The A to Z of Histological Stains

Name of the Stain

Specifically Stains

Fun Facts

4′, 6-diamidino-2-phenylindole (DAPI)

Nuclei: blue

• First synthesized in 1971 by Otto Dann’s lab as a drug to treat trypanosomiasis (1). • Selectively binds to double-stranded DNA, with no (or very little) cytoplasmic staining. • Can be easily used as a counterstain for green or red fluorescent labels. • Can be used to stain DNA in mammalian, bacterial, and metazoan cells.

Rod-shaped bacteria: red-pink

• The Ziehl-Neelsen approach for Acid Fast Red staining, which was first described in 1800s, is the most widely used. • A differential stain used to identify acid-fast bacterial organisms, such as the members of the genera

and

. • Particularly important in the diagnosis of tuberculosis.

Pluripotent cells: red/purple

• Universal stem cell membrane marker. • Commonly used to screen colonies during early stages of the reprogramming workflow (the stain maintains stem cell viability). • Can also be used as a negative selection tool at later stages to identify undifferentiated cells.

Axons, plaque neurites, and tangles: black Plaque and vascular amyloid: generally brown to dark brown Background: yellow to brown

• A silver staining method introduced by Max Bielschowsky (2), who improved the approach developed by Ramon y Cajal. • It can be used to visualize nerve fibers. • Routinely used to study Alzheimer’s disease

Amyloid fibrils: pale orange-red (apple green birefringence under polarized light)

• Although it had been around for a while, German physician, Hermann Bennhold, was the first to discover that Congo red binds to amyloid in 1923(3). • Remains the ‘gold standard’ test amongst diagnosticians to identify amyloid in tissues (most often in patients with Alzheimer’s disease)

Gram-positive bacteria: blue-black Gram-negative bacteria: red-pink

• Developed by Hans Christian Gram in 1884(4). • This stain classifies bacteria as either gram-positive cells (e.g.

.), which usually have a thicker peptidoglycan mesh, or gram-negative cells (e.g.

,

.), which usually have a lipid-polysaccharide layer external to the peptidoglycans.

Fungal elements: black (with sharp margins and cleared center) Background: light green

• A ‘broad spectrum’ fungal stain, which is better than the PAS stain (see below) at detecting even degenerated and dead fungi. • Particularly useful for staining carbohydrates. • Used for general screening for fungal infections

Nuclei: blue (hematoxylin) Endoplasmic reticulum: blue (hematoxylin) Elastic fibers: pink (eosin) Collagen fibers: pink (eosin) Reticular fibers: pink (eosin) Red blood cells: orange/red

• Most popular ‘general purpose stain’ used for routine tissue preparation. • Hematoxylin is extracted from the Haematoxylum campechianum tree; it was first used by CG Reichel in 1758, but only produced good results as a histological stain in 1865, when it was used in combination with alum by Böhmer. It binds and stains acidic structures (5). • Eosin, a yellow-red dye, was synthesized by a Polish chemist, Heinrich Caro (who named it after the nickname of a girl he liked!). Emil Fischer, a German chemist, then worked on the novel compound first publishing a paper on Eosin Y (for ‘yellowish’) in 1875 (6). Eosin binds and stains basic structures. • Hematoxylin and eosin were used in combination by a chemist, Wissowzky, in 1876 (7).

Hoechst Stain

Nuclei: blue

• Part of a family of blue fluorescent dyes developed by a German life sciences company, Hoechst AG. • Hoechst dyes specifically bind to A/T-rich regions of double stranded DNA, with no (or very little) cytoplasmic staining. • These dyes are less toxic and more cell-permeable than DAPI (see above).

Luxol Fast Blue

Myelin fibres: blue to blue/green Neurons: violet (when counterstained) Red blood cells: blue

• Created by Heinrich Klüver and Elizabeth Barrera in 1953 (8). • A copper phthalocyanine dye that is soluble in alcohol and attracted to the bases found in the lipoproteins of the myelin sheath. • Commonly used to detect (de)myelination in the central nervous system. • Often accompanied by a counterstain (e.g. hematoxylin and eosin).

Methylene Blue

Nucleus and cytoplasm: blue

• Cationic dye that binds to anions in the tissue, such as carboxylic acid, sulfuric acid, and phosphoric acid groups.

Neutral triglycerides and lipids (frozen sections), lipoproteins (paraffin sections) and lipofuscin: Bright red

• Introduced in 1926 by RW French (9). • Closely related to the Sudan dyes. • Used to demonstrate the presence of fats or lipids in fresh, frozen tissue sections. • Also used in forensic pathology to enhance latent prints produced by oily fingers.

Glycogen and other carbohydrates: magenta Nuclei: blue Collagen fibers: pink

• Useful for researching/diagnosing glycogen storage diseases or diseases of the basement membrane. • Hematoxylin is typically used as a counterstain to visualize other tissue elements. • A light green counter stain is preferred when PAS is used to demonstrate fungal organisms.

Iron deposits: Blue or purple Other tissue components: red (when counterstained with neutral red)

• Prussian blue was accidentally developed in 1704 by a chemist, but it was first introduced as a histological stain by the German pathologist, Max Perls, in 1867 (10). • Important stain to identify patients with hemosiderin (type of iron-storage complex found inside cells) deposits, for instance in some liver diseases or hemolytic anemia.

Sudan Black B

Neutral triglycerides and lipids (frozen sections) and some lipoproteins (paraffin-embedded sections): blue-black Nuclei: red

• Similar to other synthetic Sudan stains (including Oil Red O). • Usually tissue sections are counterstained (with hematoxylin/nuclear fast red). • Can also be used to stain myeloblasts, but not lymphoblasts.

Mast cell granules and polysaccharides: violet Nuclei: blue Cytoplasm: blue Red blood cells: blue Collagen fibres: blue

• Developed by William Henry Perkin in 1856 (11). • Basic dye that selectively stains acidic tissue components. • It is also useful for staining thin sections of resin-embedded tissues for electron microscopy, in order to help with the orientation and visualization of samples.

Collagen, bone: green-blue Muscle, fibrin, cytoplasm: red Red blood cells: yellow or red Nuclei: dark red-black

• Original trichrome recipe was formulated by pathologist, Claude L. Pierre Masson, in the early 1900s (12). • Technique using three (acidic) dyes to produce different coloration of (basic) tissue elements. • This stain is routinely used in diagnostic labs to evaluate liver diseases, such as cirrhosis. • Various staining approaches exist, of which Masson’s Trichrome and Gömöri’s Trichrome are the most commonly used. today.

Elastic fibers and cell nuclei: black (Verhoeff component) Collagen and muscle: Red (van Gieson component) Cell cytoplasm and other components: yellow

• Ira van Gieson first described this staining procedure in 1889 (13), which was modified by Frederick Herman Verhoeff in 1908 (14). • The Verhoeff component is an iron-hematoxylin stain, while the van Gieson component is a collagen-specific counterstain, which is comprised of picric acid and acid fuchsin. • Used to validate the presence or absence of elastic fibers in tissues.

Microorganism: dark brown to black Background: golden brown (due to lower concentrations of silver deposits)

• First developed by American pathologists, Aldred Scott Warthin and Allen Chronister Starry, in 1920 (15). • Silver nitrate-based staining method. • Considered the best approach to detect Gram-negative organisms, such as small bacilli and spirochaetes.