Ultrastructural visualization of complex carbohydrates in epiphyseal cartilage with the tannic acid-metal salt methods.

Abstract

The present study has ultrastructurally applied the tannic acid-ferric chloride (TA-Fe) and the TA-uranyl acetate (TA-UA) methods to thin sections of glutaraldehyde-fixed, unosmicated embedded epiphyseal cartilage from rat tibiae to demonstrate complex carbohydrates. The strongest TA-Fe and TA-UA staining was observed after fixation of the specimens in glutaraldehyde containing TA. TA-Fe (pH 1.5) strongly stained matrix granules presumed to be proteoglycan monomers and chondrocyte secretory granules at various maturational stages but did not stain collagen fibrils and glycogen. TA-UA (pH 4.2) strongly stained matrix granules, intracellular glycogen, and chondrocyte secretory granules, and moderately stained collagen fibrils in the cartilage matrix. Ribosomes and nuclei were not stained above background staining with UA alone. In alpha-amylase-digested specimens, all TA-UA-reactive cytoplasmic glycogen was selectively removed. Testicular hyaluronidase digestion of specimens selectively removed TA-UA staining in matrix granules and all TA-Fe staining. When the pH of the UA solution was reduced to 1.5, TA-UA staining of glycogen and collagen was markedly decreased or absent, whereas staining of anionic sites was unaltered and significantly greater than with UA staining alone. Thus the TA-metal salt methods are pH dependent and allow differential intracellular and extracellular localization of complex carbohydrates in cartilage tissues at the electron microscope level.

Ultrastructural distribution of sulfated complex carbohydrates in elastic cartilage of the young rabbit

“…Thin sections mounted on stainless steel grids were treated for 10 minutes with a filtered fresh TA solution, pH 2.6-2.8, containing 5 gm tannic acid (J.T. Baker Chemical Co., Phillipsburg, NJ) in 95 ml of distilled water, rinsed three times in distilled water, and treated for 1 minute with a filtered fresh ferric chloride (Fe) solution (pH 1.4-1.6) which was prepared by adding 5 ml of 40% ferric chloride (Fisher Scientific Co., Fair Lawn, NJ) to 95 ml of distilled water (Sannes et al, 1978;Takagi et al, 1983). Subsequently, stained sections were rinsed six times in distilled water and examined.…”

Postembedment staining of complex carbohydrates: Influence of fixation and embedding procedures

“…Sectioning was performed with a diamond knife, except for the polystyrene blocks, Gustafson and Pihl, Geyer et al, 1971Courtoy et al, 1974Revel, 1964Groot, 1981Matukas et al, 1967Thiery, 1970 Thiery and Thomopoulos et al, Ovtracht, 19791983bMonga et al, 1972Thomopoulos et al, 1983cTakagi et al, 1983 ferric chloride formaldehide Quatacker, 1985 which were sectioned with glass knives. Thin sections were placed on uncoated grids, except for the sections obtained from the tissues embedded in polystryrene and styrenemethacrylate, which required some form of support such as formvar-coated grids.…”

The presence of an extracellular matrix between cells involved in the determination of the mesoderm bands in embryos ofPatella vulgata (Mollusca, gastropoda)

“…Also, the lamina-densa-like layer looks different after the addition of tannic acid to the fixative, having a granular appearance. The diameter of the granules is about 30 nm, which is in the order of proteoglycan monomers (Takagi et al 1983). …”

“…Apparently, fixation with tannic acid leads to the preservation of one or more extra components in the lamina-rara-like layer. Particularly acidic glycosaminoglycans (GAG) are lost by the routine method of fixation with glutaraldehyde only, but they are preserved if tannic acid is included (Takagi et al 1983). The presence of the electron-dense layer adjacent to the outer surface of the cell membrane after the addition of the mordant tannic acid represents GAG (Sannes et al 1978;Singley and Solursh 1980;Spicer et al 1981;Toole 1981).…”

“…These granules had the same size (approx. 30 nm) and looked like proteoglycan monomers as observed by Takagi et al (1983) in epiphyseal cartilage and like the proteoglycan granules in the basement membrane of sea urchin embryos (Spiegel et al 1983).…”