Special Stains For Mucin Evaluation: Alcian Blue / PAS, Mucicarmine

Mucins are high molecular weight glycoproteins that are found dispersed throughout the epithelia of the gastrointestinal, respiratory and reproductive tract. Mucins are composed of a central protein core with multiple chains of carbohydrates (polysaccharides) attached. The carbohydrate content of the mucin molecule may account for 60-80% of the molecular weight of the molecule. The protein core contains a high content of the amino acids' serine and threonine. A defining structure of the mucins is the presence of tandem repeats of specific amino acid sequences within the protein core. From a molecular perspective, mucins are categorized into distinct families (Muc 1, Muc 2, Muc 3, etc.) based upon differences in the sequence and size of the tandem repeats.

There are a number of other glycoprotein molecules which share structural similarities with the mucins and often are confused with mucins. Proteoglycans are high molecular weight glycoconjugate complexes that are found in high concentrations within the extracellular matrix as well as connective tissues. In older literature, proteoglycans frequently are referred to as connective tissue mucins. However, the structure, (particularly the protein core of the proteoglycans), is different and distinct from that of mucins.

The protein core of the mucins has little or no bearing on the histochemical reactivity of the mucins with the dyes used in special stain techniques. In fact, the histochemical reactivity is dependent largely upon the carbohydrate composition of the mucins. The word “carbohydrate” was coined over a hundred years ago to describe a diverse family of molecules of the general formula Cn(H20)n. The word “carbohydrate” actually is descriptive of the 1:1 ratio of carbon molecules to water (hydrate). The glucose molecule (Figure 1) demonstrates the structure of the typical carbohydrate monosaccharide. Other similar monosaccharides are mannose and galactose. These molecules are not charged as they demonstrate no ionizable groups under normal conditions. In contrast other monosaccharides may contain acidic groups such as carboxyl (COOH) and sulphonic (SO3H) groups that are capable of ionization to confer an overall negative charge on the molecule. The carboxylated monosaccharide N-acetylneuraminic acid which is commonly known as sialic acid is shown in (Figure 2). The presence of these types of functional groups defines in part the potential reactivity with histochemical stains.

From a histochemical perspective, mucins can be placed in one of two broad categories based upon the chemistry and composition of the carbohydrate component of the mucin. The charged or “acid” mucins contain carbohydrates with carboxylate (COO-) or sulphonate (SO3) groups. Both of these groups are ionized at a physiologic pH to produce an overall negative charge on these mucin molecules. The carbohydrate chains of neutral mucins lack acidic groups and thus carry no net charge. The neutral mucins can be found primarily in the surface epithelia of the stomach, Brunner’s glands of the duodenum and in the prostatic epithelium. The acid mucins are found widely distributed throughout the gastrointestinal tract and the respiratory tract.

The typical mucin special stains contain cationic (positively charged) dye molecules in solution at a specific pH. This is true in the case of mucicarmine, Alcian blue as well as the older metachromatic techniques that utilized such dyes as azure A or toluidine blue. The cationic dye molecules bind via electrostatic forces to the anionic carboxylated or sulfated polysaccharide chains of the mucin molecules.