thiocarbohydrazide and dimethyl-4-phenylenediamine

The cytochemical reactivity of pulmonary connective tissue matrix component in neonatal and adult rat was evaluated using high iron diamine (HID) to detect sulfate ester end groups and dialyzed iron (DI) to detect sulfated and carboxylated end groups of complex carbohydrates, including glycoproteins and glycosaminoglycans at the ultrastructural level. The HID reaction product, in the form of discrete 5-12 nm silver particles following appropriate intensification with thiocarbohydrazide-silver proteinate, was found associated with cell surfaces, the elastin component of elastic fibers, and at regular intervals along the length of collagen fibers in large airways and deep lung interstitium. Staining was similar in adult and neonatal rats, except in areas where connective tissues were presumably still rapidly developing in the neonatal animals. Here large gaps or spaces containing filamentous structures were observed between collagen and elastic fibers. The distribution of DI-reactive sites was similar to that seen with HID with the exception of elastic fibers in which only the microfibrillar portion stained. The collagen-associated reaction was not regularly disposed like that stained with HID, but rather it formed a tight continuous density around the fiber. These results indicated the presence and location of glycoproteins and glycosaminoglycans in connective tissue ground substance regions prior to the full development of elastic and collagenous elements in neonatal pulmonary airways and parenchyma. They also demonstrate cytochemically the presence of a sulfate ester-containing complex sugar found associated with the elastin component of elastic fibers in the lung.

Topics: Aging; Animals; Animals, Newborn; Collagen; Connective Tissue; Elastic Tissue; Extracellular Matrix; Hydrazines; Iron; Lung; Microscopy, Electron; Phenylenediamines; Rats; Silver Proteins; Staining and Labeling; Trachea

The ultrastructural localization of acidic glycosaminoglycans, presumed to be proteoglycans, was examined during initial matrix vesicle-mediated calcification in dentine, by using ruthenium red (RR) staining, high iron diamine thiocarbohydrazide silver proteinate (HID-TCH-SP) staining, and an enzymatic digestion method. Progenitor predentine 2-10 micron width of developing mouse molar tooth germs was used throughout the present study. The outer surface membrane of the intact matrix vesicles had a strong affinity for RR. The RR positive materials appeared beaded and extended perpendicularly from the vesicle membrane. They tended to disappear with the disruption of the vesicular membrane, which resulted from overextension due to needle-like, crystal-like structures. The HID-TCH-SP stain deposits, approximately 10 nm in diameter, were densely distributed around the intact matrix vesicles, though few were found inside them. Some matrix vesicles that were presumably disrupted, however, contained smaller stain deposits. On the outer surface membrane of the disrupted vesicles, HID-TCH-SP stain deposits were fewer in number. The results obtained from enzymatic degradation studies showed that the anionic materials on the outer surface membrane of the matrix vesicles were represented by chondroitin-4-sulfate and/or chondroitin-6-sulfate. We suggest that chondroitin sulfates attached to the outer leaflet of the vesicular membrane play an important role during the incipient stage of the matrix vesicle-mediated calcification process.

Topics: Animals; Calcium; Chondroitin Sulfates; Dentin; Dentinogenesis; Extracellular Matrix; Glycosaminoglycans; Hyaluronoglucosaminidase; Hydrazines; Mice; Phenylenediamines; Proteoglycans; Ruthenium Red; Silver Proteins; Staining and Labeling; Tooth Calcification; Tooth Germ