinity for eosin.
- Nuclei = blue (with some metachromasia)
- Cytoplasm = varying shades of pink
Immunohistochemistry is a technique used to identify the location of specific molecule (usually proteins). Positive immunostaining depends on the ability of lymphocytes to generate antibodies against specific epitopes. Antibodies can be prepared against a vast array of molecules which include proteins, peptides, lipids, and specific sugar complexes. An antibody prepared against a specific antigen is the primary antibody. Binding of the primary antibody is amplified using a secondary antibody that is either conjugated to an enzyme (which generates a precipitable reaction product) or a fluorescent probe which enables the antigen to be visualized. The fluorescent antibody technique is especially useful for demonstrating co-localization of antigens using confocal microscopy.
- Molecule of interest (protein, peptide, lipid, etc.) = staining is variable, based on reaction products. With commonly-used enzymes like Horseradish-peroxidase (HRP), precipitates are generally brown, black, or blue-black. With fluorescence, shades depend on the fluorescent tag and are most often red, green, or yellow.
In situ hybridization
This technique is used to determine which cells are transcribing a specific gene. It exploits the ability of nucleotide sequences to form stable hybrids with complementary cDNA or riboprobe sequences. By tagging a specific nucleotide sequence in the probe, specific cells in tissue sections that are synthesizing a specific messenger RNA can be identified. These probes have traditionally been labeled using radioisotopes, which necessitates coating the slides with a photographic emulsion in order to detect the emitted electrons. Non-radioactive techniques (e.g. fluorescence) can be employed for abundantly expressed mRNAs.
- mRNA of interest = staining is variable, based on method and reaction products
Iron hematoxylin (Heidenhain's hematoxylin)
When iron is used as the mordant, the color of the hematoxylin is blue-black or brown. This stain is useful for demonstrating chromosomes, desmosomes, and muscle bands.
- Nuclei and other structures - blue to black
Methylene Blue (Basic)
A widely used basic dye, closely related to thionine. It is readily oxidized into lower homologues such as the azures, giving a mixture called polychrome methylene blue which is used in Wright's blood stain, and the May Grunwald-Giemsa stain. Methylene blue is also used as a nuclear stain, vital stain for nerves, and many other uses. It has metachromatic properties.
OsO4 (Osmium Tetroxide)
A routine fixative and stain for electron microscopy. Osmium tetroxide stains membrane lipid droplets black to brown depending on amount of unsaturated fatty acids present; and stains myelin, adipocytes, and and Golgi apparatus black.
- Myelin - black
- Adipocytes - black
- Golgi apparatus - black
- Cell membranes - brown/black
PAS (Periodic Acid Schiff) Method
This technique demonstrates 1, 2-diglycols in tissue sections. The stain will demonstrate neutral polysaccharides such as glycogen and certain mucoproteins of salivary glands. The brush border of tissues such as the proximal tubule of the kidney contains considerable amounts of polysaccharide material between the microvilli comprising the brush border. PAS will stain these tissue components. The staining sequence is often counterstained with hematoxylin to demonstrate nuclei.
- Glycogen/fungus = magenta
- Nuclei = blue
PTAH (Phosphotungstic Acid Hematoxylin)
This technique utilizes no counterstain and is the combination of hematoxylin and phosphotungstic acid. The most outstanding result can be seen in staining of muscle, where the muscle is stained blue and the connective tissue a delicate rose-pink. Muscle and connective tissue complex with the phosphotungstic acid in different ways, resulting in a dye that is slightly different in its resonance properties. The resulting difference in resonance structures permits distinguishing of two tissue components by means of a single dye.
- Cross-striations, glial fibers, fibrin = blue
- Nuclei = blue
- Collagen = red-brown
- Elastic fibers = purplish
Resorcin-Fuchsin (Weigert’s) Elastic Fiber Stain
For the demonstration of elastic fiber, basic fuchsin and resorcin form a complex that binds to elastic fibers and nuclear detail is stained with an iron hematoxylin. The iron hematoxylin, however, does not over-differentiate in the acidic pH of the staining solution so the nuclei stain more lightly than the elastic fibers.
- Elastic fibers = blue-black
- Collagen = pink-red
- Nuclei = pale blue-black
- Other = yellow
This silver impregnation technique is most useful in parenchymal organs (liver, spleen, etc) to highlight their microarchitecture. Reticular fibers are argyrophilic (bind to silver) and are highly organized, fine strands in a normal organ. The tissue is first oxidized, then sensitized with iron alum. The iron is replaced with silver and, upon reduction with formalin, the silver (bound to reticular fibers) becomes visible by light microscopy.
- Reticular fibers = black
- Nuclei (when counterstained with nuclear-fast red) = red
Toluidine Blue (Basic)
Useful as a stain for basophilic (i.e., acidic) structures. Toluidine blue, thionine, methylene blue, and the azures are closely related dyes. RNA stains purplish, DNA blue-greenish; and acid mucopolysaccharides purple. Toluidine blue staining exhibits metachromatic properties, that is, the color of the dye is altered, from blue to purple or pink, by certain substances. The mechanism of this effect is not definitely known, but the substances that stain metachromatically are polyanions such as RNA and mucopolysaccharides. It is theorized that the high density of negative charges binding dye molecules very close to one another is related to metachromasia.
- RNA/ mucopolysaccharides/ mast cells = violet
- DNA = blue-green
This stain is a hematoxylin variant specific for elastic fibers. The counterstain
is usually acid fuchsin which stains collagen red, and picric acid which stains cytoplasm
- Nuclei – black
- Elastic fibers – black
- Fibrin - mature – red
- Muscle – red
- Collagen – Red
This stain is an important for visualizing components of blood. The May-Grunwald-Giemsa is a closely related technique. With this stain, all basophilic components are colored blue or purple depending on whether they happen to bind methylene blue (basophilic) or azure (azurophilic); all acidophilic components are red. The reaction is not specific unless the pH is controlled.
- Erthrocytes - pink to tan
- Eosinophiles: Granules - red
- Granules - red to purple
- Cytoplasm - blue
- Neutrophils: Granules - purple to pink
Top of page
- Hey, do tell me that Acid Aniline Fuchsin is so named for my favorite color! -- LorenEvey - 14 Jul 2007
- Smarter than the average bear! -- LorenEvey - 14 Jul 2007
- "We" actually planned on adding that later. Aren't we bloody brilliant? PS: Thanks for un-Wiki-wording that osmium for me. I'd have caught it eventually... but your keen eye is clearly seeing better than mine these days! -- AshleyLPistorio - 13 Jul 2007
- I wonder if "we" could have a representative slide of each stain on this page? -- LorenEvey - 13 Jul 2007
- You had a wikiword on OsO4. I put the exclamation. -- LorenEvey - 11 Jul 2007
- 11 Jul 2007