Simultaneous Differential Staining of Phosphoproteins, Sialoglycoproteins, Hyaluronic Acid, Sulfated Glycosaminoglycans, Proteins, and Nucleic Acids in Human Breast Tissue With a Cationic Carbocyanine Dye

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Abstract
Surgical specimens of normal and malignant human breast tissue fixed in Formalin and embedded in paraffin were stained with 1-ethyl-2-[3-(1-ethylnaphtho[1,2d]thiazolin-2-ylidene)-2-ethylpropenyl]naphtho[1,2d]thiazolium bromide (Ethyl-Stains-all), a metachromatic cationic carbocyanine dye. Proteins stained red at pH 4.3 and were pink or unstained at pH 2.8. Caseins (phosphoproteins) were blue at pH 4.3 and light pink at pH 2.8. Nuclei stained purple, mast cells and plasma cells stained deep red, sulfated glycosaminoglycans stained shades of purple, hyaluronic acid stained blue and sialoglycoproteins stained green at both H ion concentrations. This simple staining method gave information concerning the types of macromolecules present in connective tissue and in secretions within cells, alveolar lumina and ducts; nuclear counterstaining was not required. Enzymatic digestions with Streptomyces hyaluronidase confirmed the presence of variable but easily detectable amounts of hyaluronic acid in the connective tissue stroma of several specimens of breast carcinoma. Hyaluronic acid was also in the stroma of a fibroadenoma from a pregnant woman. Neuraminidase digestion confirmed the presence of sialoglycoproteins in alveolar lumina, within ducts and, in some carcinomas, within cells. Sialoglycoproteins and phosphoproteins were easily detectable because of color difference from pink cytoplasm and purple nuclei. When compared with Alcian blue, periodic acid-Schiff or toluidine blue, Ethyl-Stains-all had advantages in terms of simplicity, specificity and sensitivity for the detection of these macromolecules in pathologic material. This stain was particularly useful in detecting sialoglycoprotein production by normal and malignant tissue and in showing the presence of hyaluronic acid and sulfated glycosaminoglycans in stromal tissue.