scholarly journals Surface coat on the epithelium of developing palatine shelves in the mouse as revealed by electron microscopy

Development ◽  
1974 ◽  
Vol 31 (3) ◽  
pp. 683-692
Author(s):  
R. M. Greene ◽  
D. M. Kochhar
Keyword(s):  
Electron Microscopy ◽  
Fine Structure ◽  
Ruthenium Red ◽  
Entire Length ◽  
Surface Epithelium ◽  
Positive Staining ◽  
Surface Coat ◽  
In Vitro Experiments ◽  
Acid Mucopolysaccharides

The fine structure of the surface epithelium of developing palatine shelves in the mouse was studied from days 11 through 14 of gestation. Ruthenium red, a cationic stain used as an ultrastructural indicator of acid mucopolysaccharides, was employed to detect the presence of any surface coat. Positive staining was first observed on day 12 of gestation and was seen to be present throughout the period of shelf elevation and fusion. It was seen over medial and lateral surfaces as well as the inferior tip of vertical shelves. The surface coat was found to be present along the entire length of the shelf, extending superiorly up the medial and lateral epithelial borders until it abruptly disappeared. Since this surface coat first appeared approximately 48 h prior to shelf elevation, it is suggested that its appearance may be associated with the ability of palatine shelves to undergo fusion as shown by previous in vitro experiments. The time of acquisition by the shelves of this ‘fusing potential’ is also in the range of 48 h before shelf elevation.

Cell surface saccharides of Trypanosoma lewisi. I. Polycation-induced cell agglutination and fine-structure cytochemistry

1975 ◽  
Vol 19 (3) ◽  
pp. 621-644
Author(s):  
D.M. Dwyer
Keyword(s):  
Fine Structure ◽  
Cell Surface ◽  
Alcian Blue ◽  
Chondroitin Sulphate ◽  
Surface Membrane ◽  
Ruthenium Red ◽  
Surface Coat ◽  
Cell Agglutination ◽  
Trypanosoma Lewisi ◽  
Cationic Compounds

Trypanosoma lewisi bloodstream and culture forms were agglutinated differentially with low concentrations of the cationic compounds: ruthenium red, ruthenium violet, Alcian blue chloride, 1-hexadecylpyridinium chloride, lanthanum chloride, and cationized ferritin. The bloodstream form trypanosomes gave the highest agglutination levels with each of the compounds tested. Ruthenium red was the most effective inducer of cell agglutination among the several cations used. Trypsin-treated bloodstream forms were agglutinated less in the presence of ruthenium red than untreated controls. Ruthenium red-induced cell agglutination also was lowered with chondroitin sulphate and dextran sulphate, but not with alpha-D-glucose, alpha-D-mannose or with several methyl glycosides. Treatment of the bloodstream trypanosomes with alpha-amylase, dextranase, or neuraminidase had little effect on agglutination levels obtained with ruthenium red. Fine-structure cytochemical staining with ruthenium red, ruthenium violet, and Alcian blue-lanthanum nitrate was used to ascertain the presence and distribution of presumptive carbohydrates in the trypanosome cell surface. The extracellular surface coat of the bloodstream forms stained densely with each of the polycationic dyes. Trypsin treatment removed the surface coat from bloodstream trypanosomes; however, the surface membranes of the organisms were stained densely with the several dyes. Similar surface-membrane staining was obtained with the cationic compounds and the culture forms, which lack a cell surface coat. Cationized ferrin was used at the fine-structure level to visualize the negative surface charge present in the cell surface coat and external membrane of the several trypanosome stages. Results obrained from the agglutination and cytochemistry experiments indicate that complex polysaccharides are present in the surface membranes and cell surface coat of T. lewisi bloodstream forms. Similar conclusions also pertain to the surface membranes of the T. lewisi culture from trypanosomes. The carbohydrates probably represent glycopeptide and glycoprotein structural components of the surface membrane of this organism.

scholarly journals Method combining BAC film and positive staining for the characterization of DNA intermediates by dark-field electron microscopy

2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Yann Benureau ◽  
Eliana Moreira Tavares ◽  
Ali-Akbar Muhammad ◽  
Sonia Baconnais ◽  
Eric Le Cam ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Dark Field ◽  
In Vitro Assays ◽  
Positive Staining ◽  
Synthetic Dna ◽  
Dark Field Imaging ◽  
Alkyl Ammonium

