Molecular Hybridization of Mouse Satellite DNA-Complementary RNA in Ultrathin Sections Prepared for Electron Microscopy

1974 ◽  
Vol 14 (2) ◽  
pp. 253-261
Author(s):  
J. JACOB ◽  
KATHERINE GILLIES ◽  
D. MACLEOD ◽  
K. W. JONES
Keyword(s):  
Electron Microscopy ◽  
Satellite Dna ◽  
Similar Distribution ◽  
Tissue Sections ◽  
Satellite Sequences ◽  
Interphase Cells ◽  
Nucleolar Chromatin ◽  
Ultrathin Sections ◽  
Mouse Satellite Dna

The feasibility of in situ hybridization in tissue sections prepared for electron microscopy has been examined using mouse satellite DNA-complementary RNA and mouse L cells. The results obtained are encouraging, although certain technical aspects require further clarification. In interphase cells, hybrid-forming sites occur in chromatin patches positioned along the nuclear envelope. It is also confirmed that satellite DNA occurs in nucleolus-associated chromatin. The results suggest that satellite sequences are present in intranucleolar and peri-nucleolar chromatin. A similar distribution is indicated for ribosomal cistrons.

scholarly journals Mouse satellite DNA, centromere structure, and sister chromatid pairing.

1986 ◽  
Vol 103 (4) ◽  
pp. 1145-1151 ◽  
Author(s):  
L M Lica ◽  
S Narayanswami ◽  
B A Hamkalo
Keyword(s):  
Electron Microscopy ◽  
Sister Chromatid ◽  
Satellite Dna ◽  
Marker Chromosome ◽  
Restriction Enzymes ◽  
Centromeric Heterochromatin ◽  
Mitotic Chromosomes ◽  
Metaphase Chromosomes ◽  
Sister Chromatids ◽  
Mouse Satellite Dna

The experiments described were directed toward understanding relationships between mouse satellite DNA, sister chromatid pairing, and centromere function. Electron microscopy of a large mouse L929 marker chromosome shows that each of its multiple constrictions is coincident with a site of sister chromatid contact and the presence of mouse satellite DNA. However, only one of these sites, the central one, possesses kinetochores. This observation suggests either that satellite DNA alone is not sufficient for kinetochore formation or that when one kinetochore forms, other potential sites are suppressed. In the second set of experiments, we show that highly extended chromosomes from Hoechst 33258-treated cells (Hilwig, I., and A. Gropp, 1973, Exp. Cell Res., 81:474-477) lack kinetochores. Kinetochores are not seen in Miller spreads of these chromosomes, and at least one kinetochore antigen is not associated with these chromosomes when they were subjected to immunofluorescent analysis using anti-kinetochore scleroderma serum. These data suggest that kinetochore formation at centromeric heterochromatin may require a higher order chromatin structure which is altered by Hoechst binding. Finally, when metaphase chromosomes are subjected to digestion by restriction enzymes that degrade the bulk of mouse satellite DNA, contact between sister chromatids appears to be disrupted. Electron microscopy of digested chromosomes shows that there is a significant loss of heterochromatin between the sister chromatids at paired sites. In addition, fluorescence microscopy using anti-kinetochore serum reveals a greater inter-kinetochore distance than in controls or chromosomes digested with enzymes that spare satellite. We conclude that the presence of mouse satellite DNA in these regions is necessary for maintenance of contact between the sister chromatids of mouse mitotic chromosomes.

