Ultrastructural methods for localizing complex carbohydrates

Abstract

Carbohydrate-rich macromolecules in mammalian tissue fall into two main groups, glycoproteins and glycosaminoglycans. Although glycoproteins occur mainly in epithelial cells and glycosaminoglycans in connective tissue interstices, exceptions are known. Chemical constituents that are characteristic of glycoproteins and provide a basis for their selective demonstration cytochemically include the sulfate ester, presumably bound to an internal sugar, the carboxyl group of terminal sialic acid, and the vicinal glycol of hexose (galactose or mannose) and deoxyhexose (fucose). Characteristic chemical components of glycosaminoglycans that are detectable cytochemically include the sulfate ester, usually linked to an aminosugar, and the carboxyl group of glucuronic or iduronic acid. Individual sugars of glycoconjugates, particularly those in a terminal positions, are also identifiable cytochemically. Cytochemical methods are available to detect specifically one or more of the moieties characteristic of glycoproteins or glycosaminoglycans at the electron microscopic level. The sulfate esters can be localized specifically by pre-embedment staining with the high-iron diamine (HID) method. Dialyzed iron (DI) and cationized ferritin stain both carboxyl groups and sulfate esters when similarly applied before embedment. In addition, these cationic reagents can be utilized to stain at least some sulfated glycocon-jugates in post-embedment procedures carried out on ultrathin sections. Ruthenium red in the fixative solution localizes glycoconjugate and possibly other constituents on the cell surface or in the extracellular space. The periodic acid-thiocarbohydrazide-silver proteinate (PA-T-SP) method and the tannic acid-uranyl acetate sequence carried out after embedment on epoxy thin sections demonstrate sugar residues of glycoconjugates, extending light microscopic periodic acid-Schiff (PAS) staining to the ultrastructural level. Further specificity can be achieved by pre- or post-embedment staining with conjugates of horseradish peroxidase or ferritin to a lectin with affinity for a specific sugar in a complex carbohydrate and by enzymatic digestion with specific glycosidase to decrease or increase staining with the above mentioned methods. DI-positive, HID-negative sites can be interpreted as containing nonsulfated, carboxylated complex carbohydrate, and HID-positive sites as containing sulfated mucosubstance. DI- or HID-positive sites lacking PA-T-SP reactivity, as a rule, consist of glycosaminoglycans, but infrequently contain periodate-negative glycoprotein. On the other hand, PA-T-SP staining and DI or HID non-reactivity evidence the presence of neutral glycoprotein, whereas PA-T-SP positivity plus basophilia indicates acidic glycoprotein. Examples of applications of these methods are presented. The applications to normal tissue provide a basis for comparison with diseased specimens and point to the potential of carbohydrate histochemistry for investigating and diagnosing pathologic change.