Abstract DNA intermediate structures are formed in all major pathways of DNA metabolism. Transmission electron microscopy (TEM) is a tool of choice to study their choreography and has led to major advances in the understanding of these mechanisms, particularly those of homologous recombination (HR) and replication. In this article, we describe specific TEM procedures dedicated to the structural characterization of DNA intermediates formed during these processes. These particular DNA species contain single-stranded DNA regions and/or branched structures, which require controlling both the DNA molecules spreading and their staining for subsequent visualization using dark-field imaging mode. Combining BAC (benzyl dimethyl alkyl ammonium chloride) film hyperphase with positive staining and dark-field TEM allows characterizing synthetic DNA substrates, joint molecules formed during not only in vitro assays mimicking HR, but also in vivo DNA intermediates.

scholarly journals THE FINE STRUCTURE OF EMBRYONIC CHICK SKELETAL MUSCLE CELLS DIFFERENTIATED IN VITRO

1967 ◽  
Vol 35 (2) ◽  
pp. 445-453 ◽  
Author(s):  
Y. Shimada ◽  
D. A. Fischman ◽  
A. A. Moscona
Keyword(s):  
Electron Microscopy ◽  
Skeletal Muscle ◽  
Fine Structure ◽  
Muscle Cells ◽  
Skeletal Muscle Cells ◽  
Chick Embryos ◽  
Embryonic Chick ◽  
Developing Muscle

Dissociated myoblasts from 12-day chick embryos were cultured in monolayer, and the differentiation of skeletal muscle cells was studied by electron microscopy. The results have revealed a striking ultrastructural similarity between the in vivo and the in vitro developing muscle, particularly with respect to the myofibrils and sarcoplasmic reticulum. This study demonstrates that all the characteristic organelles of mature skeletal muscle can develop in vitro in the absence of nerves.

scholarly journals Effect of crosslinking with riboflavin and ultraviolet a (UVA) on the scleral tissue structure

2017 ◽  
Vol 10 (2) ◽  
pp. 6-12
Author(s):  
Mukharram M Bikbov ◽  
Valentina K Surkova ◽  
Emin L Usubov ◽  
Nikolaj A Nikitin ◽  
Mikhail Nikolaevich Astrelin
Keyword(s):  
Electron Microscopy ◽  
Aqueous Solution ◽  
Average Diameter ◽  
Tissue Structure ◽  
In Vitro Experiments ◽  
Pathological Changes ◽  
Ultraviolet A ◽  
Intermediate Space ◽  
Special Software

Purpose. To evaluate the effect of the scleral crosslinking with riboflavin and ultraviolet A (UVA) on the scleral tissue structure in vitro experiments. Material and methods. The study was conducted on 7 porcine cadaver eyes. Two parallel scleral strips were excised from each eyeball, one of which was subjected to crosslinking procedure (instillation of 0.1% aqueous solution of riboflavin mononucleotide for 20 minutes, ultraviolet irradiation of 3 mW /cm2 for 30 minutes), second was used as control. Scleral structure was evaluated by light (Van Gieson’s stain) and electron microscopy. Morphometric analysis of the microphotographs was performed using special software. Results. As a result of crosslinking, the average packing density of collagen fibers increased by 8.2%, the area of the intermediate space decreased by 5.2%, the average diameter of collagen fibrils increased by 12%. There were no pathological changes in scleral structures. Conclusion. Obtained results confirm the efficacy of scleral crosslinking with riboflavin/UVA in the formation of additional crosslinks, and the procedure safety for the scleral tissue.

scholarly journals The cercarial glycocalyx of Schistosoma mansoni.

1985 ◽  
Vol 100 (5) ◽  
pp. 1423-1434 ◽  
Author(s):  
J C Samuelson ◽  
J P Caulfield
Keyword(s):  
Electron Microscopy ◽  
Molecular Weight ◽  
Schistosoma Mansoni ◽  
High Molecular Weight ◽  
Membrane Surface ◽  
Periodate Oxidation ◽  
Apparent Molecular Weight ◽  
Ruthenium Red ◽  
Unit Membrane