Uranyl Acetate as a Fixative—from pH 2.0 to 8.0

1971 ◽  
Vol 29 ◽  
pp. 490-491
Author(s):  
V. R. Mumaw ◽  
B. L. Munger
Keyword(s):  
Electron Microscopy ◽  
Oxalic Acid ◽  
Osmium Tetroxide ◽  
Uranyl Acetate ◽  
En Bloc ◽  
Tissue Sections ◽  
Normal Rat ◽  
Lead Hydroxide ◽  
Ultrathin Sections ◽  
Rat Tissue

Numerous applications utilizing uranyl acetate as an electron stain for electron microscopy have been described. Uranyl acetate has become a routine stain used in conjunction with lead hydroxide for staining ultrathin sections. En bloc staining with uranyl acetate following osmium tetroxide post-fixation produces undesirable effects on some cytoplasmic components, especially glycogen. Recent studies using uranyl acetate as a fixative and en bloc stain at pH 7.2 before osmification has shown uranyl acetate to have desirable fixation and staining qualities. Tissues treated with uranyl acetate at a pH of 2.0-8.0 were studied. Normal rat tissue was fixed in Karnovsky's paraformaldehyde-glutaraldehyde fixative. The tissue was post-fixed in 0.5% uranyl acetate in water at pH 2.0 and 0.5% uranyl acetate in 0.1M s-collidine with 0.01M oxalic acid at pH 4, pH 6.0, pH 7.2, and pH 8.0 for 1 hour at 4°C. Following several rinses of 0.1M s-collidine buffer, the tissues were treated with 1.33% osmium tetroxide 1 hour at 4°C followed by rapid dehydration in ethanol and embedded in Durcupan ACM. Tissue sections were stained with lead hydroxide.

scholarly journals In situ hybridization at the electron microscope level: hybrid detection by autoradiography and colloidal gold

1982 ◽  
Vol 95 (2) ◽  
pp. 609-618 ◽  
Author(s):  
NJ Hutchison ◽  
PR Langer-Safer ◽  
DC Ward ◽  
BA Hamkalo
Keyword(s):  
In Situ Hybridization ◽  
Electron Microscope ◽  
Satellite Dna ◽  
Light Microscope ◽  
Test System ◽  
Microscope Level ◽  
Metaphase Chromosomes ◽  
Light Microscope Level ◽  
Mouse Satellite Dna

In situ hybridization has become a standard method for localizing DNA or RNA sequences in cytological preparations. We developed two methods to extend this technique to the transmission electron microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The first method devised is a direct extension of standard light microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The first method devised is a direct extension of standard light microscope in situ hybridization. Radioactively labeled complementary RNA (cRNA) is hybridized to metaphase chromosomes deposited on electron microscope grids and fixed in 70 percent ethanol vapor; hybridixation site are detected by autoradiography. Specific and intense labeling of chromosomal centromeric regions is observed even after relatively short exposure times. Inerphase nuclei present in some of the metaphase chromosome preparations also show defined paatterms of satellite DNA labeling which suggests that satellite-containing regions are associate with each other during interphase. The sensitivity of this method is estimated to at least as good as that at the light microscope level while the resolution is improved at least threefold. The second method, which circumvents the use of autoradiogrphic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction is improved at least threefold. The second method, which circumvents the use of autoradiographic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction with an antibody against biotin and secondary antibody adsorbed to the surface of over centromeric heterochromatin and along the associated peripheral fibers. Labeling is on average ten times that of background binding. This method is rapid and possesses the potential to allow precise ultrastructual localization of DNA sequences in chromosomes and chromatin.

The Progression from Optical Light Microscopy to Transmission Electron Microscopy in the Study of Soils

Clay Minerals ◽  
1994 ◽  
Vol 29 (2) ◽  
pp. 247-254 ◽  
Author(s):  
F. Van Oort ◽  
A. G. Jongmans ◽  
A. M. Jaunet
Keyword(s):  
Electron Microscopy ◽  
Clay Mineralogy ◽  
Tem Analysis ◽  
Undisturbed Soil ◽  
Thin Sections ◽  
Transmission Electron ◽  
Ultrathin Sections ◽  
Optical Light Microscopy ◽  
The Impact

