The terms histochemistry and cytochemistry are used to indicate methods for localizing substances in tissue sections. Several procedures are used to obtain this type of information, most of them based on specific chemical reactions or on high-affinity interactions between macromolecules. These methods usually produce insoluble colored or electron-dense compounds that enable the localization of specific substances by means of light or electron microscopy.
Several ions (eg, calcium, iron, phosphate) have been localized in tissues with these methods, using chemical reactions that produce a dark insoluble product (Figure 1–16).
Photomicrograph of a bone section treated with a histochemical technique to demonstrate calcium ions. The dark precipitate indicates the presence of calcium phosphate in calcified bone and cartilage. Noncalcified cartilage tissue (stained in pink) is in the upper portion of the figure. Medium magnification. (Courtesy of PA Abrahamsohn.)
DNA can be identified and quantified in cell nuclei using the Feulgen reaction, which produces a red color in DNA. DNA and RNA can also be analyzed by staining cells or tissue sections with a basic stain.
Although there are general methods to detect proteins in tissue sections, the histochemical methods usually do not permit identification of specific proteins in cells and tissues. Immunocytochemistry, presented later in this chapter, can do so.
Several histochemical methods, however, can be used to reveal, more or less specifically, a large group of proteins, the enzymes. These methods usually make use of the capacity of the enzymes to react with specific chemical bonds. Most histoenzymatic methods work in the following way: (1) tissue sections are immersed in a solution that contains the substrate of the enzyme intended for study; (2) the enzyme is allowed to act on its substrate; (3) at this stage or at a later stage in the method, the section is put in contact with a marker compound; (4) this compound reacts with a molecule that results from the degradation or transformation of the substrate; (5) the final reaction product, which must be insoluble and visible by light or electron microscopy, precipitates over the sites that contain the enzyme. When examining such a section in the microscope, it is possible to see the cells (or organelles) covered with a colored or electron-dense material.
Examples of enzymes that can be detected include the following:
Phosphatases are enzymes widely found in the body. They split the bond between a phosphate group and an alcohol residue of phosphorylated molecules. The colored insoluble reaction product of phosphatases is usually lead phosphate or lead sulfide. Alkaline phosphatases, which have their maximum activity at an alkaline pH, can be detected (Figure 1–17). Acid phosphatases are frequently used to demonstrate lysosomes, cytoplasmic organelles that contain acid phosphatase (Figure 1–18).
Photomicrograph of a rat kidney section treated by the Gomori method to demonstrate the enzyme alkaline phosphatase. The sites where this enzyme is present are covered by a black precipitate (arrows). Medium magnification.
Detection of acid phosphatase. Electron micrograph of a rat kidney cell showing three lysosomes (ly) close to the nucleus (N). The dark material on the lysosomes is lead phosphate that precipitated on places where acid phosphatase was present. (Courtesy of E Katchburian.)
Dehydrogenases remove hydrogen from one substrate and transfer it to another. There are many dehydrogenases in the body, and they play an important role in several metabolic processes. Dehydrogenases are detected histochemically by incubating nonfixed tissue sections in a substrate solution containing a molecule that receives hydrogen and precipitates as an insoluble colored compound. By this method, succinate dehydrogenase—a key enzyme in the citric acid (Krebs) cycle—can be localized in mitochondria.
Peroxidase, which is present in several types of cells, is an enzyme that promotes the oxidation of certain substrates with the transfer of hydrogen ions to hydrogen peroxide, forming molecules of water.
In this method, sections of adequately fixed tissue are incubated in a solution containing hydrogen peroxide and 3,3'-diaminoazobenzidine. The latter compound is oxidized in the presence of peroxidase, resulting in an insoluble, brown, electron-dense precipitate that permits the localization of peroxidase activity by light and electron microscopy. Peroxidase activity in blood cells, which is important in the diagnosis of leukemias, can be detected by this method.
Because peroxidase is extremely active and produces an appreciable amount of insoluble precipitate in a short time, it has also been used for an important practical application: tagging other compounds. Molecules of peroxidase can be purified, isolated, and coupled with another molecule. Later in this chapter, applications of tagging molecules with peroxidase are presented.
Polysaccharides & Oligosaccharides
Polysaccharides in the body occur either in a free state or combined with proteins and lipids. In the combined state, they constitute an extremely complex heterogeneous group. Polysaccharides can be demonstrated by the periodic acid–Schiff (PAS) reaction, which is based on the transformation into aldehyde of 1,2-glycol groups present in the sugar molecules. These aldehydes are then revealed by Schiff's reagent, which produces a purple or magenta color in areas of the section with an accumulation of polysaccharides.
A ubiquitous free polysaccharide in the body is glycogen, which can be demonstrated by the PAS reaction in liver, striated muscle, and other tissues where it accumulates.
Glycoproteins are protein molecules associated with small, branched chains of sugars (oligosaccharides). The protein chain predominates in weight and volume over the oligosaccharide chain. Because both glycogen and neutral glycoproteins are PAS positive, the specificity of the PAS reaction can be improved by comparing the staining of regular sections with that of sections pretreated with an enzyme that breaks glycogen (eg, amylase present in the saliva). Structures that stain intensely with the PAS reaction but do not stain after treatment with amylase contain glycogen. Figure 1–19 shows examples of structures stained by the PAS reaction.
Photomicrograph of an intestinal villus stained by PAS. Staining is intense in the cell surface brush border (arrows) and in the secretory product of goblet cells (G) because of their high content of polysaccharides. The counterstain was hematoxylin. High magnification.
Glycosaminoglycans are strongly anionic, unbranched long-chain polysaccharides containing aminated monosaccharides (amino sugars). The complex molecules formed by the attachment of glycosaminoglycan chains to a protein core constitute the proteoglycans. Some of the significant constituents of connective tissue matrices are proteoglycans (see Chapter 5: Connective Tissue and Chapter 7: Cartilage). Unlike the glycoproteins, the carbohydrate chains in proteoglycans constitute the major component of the molecule. Glycosaminoglycans and acidic glycoproteins are strongly anionic because of their high content of carboxyl and sulfate groups. For this reason, they react strongly with the alcian blue dye.
Lipids are best revealed with dyes that are soluble in lipids. Frozen sections are immersed in alcohol solutions saturated with the dye. Sudan IV and Sudan black are the most commonly used dyes. The dye dissolves in the cellular lipid droplets, which become stained in red or black. Additional methods used for the localization of cholesterol and its esters, phospholipids, and glycolipids are useful in diagnosing metabolic diseases in which there are intracellular accumulations of different kinds of lipids.
Several histochemical procedures are frequently used in laboratory diagnosis of diseases that result in the storage of iron, glycogen, glycosaminoglycans, and other substances. Examples are Perls' reaction for iron (eg, hemochromatosis, hemosiderosis), the PAS-amylase reaction for glycogen (glycogenosis), alcian blue staining for glycosaminoglycans (mucopolysaccharidosis), and lipid staining (sphingolipidosis).
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