Biological tissue has little inherent contrast in either the light or electron microscope. Staining is employed to give both contrast to the tissue as well as highlighting particular features of interest. Where the underlying mechanistic chemistry of staining is understood, the term histochemistry is used. Hematoxylin and eosin (H&E stain) is the most commonly used light microscopical stain in histology and histopathology. Hematoxylin, a basic dye, stains nuclei blue due to an affinity to nucleic acids in the cell nucleus; eosin, an acidic dye, stains the cytoplasm pink. Uranyl acetate and lead citrate are commonly used to impart contrast to tissue in the electron microscope.
There are many other staining techniques that have been used to selectively stain cells and cellular components. One of these techniques involves marking peripheral tumors or surgical margins, in which a certain color of dye is applied to the posterior border of a sample, another to the anterior, etc., so that one can identify the location of a tumor or other pathology within a specimen. Other compounds used to color tissue sections include safranin, Oil Red O, Congo red, Fast green FCF, silver salts, and numerous natural and artificial dyes that usually originated from the development of dyes for the textile industry.
Histochemistry refers to the science of using chemical reactions between laboratory chemicals and components within tissue. A commonly performed histochemical technique is the Perls Prussian blue reaction, used to demonstrate iron deposits in diseases like hemochromatosis.
Histology samples have often been examined by radioactive techniques. In historadiography, a slide (sometimes stained histochemically) is X-rayed. More commonly, autoradiography is used to visualize the locations to which a radioactive substance has been transported within the body, such as cells in S phase (undergoing DNA replication) which incorporate tritiated thymidine, or sites to which radiolabeled nucleic acid probes bind in in situ hybridization. For autoradiography on a microscopic level, the slide is typically dipped into liquid nuclear tract emulsion, which dries to form the exposure film. Individual silver grains in the film are visualized with dark field microscopy.
Recently, antibodies have been used to specifically visualize proteins, carbohydrates, and lipids. This process is called immunohistochemistry, or when the stain is a fluorescent molecule, immunofluorescence. This technique has greatly increased the ability to identify categories of cells under a microscope. Other advanced techniques, such as nonradioactive in situ hybridization, can be combined with immunochemistry to identify specific DNA or RNA molecules with fluorescent probes or tags that can be used for immunofluorescence and enzyme-linked fluorescence amplification (especially alkaline phosphatase and tyramide signal amplification). Fluorescence microscopy and confocal microscopy are used to detect fluorescent signals with good intracellular detail. Digital cameras are increasingly used to capture histological and histopathological image
Common laboratory stains Edit
Stain Common use Nucleus Cytoplasms Red blood cell (RBC) Collagen fibers Specifically stains
Haematoxylin General staining when paired with eosin (i.e. H&E) Orange, Cyan Blue or Green Blue/Brown/Black N/A N/A Nucleic acids—blue
ER (endoplasmic reticulum)—blue
Eosin General staining when paired with haematoxylin (i.e. H&E) N/A Pink Orange/red Pink Elastic fibers—pink
Collagen fibers—pink
Reticular fibers—pink
Toluidine blue General staining Blue Blue Blue Blue Mast cells granules—purple
Masson's trichrome stain Connective tissue Black Red/pink Red Blue/green Cartilage—blue/green
Muscle fibers—red
Mallory's trichrome stain Connective tissue Red Pale red Orange Deep blue Keratin—orange
Cartilage—blue
Bone matrix—deep blue
Muscle fibers—red
Weigert's elastic stain Elastic fibers Blue/black N/A N/A N/A Elastic fibers—blue/black
Heidenhain's AZAN trichrome stain Distinguishing cells from extracellular components Red/purple Pink Red Blue Muscle fibers—red
Cartilage—blue
Bone matrix—blue
Silver stain Reticular fibers, nerve fibers, fungi N/A N/A N/A N/A Reticular fibers—brown/black
Nerve fibers—brown/black
Fungi—black
Wright's stain Blood cells Bluish/purple Bluish/gray Red/pink N/A Neutrophil granules—purple/pink
Eosinophil granules—bright red/orange
Basophil granules—deep purple/violet
Platelet granules—red/purple
Orcein stain Elastic fibres Deep blue N/A Bright red Pink Elastic fibres—dark brown
Mast cells granules—purple
Smooth muscle—light blue
Periodic acid-Schiff stain (PAS) Basement membrane, localizing carbohydrates Blue N/A N/A Pink Glycogen and other carbohydrates—magenta
Table sourced from Michael H. Ross, Wojciech Pawlina, (2006). Histology: A Text and Atlas. Hagerstown, MD: Lippincott Williams & Wilkins. ISBN 0-7817-5056-3.
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