AbstractThe use of electron microscopy to study clay microfabrics in thin-sections is discussed. A technique is described to isolate undisturbed microparts of pedofeatures from thin-sections, which are subsequently used for TEM analysis. Re-embedding with a polyester resin of undisturbed, in situ, neoformed clay microfabrics, obtained by microdrilling and preparation of ultrathin sections by microtoming with a diamond knife are emphasized; these steps enable micromorphology, clay mineralogy, microchemical and HRTEM analysis to be performed on one unique microsample of clay fabrics, with conserved micro-organization. Two examples on clay neoformation are presented to demonstrate that this technique can successfully be applied to unravel the impact of mineral alteration and clay neoformation in undisturbed soil samples on a micro- and a nanometer scale.

Platinum Compounds as Stains for Electron Microscopy

1974 ◽  
Vol 32 ◽  
pp. 230-231
Author(s):  
S. K. Aggarwal ◽  
P. McAllister ◽  
R. W. Wagner ◽  
B. Rosenberg
Keyword(s):  
Electron Microscopy ◽  
Hela Cells ◽  
Uranyl Acetate ◽  
Sarcoma 180 ◽  
Platinum Compounds ◽  
Kb Cells ◽  
En Bloc ◽  
Human Lymphoma ◽  
Ultrathin Sections ◽  
Blue Coloration

Uranyl acetate has been used as an electron stain for en bloc staining as well as for staining ultrathin sections in conjunction with various lead stains (Fig. 1). Present studies reveal that various platinum compounds also show promise as electron stains. Certain platinum compounds have been shown to be effective anti-tumor agents. Of particular interest are the compounds with either uracil or thymine as one of the ligands (cis-Pt(II)-uracil; cis-Pt(II)-thymine). These compounds are amorphous, highly soluble in water and often exhibit an intense blue coloration. These compounds show enough electron density to be used as stains for electron microscopy. Most of the studies are based on various cell lines (human AV, cells, human lymphoma cells, KB cells, Sarcoma-180 ascites cells, chick fibroblasts and HeLa cells) while studies on tissue blocks are in progress.

Immunocytochemistry. A Review of Methodology for Electron Microscopic Applicaiton

1974 ◽  
Vol 32 ◽  
pp. 328-329
Author(s):  
Joseph E. Mazurkiewicz
Keyword(s):  
Electron Microscopy ◽  
Light Microscopy ◽  
Ultrastructural Level ◽  
Electron Microscopic ◽  
Biological Interest ◽  
Investigative Approach ◽  
Immunologic Activity ◽  
Dense Label

Immunocytochemistry is a powerful investigative approach in which one of the most exacting examples of specificity, that of the reaction of an antibody with its antigen, isused to localize tissue and cell specific molecules in situ. Following the introduction of fluorescent labeled antibodies in T950, a large number of molecules of biological interest had been studied with light microscopy, especially antigens involved in the pathogenesis of some diseases. However, with advances in electron microscopy, newer methods were needed which could reveal these reactions at the ultrastructural level. An electron dense label that could be coupled to an antibody without the loss of immunologic activity was desired.

Electron Microscopy of Mycoplasma Pneumoniae

1971 ◽  
Vol 29 ◽  
pp. 258-259 ◽  
Author(s):  
E. S. Boatman ◽  
G. E. Kenny
Keyword(s):  
Electron Microscopy ◽  
Light Microscopy ◽  
Growth Phase ◽  
Hela Cells ◽  
Sulfonic Acid ◽  
Gamma Globulin ◽  
Horse Serum ◽  
Morphological Aspects ◽  
The Family

