The light microscopic study of cells, tissues, and organs requires that the material to be examined be sectioned thinly enough to permit light to penetrate it and that the light be sufficient to be collected by the lenses of the microscope and to reach the retina of the examiner. Moreover, the tissue has to maintain its natural, living characteristics; otherwise, the viewer will have a distorted picture of the tissue. Over the years, numerous procedures were developed and refined to make sure that there is a close resemblance between the image under the microscope and the properties of the tissue while it was in its living state. These procedures include fixation, dehydration, clearing, embedding, sectioning, mounting, staining, and the affixing of a coverslip over the section.

•Fixation is the use of chemicals that inhibits tissue necrolysis and prevents alteration of its normal morphology. For light microscopy, the fixative of choice is neutral buffered formalin, although many other fixatives are commonly used.

•Dehydration and clearing are accomplished by using an increasing concentration of ethanol (from 50% to 100%) followed by a clearing agent such as Xylene to make the tissue transparent and miscible with an embedding material.

•Embedding and sectioning is the process that permeates the tissue with an agent, such as paraffin or

a plastic polymer, that can be sliced into sections thin enough to be transparent to visible light. Tissues embedded in paraffin are usually sectioned 5 to 10 μm in thickness, whereas those embedded in plastic are sectioned much thinner (≤0.1 μm). Many other embedding media and sectioning techniques are also available.

• Sections obtained from paraffin or plastic blocks are mounted on glass slides coated with an adhesive

material, such as albumin, to ensure that the sections adhere to the glass slides.

•Staining of the sections is necessary because the optical densities of the various tissue elements are so similar that they are indistinguishable from one another without being treated with various dyes. Because many of the stains used are water miscible, the sections must be deparaffinized and rehydrated before they can be stained.

•The stained sections have to be dehydrated once again to permit them to be made permanent by the placing and affixing of a coverslip over the tissue section.

Terminology of Staining

Frequently, when staining histological sections, a principal stain is used in conjunction with a counterstain, a contrasting color that stains those components of the tissue that are not stained well with the principal stain. Usually, the stains are either acidic (anionic) or basic (cationic) and are attracted to those components of the cell or tissue that are basic or acidic, respectively. Therefore, the acidic components of the cell, such as nucleic acids, attract the basic stains and are said to be basophilic. Those components of the cell whose pH is greater than 7, such as many cytoplasmic proteins, attract acidic stains and are said to be acidophilic.

Common Stains Used in Histology

Although a great number of histologic and histopathologic stains have been developed, only the most commonly used stains are listed in this Appendix.

480 A P P E N D I X

Hematoxylin and Eosin

Hematoxylin, in conjunction with the counterstain eosin, is one of the most commonly used stains in histological and histopathological preparations. Hematoxylin is a basic stain that dyes nuclei, nucleoli, and ribosomes blue to purple in color. Eosin stains basic components of the cell, including myofilaments of muscle, pink to light red in color. Red blood cells stain orange to bright red in color. Additionally, extracellular matrix proteins, such as collagen, are also stained pink to light red.

Wright Stain

Wright stain and the related Giemsa stain are designed specifically for staining cells of blood. It stains erythrocytes salmon pink; nuclei of leukocytes and granules of platelets stain dark blue to purple, whereas the specific granules of eosinophils stain salmon pink, and those of basophils stain dark blue to black. The cytoplasm of lymphocytes and monocytes stains a light blue in color.

Weigert Method for Elastic Fibers and Elastic van Gieson Stain

Weigert method and van Gieson stain for elastic fibers are both used commonly to stain elastic fibers. They both dye elastic fibers dark blue to black. Since nuclei also stain dark gray to black, the fibroblasts present among the elastic fibers are very difficult to see.

Silver Stain

Silver stain uses silver salts in solution that precipitate out as silver metal on the surfaces of type III collagen fibers (reticular fibers), staining them black. Some cells, such as diffuse neuroepithelial cells, also stain with silver stains and were called argentaffin or argyrophilic cells. Their granules stain brown to black with silver stains.

Iron Hematoxylin

Bielschowsky Silver Stain

Bielschowsky stain uses silver salts to permeate the tissue, and then the silver is reduced so that it stains dendrites and axons black. The surrounding tissues are golden brown yellow with a tinge of red in the cytoplasm, and the nucleoli are black.

Masson Trichrome

As the name implies, this stain produces three colors and is used to differentiate collagen of connective tissues from muscle and other living cells. Depending on the variant used, collagen is stained blue or green, muscle cells are red, cytoplasm of non-muscle cells are a pink to light red, and nuclei stain black. (Reprinted with permission from Mills SE, Carter D, et al., eds. Sternberg’s Diagnostic Surgical Pathology, 5th ed., Philadelphia: Lippincott, Williams & Wilkins, 2010. p. 1694.)

Periodic Acid-Schiff Reaction (PAS)

PAS reaction stains glycogens, glycoproteins, mucins, and glycolipids. Thus, basement membranes stain pinkish red, whereas mucins of goblet cells and of mucous salivary glands stain a deep red to magenta. (Reprinted with permission from Mills SE, ed. Histology for Pathologists, 3rd ed., Philadelphia: Lippincott,Williams & Wilkins, 2007. p. 608.)

482 A P P E N D I X

Alcian Blue

Alcian blue is specific for staining mucins, glycoproteins, and the matrix of cartilage blue in color, whereas the cytoplasm stains a light pink and nuclei stain red. (Reprinted with permission from Mills SE, ed. Histology for Pathologists, 3rd ed., Philadelphia: Lippincott, Williams & Wilkins, 2007. p. 415.)

von Kossa Stain

von Kossa stain uses silver salts that become reduced to demonstrate calcification and calcified tissues that stain black. (Reprinted with permission from Rubin R, Strayer D, et al., eds. Rubin’s Pathology. Clinicopathologic Foundations of Medicine, 5th ed., Baltimore: Lippincott, Williams & Wilkins, 2008, p. 1113.)

Sudan Red

Sudan red is used to stain lipids, phospholipids, lipoproteins, and triglycerides, all of which stain an intense red with this dye. (Reprinted with permission from Rubin R, Strayer D, et al., eds. Rubin’s Pathology. Clinicopathologic Foundations of Medicine, 5th ed., Baltimore: Lippincott Williams & Wilkins, 2008, p. 239.)

Mucicarmine Stain

As its name implies, mucicarmine is used to localize mucin, which it stains a deep red color. The cytoplasm appears a light, salmon pink, nuclei are stained bluish black, and connective tissue is stained a yellowishorange color. (Reprinted with permission from Rubin R, Strayer D, et al., eds. Rubin’s Pathology. Clinicopathologic Foundations of Medicine, 5th ed., Baltimore: Lippincott, Williams & Wilkins, 2008, p. 541.)