Cercariae, the freshwater stage of Schistosoma mansoni infectious to man, are covered by a single unit membrane and an immunogenic glycocalyx. When cercariae penetrate the host skin, they transform to schistosomula by shedding tails, secreting mucous and enzymes, and forming microvilli over their surface. Here the loss of the glycocalyx from cercariae transforming in vitro was studied morphologically and biochemically. By scanning electron microscopy, the glycocalyx was a dense mesh composed of 15-30 nm fibrils that obscured spines on the cercarial surface. The glycocalyx was absent on organisms fixed without osmium and was partially lost when parasites aggregated in their own secretions before fixation. By transmission electron microscopy, a 1-2 microns thick mesh of 8-15-nm fibrils was seen on parasites incubated with anti-schistosomal antibodies or fixed in aldehydes containing tannic acid or ruthenium red. Cercariae transformed to schistosomula when tails were removed mechanically and parasites were incubated in saline. Within 5 min of transformation, organisms synchronously formed microvilli which elongated to 3-5 microns by 20 min and then were shed. However, considerable fibrillar material remained adherent to the double unit membrane surface of schistosomula. For biochemical labeling, parasites were treated with eserine sulfate, which blocked cercarial swimming, secretion, infectivity, and transformation to schistosomula. Material labeled by periodate oxidation and NaB3H4 was on the surface as shown by autoradiography and had an apparent molecular weight of greater than 10(6) by chromatography. Periodate-NaB3H4 glycocalyx had an isoelectric point of 5.0 +/- 0.4 and was precipitable with anti-schistosomal antibodies. More than 60% of the radiolabeled glycocalyx was released into the medium by transforming parasites in 3 h and was recovered as high molecular weight material. Parasites labeled with periodate and fluorescein-thiosemicarbazide and then transformed had a corona of fluorescence containing microvilli, much of which was shed onto the slide. Material on cercariae labeled by lodogen-catalyzed iodination was also of high molecular weight and was antigenic. In conclusion, the cercarial glycocalyx appears to be composed of acidic high molecular weight fibrils which are antigenic and incompletely cleared during transformation.

Ruthenium red-osmium bridging with TCH: new technique to stain biological specimens for light and TEM and to coat them for SEM

1988 ◽  
Vol 46 ◽  
pp. 20-21
Author(s):  
B. Giammara ◽  
J. Hanker
Keyword(s):  
Electron Microscopy ◽  
Microscopic Study ◽  
Osmium Tetroxide ◽  
Light And Electron Microscopy ◽  
Ruthenium Red ◽  
Surface Coat ◽  
Repeated Application ◽  
Electron Microscopic ◽  
Sputter Coating ◽  
Selection Of

The demonstration and coating of glycomacromolecular surface coat components of biological specimens (1) with ruthenium red (RR, Fig. 1) is improved by treating with osmium tetroxide (2) probably due to its attachment to glycolipids. Since 1966 studies have shown how bridging osmium to osmium with thiocarbohydrazide (TCH, Fig. 2) can result in improvement in contrast of biological specimens (3,4) for light and electron microscopy. Since 1973 this bridging procedure has widely been applied (5,6) to obtain a conductive coating for biological specimens for SEM eliminating the need for sputter coating. Improvement in conductance of uncoated specimens for EM has also been obtained (6) by bridging osmium with p-phenylenediamine hydrochloride (PPD). The improvement in conductance of RR coated biological specimens for SEM by OsO4 treatment without TCH (2) required repeated application of the reagent solutions and did not result in sufficient staining of the glycomacromolecules and glycolipids for the light microscopic selection of areas for electron microscopic study.

Characterization of sea urchin primary mesenchyme cells and spicules during biomineralization in vitro

Development ◽  
1987 ◽  
Vol 101 (2) ◽  
pp. 297-312 ◽  
Author(s):  
G.L. Decker ◽  
J.B. Morrill ◽  
W.J. Lennarz
Keyword(s):  
Electron Microscopy ◽  
Sea Urchin ◽  
Secretory Pathway ◽  
Divalent Cations ◽  
Ruthenium Red ◽  
Coated Pits ◽  
Residual Matrix ◽  
Mesenchyme Cells ◽  
Primary Mesenchyme Cells