Information concerning the morphology and replication of organism of the family Mycoplasmataceae remains, despite over 70 years of study, highly controversial. Due to their small size observations by light microscopy have not been rewarding. Furthermore, not only are these organisms extremely pleomorphic but their morphology also changes according to growth phase. This study deals with the morphological aspects of M. pneumoniae strain 3546 in relation to growth, interaction with HeLa cells and possible mechanisms of replication.The organisms were grown aerobically at 37°C in a soy peptone yeast dialysate medium supplemented with 12% gamma-globulin free horse serum. The medium was buffered at pH 7.3 with TES [N-tris (hyroxymethyl) methyl-2-aminoethane sulfonic acid] at 10mM concentration. The inoculum, an actively growing culture, was filtered through a 0.5 μm polycarbonate “nuclepore” filter to prevent transfer of all but the smallest aggregates. Growth was assessed at specific periods by colony counts and 800 ml samples of organisms were fixed in situ with 2.5% glutaraldehyde for 3 hrs. at 4°C. Washed cells for sectioning were post-fixed in 0.8% OSO4 in veronal-acetate buffer pH 6.1 for 1 hr. at 21°C. HeLa cells were infected with a filtered inoculum of M. pneumoniae and incubated for 9 days in Leighton tubes with coverslips. The cells were then removed and processed for electron microscopy.

Laboratory and in-situ analysis of comet dust

1988 ◽  
Vol 46 ◽  
pp. 8-9
Author(s):  
D.E. Brownlee ◽  
A.L. Albee
Keyword(s):  
Electron Microscopy ◽  
Solar System ◽  
Laboratory Study ◽  
Isotopic Analysis ◽  
Building Blocks ◽  
Sem Analysis ◽  
Early Solar System ◽  
Cometary Material ◽  
Comet Dust

Comets are primitive, kilometer-sized bodies that formed in the outer regions of the solar system. Composed of ice and dust, comets are generally believed to be relic building blocks of the outer solar system that have been preserved at cryogenic temperatures since the formation of the Sun and planets. The analysis of cometary material is particularly important because the properties of cometary material provide direct information on the processes and environments that formed and influenced solid matter both in the early solar system and in the interstellar environments that preceded it.The first direct analyses of proven comet dust were made during the Soviet and European spacecraft encounters with Comet Halley in 1986. These missions carried time-of-flight mass spectrometers that measured mass spectra of individual micron and smaller particles. The Halley measurements were semi-quantitative but they showed that comet dust is a complex fine-grained mixture of silicates and organic material. A full understanding of comet dust will require detailed morphological, mineralogical, elemental and isotopic analysis at the finest possible scale. Electron microscopy and related microbeam techniques will play key roles in the analysis. The present and future of electron microscopy of comet samples involves laboratory study of micrometeorites collected in the stratosphere, in-situ SEM analysis of particles collected at a comet and laboratory study of samples collected from a comet and returned to the Earth for detailed study.

Application of analytical electron microscopy to the study of partitioning of silicon in a 2 wt% Si eutectoid steel

1978 ◽  
Vol 36 (1) ◽  
pp. 616-617
Author(s):  
N. Ridley ◽  
S.A. Al-Salman ◽  
G.W. Lorimer
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Analytical Electron Microscopy ◽  
Eutectoid Steel ◽  
Minimum Diameter ◽  
Thin Foils ◽  
Analytical Electron ◽  
Analytical Electron Microscope ◽  
Transformation Front

The application of the technique of analytical electron microscopy to the study of partitioning of Mn (1) and Cr (2) during the austenite-pearlite transformation in eutectoid steels has been described in previous papers. In both of these investigations, ‘in-situ’ analyses of individual cementite and ferrite plates in thin foils showed that the alloying elements partitioned preferentially to cementite at the transformation front at higher reaction temperatures. At lower temperatures partitioning did not occur and it was possible to identify a ‘no-partition’ temperature for each of the steels examined.In the present work partitioning during the pearlite transformation has been studied in a eutectoid steel containing 1.95 wt% Si. Measurements of pearlite interlamellar spacings showed, however, that except at the highest reaction temperatures the spacing would be too small to make the in-situ analysis of individual cementite plates possible, without interference from adjacent ferrite lamellae. The minimum diameter of the analysis probe on the instrument used, an EMMA-4 analytical electron microscope, was approximately 100 nm.

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