Although H&E–stained sections of formalin-fixed specimens are convenient to use because they adequately display general structural features, they cannot elucidate the specific chemical composition of cell components. Also, many components are lost in the preparation of the specimen. To retain these components and structures, other fixation methods must be used. These methods are generally based on a clear understanding of the chemistry involved. For instance, the use of alcohols and organic solvents in routine preparations removes neutral lipids.
To retain neutral lipids, such as those in adipose cells, frozen sections of formalin-fixed tissue and dyes that dissolve in fats must be used; to retain membrane structures, special fixatives
FIGURE 1.1 • Hematoxylin and eosin (H&E) staining. This series of specimens from the pancreas are serial (adjacent) sections that demonstrate the effect of hematoxylin and eosin used alone and hematoxylin and eosin used in combination. a. This photomicrograph reveals the staining with hematoxylin only. Although there is a general overall staining of the specimen, those components and structures that have a high affinity for the dye are most heavily stained−for example, the nuclear DNA and areas of the cell containing cytoplasmic RNA. b. In this photomicrograph, eosin, the counterstain, likewise has an overall staining effect when used alone. Note, however, that the nuclei are less conspicuous than in the specimen stained with hematoxylin alone. After the specimen is stained with hematoxylin and then prepared for staining with eosin in alcohol solution, the hematoxylin that is not tightly bound is lost, and the eosin then stains those components to which it has a high affinity. c. This photomicrograph reveals the combined staining effect of H&E. 480.
containing heavy metals that bind to the phospholipids, such as permanganate and osmium, are used (Folder 1.1). The routine use of osmium tetroxide as a fixative for electron microscopy is the primary reason for the excellent preservation of membranes in electron micrographs.
Hematoxylin and eosin are used in histology primarily to display structural features.
Despite the merits of H&E staining, the procedure does not adequately reveal certain structural components of histologic sections such as elastic material, reticular fibers, basement membranes, and lipids. When it is desirable to display these components, other staining procedures, most of them selective, can be used. These procedures include the use of orcein and resorcin-fuchsin for elastic material and silver impregnation for reticular fibers and basement membrane material. Although the chemical bases of many staining methods are not always understood, they work. Knowing the components that a procedure reveals is more important than knowing precisely how the procedure works.
Specific chemical procedures can provide information about the function of cells and the extracellular components of tissues.
Histochemical and cytochemical procedures may be based on specific binding of a dye, use of a ﬂuorescent dye–labeled antibody with a particular cell component, or the inherent enzymatic activity of a cell component. In addition, many large molecules found in cells can be localized by the process of autoradiography, in which radioactively tagged precursors of the molecule are incorporated by cells and tissues before fixation. Many of these procedures can be used with both light microscopic and electron microscopic preparations.
Before discussing the chemistry of routine staining and histochemical and cytochemical methods, it is useful to examine briefly the nature of a routinely fixed and embedded section of a specimen.
Chemical Composition of Histologic Samples
The chemical composition of a tissue ready for routine staining differs from living tissue.