Histological Stains

Staining Reactions Staining reactions have both physical and chemical characteristics. The mechanisms involved in staining include the following: The dye may actually be dissolved in the stained substance. Most fat staining is accomplished in this fashion. A dye may be absorbed on the surface of a structure, or dyes may be precipitated within the structure, simply because environmental factors (pH, ionic strength, temperature, etc.) favor absorption or precipitation. Most staining reactions involve a chemical union between dye and stained substance through salt linkages, hydrogen bonds, or others. Staining with these dyes results in a predictable color pattern based in part on the acid base characteristics of the tissue. However, color and color distribution are not absolutely reliable for discrimination between tissue components. Color will vary not only with specific stains used, but also with the conditions that exist during preparation of the slide. These include everything from the initial fixing solution to the ionic strength of the staining solution and the differentiating solvents utilized after staining. Acid and Basic Dyes

substance that is stained by an acid dye is referred to as acidophilic; it carries basic groups which bind the acid dye. With eosin, acidophilic structures appear in various shades of pink. Since eosin is a widely used acid dye, acidophilic substances are frequently referred to as eosinophilic. Trichrome Stains

In the trichrome stains, which commonly employ more than one acid dye, use is made of dye competition. For instance, acid fuchsin and picric acid are used in Van Gieson's trichrome stain. In the picric acid-fuchsin mixture, the small picric acid molecule reaches and stains the available sites in muscle before the larger fuchsin molecules can enter. Used by itself, acid fuchsin has no difficulty in staining muscle. Neutral Stains

Most histologic dyes are classified either as acid or as basic dyes. An acid dye exists as an anion (negatively charged) in solution, while a basic dye exists as a cation (positive charge). For instance, in the hematoxylin-eosin stain (H&E), the hematoxylin-metal complex acts as a basic dye. The eosin acts as an acid dye. Any substance that is stained by the basic dye is considered to be basophilic; it carries acid groups which bind the basic dye through salt linkages. When using hematoxylin, basophilic structures in the tissue appear blue (or purple or brown; this varies according to the stain that is being used). A

These are compounds of an acid dye and basic dye. For instance, aqueous solutions of acid fuchsin may be neutralized by addition of aqueous methyl green. The resulting neutral product is water insoluble, but may be kept in solution by the presence of excess amounts of either component. The tissue stained with such a solution may show affinity for the acid dye, the basic dye, and for the whole compound. Some blood stains are "neutral stains." Wright's Stain, for instance, is formed by the combination of partially oxidized and demethylated methylene blue and eosin. Such a stain can be used to differentiate between blood cells that contain acidic, basic, and neutral granules.

Characteristics of Commonly used Stains Hematoxyl and Eosin (H&E)

Stains elastic fibers purple to black. Can be counter-stained with a dye of contrasting color, such as metanil yellow. Example: slide 42d. Verhoeff's Hematoxylin

This is a good general stain and is widely used. Most of your slides are stained with H&E. A hematoxylin-metal complex acts a as a basic dye, staining nucleic acids in the nucleus and the cytoplasm blue, brown, or black. Eosin is an acid aniline dye which stains the more basic proteins within cells (cytoplasm) and in extracellular spaces (collagen) pink to red. Cartilage and mucus may stain light blue. Example: slide 42c. Masson Trichrome Stain

Another variant of the versatile hematoxylin stains. This method stains elastic fibers black in addition to nuclei. Example: slide 71d. Reticular Fiber Stain - Weigert

A staining sequence involving iron hematoxylin, acid fuchsin, and light green. It is a good stain for distinguishing cellular from extracellular components. Collagen fibers stain an intense green. Black or brown nuclei; mucus and ground substances take on varying shades of green. Cytoplasm stains red. Elastic fibrils, red blood cells and nucleoli stain pink. Example: slide 56-l. Aldehyde Fuchsin

Reticular fibers are impregnated with a silver salt and appear as sharp black. Collagenous fibers usually stain purple. This stain can be used with a counterstain (such as slide 21c) or without, if the silver stain turned out very dark (such as slide 22b). Wright's/Giemsa Stain

This and similar stains for blood and bone marrow smears are mixtures of basic (methylene blue derivatives) and acid dyes (usually eosin). According to the number of acid and basic groups present, cell components take up the dyes

from the mixture in various proportions. Example: slide 17b. Some blood stains use acid and basic dyes in separate dye baths. Metachromatic Stain

also known as the Schiff's Reagent. Periodic Acid Schiff (PAS)

Certain basic dyes, such as toluidine blue, stain nucleic acids blue (the orthochromatic color), but sulfated polysaccharides purple (the metachromatic color). When dye molecules bound to sulfate groups are stacked closely together, the dye experiences a color shift from blue to purple. Thus, a metachromatic reaction often indicates the presence of numerous closely packed sulfate groups. Plastic Sections Stained with Toluidine Blue or with H&E

Adjacent hydroxyl groups (1, 2 glycols) or amino and hydroxyl groups are oxidized to aldehyde groups with periodic acid. Schiff's Reagent then produces a red or magenta addition product with the aldehyde groups and this technique identifies a number of polysaccharides and carbohydratecontaining compounds. The slide may also be counter stained with hematoxylin. Example: slide 56b. Feulgen Reaction: Mild hydrolysis with HCl frees the aldehyde group of deoxyribose, which is then reacted with the Schiff's reagent. This reaction is highly specific for DNA and may also be used with a counter stain for the cytoplasm. Enzyme-histochemical Techniques These techniques localize various enzymes within cells and tissues by making use of the enzyme activity itself. The enzyme is made to react with a specific substrate. The product of this reaction may itself be visible in the microscope and thus demonstrate the presence of the enzyme at a specific location, or the reaction product is subsequently reacted to form a visible secondary reaction product. Example: Reaction for acid phosphatase. 1) The tissue is gently fixed in order to preserve acid phosphatase activity. 2) Sections are incubated in a medium consisting of betaglycerophosphate (the specific substrate), lead nitrate ("the capturing agent" for the secondary reaction), buffer at pH 5 (the pH for optimal activity of the acid phosphatase). During incubation, the acid phosphatase splits phosphate groups off the betaglycerophosphate. The phosphate groups immediately react with the lead ions to form insoluble lead phosphate, which precipitates at the location of the enzyme. Lead phosphate is visible in the electron microscope or can be made visible in the light microscope with a further reaction. This type of reaction can be modified to demonstrate a wide variety of enzymes.

Plastic embedded tissues can be cut as thin as 0.1 m with a glass knife. These sections are then stained with toluidine blue in an alkaline solution. Almost all tissue components are stained more or less deeply (usually a bluish-purple) and structural detail is very sharp. For the knowledgeable observer, this type of preparation may be very informative. Example: slide 56c. Histochemical Techniques In contrast to the staining techniques described above, the mechanism of a histochemical technique is usually well understood. If properly controlled, the technique is chemically specific, highly reproducible and capable of providing information that can then be quantitated. The following two methods make use of leuko-fuchsin,

Immunohistochemical Techniques These techniques are well described in the text. They are the most specific and sensitive methods available for identifying specific substances (antigens) within cells and tissues. They utilize antibodies that are made against specific antigens and enable us to localize specific substances within cells and tissues.