An in vitro culture system for primary mesenchyme cells of the sea urchin embryo has been used to study the cellular characteristics of skeletal spicule formation. As judged initially by light microscopy, these cells attached to plastic substrata, migrated and fused to form syncytia in which mineral deposits accumulated in the cell bodies and in specialized filopodial templates. Subsequent examination by scanning electron microscopy revealed that the cell bodies and the filopodia and lamellipodia formed spatial associations similar to those seen in the embryo and indicated that the spicule was surrounded by a membrane-limited sheath derived by fusion of the filopodia. The spicules were dissolved from living or fixed cells by a chelator of divalent cations or by lowering the pH of the medium. However, granular deposits found in the cell bodies appeared relatively refractory to such treatments, indicating that they were inaccessible to agents that dissolved the spicules. Use of rapid freezing and an anhydrous fixative to preserve the syncytia for transmission electron microscopy and X-ray microprobe analysis, indicated that electron-dense deposits in the cell bodies contain elements (Ca, Mg and S) common to the spicule. Examination of the spicule cavity after dissolution of the spicule mineral revealed openings in the filopodia-derived sheath, coated pits within the limiting membrane and a residual matrix that stained with ruthenium red. Concanavalin A—gold applied exogenously entered the spicule cavity and bound to matrix glycoproteins. Based on these observations, we conclude that components of the spicule initially are sequestered intracellularly and that spicule elongation occurs in an extracellular cavity. Ca2+ and associated glycoconjugates may be routed in this cavity via a secretory pathway.

Surface Coat of Entamoeba histolytica Trophozoites. - Ultrastructural Cytochemical Study

1972 ◽  
Vol 30 ◽  
pp. 158-159
Author(s):  
Alfredo Feria-Velasco ◽  
Norberto Treviño ◽  
Isabel Ruiz de Chávez
Keyword(s):  
Electron Microscopy ◽  
Entamoeba Histolytica ◽  
Alcian Blue ◽  
Ruthenium Red ◽  
Surface Coat ◽  
Free Living ◽  
Cytochemical Study ◽  
Free Living Amoeba ◽  
Ruthenium Dyes ◽  
Axenic Conditions

The surface or extraneous coat has been investigated in some free-living amoeba such as A. proteus and Chaos chaos using ruthenium dyes and alcian blue. Although the surface coat of E. histolytica trophozoites can be visualized with routine techniques for electron microscopy, its details and various elements appear ill-defined (Fig. 1). By using lanthanum staining after aldehyde fixation with alcian blue according to Shea's method, or ruthenium red during postfixation with OsO4 we were able to observe the extraneous coat of HK-9:NIH strain trophozoites of E. histolytica cultured in axenic conditions. Some trophozoites were incubated with neuraminidase at pH 5.0 for 60 minutes at 37°C before staining with alcian blue and lanthanum in the fixatives. The samples in all experiments were dehydrated and embedded in araldite and the sections were observed unstained.

scholarly journals Transmission electron microscopy studies of the zona reaction in pig oocytes fertilized in vivo and in vitro

Reproduction ◽  
2001 ◽  
pp. 443-452 ◽  
Author(s):  
H Funahashi ◽  
H Ekwall ◽  
K Kikuchi ◽  
H Rodriguez-Martinez
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Zona Pellucida ◽  
Ruthenium Red ◽  
Transmission Electron ◽  
Sperm Penetration ◽  
Outer Area ◽  
Fibrillar Network

The aim of this study was to determine the ultrastructure of cross-sectioned zonae pellucidae of in vitro-matured and ovulated pig oocytes before or after sperm penetration in vitro and in vivo, respectively. The in vitro and in vivo (ovulated) oocytes and zygotes (fertilized in vitro and in vivo) were fixed with glutaraldehyde either directly or after pretreatment with ruthenium red and saponin, processed and then examined using transmission electron microscopy. The thickness of the zona pellucida, as measured on the section of the specimens with largest diameter fixed with glutaraldehyde, differed between the in vivo (9.19 +/- 0.47 microm) and in vitro (5.95 +/- 0.51 microm) oocytes. The in vivo oocytes had a rather thick external mesh-like structure, whereas it was much thinner in the in vitro oocytes. This mesh-like external rim was less apparent in both in vivo and in vitro zygotes. Obvious differences in the density of the lattice formed by the fixed zonae pellucidae were visible between the outer and inner (ad-oolemmal) zonae. The outer area always formed a concentrically arrayed fibrillar network, whereas the inner area showed a much more compact, trabecule-like mesh. However, both areas, but particularly the outer network, were much more compacted after the zona reaction. Clear differences in the degree of fibrillar aggregation of the inner zona area were also observed between in vitro and in vivo zygotes, being much higher in the latter. This fibrillar network was more clearly visible in the zygotes pretreated with ruthenium red and saponin; the in vitro zygotes had a fibrillar, radially oriented set of parallel fibrils, whereas it was much more aggregated and trabecule-like in the in vivo zygotes. These results demonstrate that the fine structure of the zona pellucida and the zona reaction at sperm penetration differ between pig oocytes fertilized in vivo and in vitro. Moreover, the ultrastructure of the outer and inner pig zonae pellucidae has a different network organization.

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