Staining of Mast Cell Acid Glycosaminoglycans in Ultrathin Sections by Ruthenium Red

Gustafson, Gunnar T.; Pihl, Erik

doi:10.1038/216697a0

Cited by 39 publications

(8 citation statements)

References 5 publications

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“…The use of a hydrophilic embedding medium (LR-White) can result in the effective penetration of histochemical reagents into cells and tissues (16), and non-specific RR-TT reactions of other anionic substances than carbohydrates are unlikely by virtue of the low pH levels (17,23). Further, the electron density of reaction products obtained by the present method is much higher than that yielded by the previous techniques (6,15).…”

Section: Resultsmentioning

confidence: 61%

“…The RR-TT staining method developed in the present study is free from the weaknesses inherent in the previous methods (6,15). The use of a hydrophilic embedding medium (LR-White) can result in the effective penetration of histochemical reagents into cells and tissues (16), and non-specific RR-TT reactions of other anionic substances than carbohydrates are unlikely by virtue of the low pH levels (17,23).…”

Section: Resultsmentioning

confidence: 99%

“…4, 5, 7-9) and silicotungstic ( Fig. 1) RR is known to be one of the cationic dyes used for the histochemical detection of anionic sites in tissues (2,3,6,14,15,28). Gustafson and Pihl (6) employed glycol methacrylate (GMA)-embedded tissue sections for the postembedding RR staining at a neutral pH levels.…”

Section: Resultsmentioning

confidence: 99%

“…The majority of these staining procedures employed staining reagents applied to tissue specimens at preembedding stages of tissue preparations, and such procedures suffered from the limited penetration of staining reagents into cells. To overcome such weaknesses, a series of staining reagents were applied to tissue specimens at postembedding stages of tissue preparations (4,6,9,15,18,30). At first glance, these postembedding staining methods appeared to be satisfactory for visualizing acidic glycoconjugate-containing ultrastructures in electron microscopy.…”

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confidence: 99%

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Electron microscopic visualization of acidic glycoconjugates by means of postembedding procedures using ruthenium red and tungstate.

Hirabayashi

Yamada

1990

Acta Histochem. Cytochem

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Postembedding staining methods using ruthenium red (RR) incombination with tungstate (TT) (phospho-or silicotungstic acid) (RR-TT) were tested for the effective visualization of acidic glycoconjugates in electron microscopy. Tissue blocks of trachea and colon from adult male Wistar rats were fixed in phosphatebuffered glutaraldehyde-paraformaldehyde solution and embedded in LR-White. Ultrathin sections were cut, stained first with RR (pH 2.5) and then incubated in TT. In the tissues examined, all the ultrastructures containing acidic glycoconjugates exhibited apparent positive reaction for RR-TT. If stained singly with RR or TT under the same staining conditions, most of these ultrastructures failed to give any distinct electron densities. In all the acidic glycoconjugate-containing ultrastructures tested, digestion with testicular hyaluronidase greatly diminished or abolished their RR-TT reactions. In view of these results, the present RR-TT methods can be regarded as a useful technique for the efficient visualization of acidic glycoconjugates in electron microscopy.In different mammalian species, it has been well known that intra-and extracellular ultrastructures contain acidic glycoconjugates which play an important role in living organisms. To visualize such acidic glycoconjugates, numerous attempts have been made using electron microscopic histochemical methods with basic dyes [ruthenium red (2, 3, 14), alcian bule (1, 25), cuprolinic blue (21, 26), high and low iron diamine (20,22,24)] and colloidal ions [dialyzed iron, (7, 12, 29), colloidal iron (27), colloidal thorium (18)]. The majority of these staining procedures employed staining reagents applied to tissue specimens at preembedding stages of tissue preparations, and such procedures suffered from the limited penetration of staining reagents into cells. To overcome such weaknesses, a series of staining reagents were applied to tissue specimens at postembedding stages of tissue preparations (4,6,9,15,18,30). At first glance, these postembedding staining methods appeared to be satisfactory for visualizing acidic glycoconjugate-containing ultrastructures in electron microscopy. If closely examined, however, these acidic glycoconjugate-containing ultrastructures were found to be contaminated with varying degrees of non-specific background stain- 165 Correspondence to:

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Section: Resultsmentioning

confidence: 61%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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Electron microscopic visualization of acidic glycoconjugates by means of postembedding procedures using ruthenium red and tungstate.

