Cell and tissue structures that have high concentrations of ionized sulfate and phosphate groups—such as the ground substance of cartilage, heparin-containing granules of mast cells, and rough endoplasmic reticulum of plasma cells—exhibit metachromasia. Therefore, toluidine blue will appear purple to red when it stains these components.
Aldehyde Groups and the Schiff Reagent
The ability of bleached basic fuchsin (Schiff reagent) to react with aldehyde groups results in a distinctive red color and is the basis of the periodic acid–Schiff and Feulgen reactions.
The periodic acid–Schiff (PAS) reaction stains carbohydrates and carbohydrate-rich macromolecules. It is used to demonstrate glycogen in cells, mucus in various cells and tissues, the basement membrane that underlies epithelia, and reticular fibers in connective tissue. The Feulgen reaction, which relies on a mild hydrochloric acid hydrolysis, is used to stain DNA.
The PAS reaction is based on the following facts:  Hexose rings of carbohydrates contain adjacent carbons, each of which bears a hydroxyl (–OH) group.  Hexosamines of glycosaminoglycans contain adjacent carbons, one of which bears an –OH group, whereas the other bears an amino (–NH2) group.  Periodic acid cleaves the bond between these adjacent carbon atoms and forms aldehyde groups. These aldehyde groups react with the Schiff reagent to give a distinctive magenta color.
The PAS staining of basement membrane (Fig. 1.2) and reticular fibers is based on the content or association of proteoglycans (complex carbohydrates associated with a protein core). PAS staining is an alternative to silver-impregnation methods, which are also based on reaction with the sugar molecules in the proteoglycans.
The Feulgen reaction is based on the cleavage of purines from the deoxyribose of DNA by mild acid hydrolysis; the sugar ring then opens with the formation of aldehyde groups. Again, the newly formed aldehyde groups react with the
FIGURE 1.2 • Photomicrograph of kidney tissue stained by the PAS method. This histochemical method demonstrates and localizes carbohydrates and carbohydrate-rich macromolecules. The basement membranes are PAS positive as evidenced by the magenta staining of these sites. The kidney tubules (T ) are sharply delineated by the stained basement membrane surrounding the tubules. The glomerular capillaries (C) and the epithelium of Bowman’s capsule (BC) also show PAS-positive basement membranes. 360.
 FOLDER 1.2 Functional Considerations: Feulgen Microspectrophotometry is a technique devel-sion. Currently, Feulgen microspectrophotometry is used to study changes in the DNA content in dividing cells undergoing differentiation. It is also used clinically to analyze abnormal chromosomal number (i.e., ploidy patterns) in malignant cells. Some malignant cells that have a largely diploid pattern are said to be well differentiated; tumors with these types of cells have a better prognosis than tumors with aneuploid (nonintegral multiples of the haploid amount of DNA) and tetraploid cells. Feulgen microspectrophotometry has been particularly useful in studies of specific adenocarcinomas (epithelial cancers), breast cancer, kidney cancer, colon and other gastrointestinal cancers, endometrial (uterine epithelium) cancer, and ovarian cancer. It is one of the most valuable tools for pathologists in evaluating the metastatic potential of these tumors and in making prognostic and treatment decisions.