Hirabayashi

Yamada

1990

Acta Histochem. Cytochem

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“…Peritoneal rat mast cells were isolated from ~6-week old adult rats based on a modification of widely used methods for mast cell isolation and staining (Gustafson and Pihl, 1967; Nemeth and Rohlich, 1980; Martynova et al, 2005). After rats were sacrificed by CO 2 fixation and decapitation, their abdomens were skinned, followed by injection of ~60 ml of sterile phosphate buffered saline (PBS).…”

Section: Methodsmentioning

confidence: 99%

Metabolic engineering of Chinese hamster ovary cells: Towards a bioengineered heparin

Baik

Gasimli

Yang

et al. 2012

Metabolic Engineering

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Heparin is the most widely used pharmaceutical to control blood coagulation in modern medicine. A health crisis that took place in 2008 led to a demand for production of heparin from non-animal sources. Chinese hamster ovary (CHO) cells, commonly used mammalian host cells for production of foreign pharmaceutical proteins in the biopharmaceutical industry, are capable of producing heparan sulfate (HS), a related polysaccharide naturally. Since heparin and HS share the same biosynthetic pathway, we hypothesized that heparin could be produced in CHO cells by metabolic engineering. Based on the expression of endogenous enzymes in the HS / heparin pathways of CHO-S cells, human N-deacetylase/N-sulfotransferase (NDST2) and mouse heparan sulfate 3-O-sulfotransferase 1 (Hs3st1) genes were transfected sequentially into CHO host cells growing in suspension culture. Transfectants were screened using quantitative RT-PCR and Western blotting. Out of 120 clones expressing NDST2 and Hs3st1, 2 clones, Dual-3 and Dual-29, were selected for further analysis. An antithrombin III (ATIII) binding assay using flow cytometry, designed to recognize a key sugar structure characteristic of heparin, indicated that Hs3st1 transfection was capable of increasing ATIII binding. An anti-factor Xa assay, which affords a measure of anticoagulant activity, showed a significant increase in activity in the dual-expressing cell lines. Disaccharide analysis of the engineered HS showed a substantial increase in N-sulfo groups, but did not show a pattern consistent with pharmacological heparin, suggesting that further balancing the expression of transgenes with the expression levels of endogenous enzymes involved in HS / heparin biosynthesis might be necessary.

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Ruthenium red and violet. II. Fine structural localization in animal tissues

1971

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The inorganic dye, ruthenium red, stains extracellular materials in animal tissues which probably are acidic mucopolysaccharides. It complements other techniques, its advantages being fine grain, high resolution and good contrast. Localization is shown in mouse and rat muscle, heart, lung and intestine, frog cartilage and cells scraped from oral epithelium of human beings. Attention is paid to collagen bundles, the cell/collagen interface and particularly the myotendinal junction, cartilage matrix and agar gel, desmosomes, intestinal microvilli, erythrocytes and vascular endothelium, nerve fibers and the T-system of striated muscle. Although ruthenium red generally is excluded by plasma membranes, it penetrates giving intracellular density, if the membrane is broken. Even when the cell membrane is intact, exceptions occur with selective staining of the T-tubules or the sarcoplasmic sacs depending upon the state of contraction of the muscle cell, and with intracellular staining of certain nuclei and epithelial cells. Ruthenium red stains intracellular lipid droplets revealing lamelJae, and stains myelin forms grown from crude egg lecithin but cannot penetrate deeply. It is localized in extracellular materials which have an important mechanical function. Its exclusion by cell membranes permits tracing tortuous cellular invaginations and those exceptions to its exclusion invite a comparison of the localization of the dye with the function of the cell.Cells advertise themselves to the outside world and to each other by their surfaces. Scientists, however, are better acquainted with cells in terms of their internal organelles and molecules, having violated their privacy with microscopes, homogenizers, and separation procedures. The research directed toward this outer surface, called the plasma membrane, has been concerned mainly with the lipids and proteins which are known to reside there. However, during the 1960's evidence accumulated prompting consideration of another component which is minor in terms of dry mass but major in terms of function. This third component is polysaccharide, usually present as a glycoprotein. Its importance was strongly emphasized from a morphological standpoint, perhaps f m t by Bennett, who, in 1963, coined the general term "glycocalyx," although Chambers by 1940 had designated similar layers in other cells as "extraneous coats," and their history can ANAT. REC., 171: 369-416.be traced much earlier. In 1966, Rambourg, Neutra and Leblond published an important paper after examining a wide variety of tissues with several histochemical tests for polysaccharides. They concluded that "nearly all cells investigated are covered by a thin but definite band of stained material indicating the existence of a surface layer." They also felt that "the layer is not part of the plasma membrane itself but is a surface coat." The biochemical difficulties which hindered work on these substances as well as their general importance in biology has been elegantly reviewed recently by G. M. W. Cook ('6...

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Staining of Mast Cell Acid Glycosaminoglycans in Ultrathin Sections by Ruthenium Red

Cited by 39 publications

References 5 publications

Electron microscopic visualization of acidic glycoconjugates by means of postembedding procedures using ruthenium red and tungstate.

Electron microscopic visualization of acidic glycoconjugates by means of postembedding procedures using ruthenium red and tungstate.

Metabolic engineering of Chinese hamster ovary cells: Towards a bioengineered heparin

Ruthenium red and violet. II. Fine structural localization in animal tissues

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