scholarly journals In situ nick translation at the electron microscopic level: a tool for studying the location of DNAse I-sensitive regions within the cell.

1991 ◽  
Vol 39 (6) ◽  
pp. 871-874 ◽  
Author(s):  
M Thiry
Keyword(s):  
Ultrastructural Level ◽  
Dnase I ◽  
Electron Microscopic Level ◽  
Nick Translation ◽  
Dna Polymerase I ◽  
Electron Microscopic ◽  
Thin Sections ◽  
In Situ Nick Translation ◽  
Polymerase I

The in situ nick translation method was adapted to the ultrastructural level, to study the location of DNAse I-sensitive sequences within the cell. Ultra-thin sections of Lowicryl-embedded cells were incubated in a medium containing DNAse I, DNA polymerase I, and all four deoxyribonucleotides, some being biotinylated. The nick-translated sites were then visualized by an indirect immunogold labeling technique. The resulting labeling pattern is closely dependent on the DNAse I concentration in the nick-translation medium. The method reveals with great precision the specific DNAse I-sensitive regions within the nucleus. This technique can be used to discriminate between active and inactive regions of interphase chromatin.

The distribution of genes on human chromosomes as studied by in situ nick translation

Genome ◽  
1992 ◽  
Vol 35 (5) ◽  
pp. 890-894 ◽  
Author(s):  
J. de la Torre ◽  
A. T. Sumner ◽  
J. Gosalvez ◽  
L. Stuppia
Keyword(s):  
Cpg Islands ◽  
Dnase I ◽  
Human Chromosomes ◽  
Nick Translation ◽  
X Chromosomes ◽  
Dnase I Hypersensitivity ◽  
In Situ Nick Translation ◽  
Inactive X Chromosome ◽  
Active Genes

We have studied the distribution of potentially active genes on human chromosomes, using two methods: DNAse I hypersensitivity and restriction enzyme – nick translation with enzymes sensitive to methylation of CpG doublets. DNAse hypersensitivity is known to be associated with potentially active genes, and, when the reaction is detected by "in situ" nick translation, produces an R-banding pattern. Digestion of chromosomes with HpaII or CfoI, both of which should preferentially cut unmethylated sequences in the CpG islands associated with the majority of genes, also produces R-banding patterns. Deviations are attributable to overdigestion of the chromosomes, leading to extraction of DNA and loss of the specific sites that were to be detected. Contrary to the results of a number of previous workers, we have failed to demonstrate any differences between the DNAse I hypersensitivity or the degree of methylation of the active and inactive X chromosomes in metaphases from females.Key words: human chromosomes, gene distribution, DNAse I hypersensitivity, in situ nick translation, R-banding, CpG islands, DNA methylation, inactive X chromosome.

scholarly journals Usefulness of the immunogold technique in quantitation of a soluble protein in ultra-thin sections.

1987 ◽  
Vol 35 (4) ◽  
pp. 405-410 ◽  
Author(s):  
G Posthuma ◽  
J W Slot ◽  
H J Geuze
Keyword(s):  
Model System ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Embedded Model ◽  
Intracellular Compartments ◽  
Embedded Blocks ◽  
Gelatin Concentration ◽  
Lowicryl K4m

We used a model system to study whether measurements of absolute local antigen concentrations at the electron microscopic level are feasible by counting immunogold labeling density in ultra-thin sections. The model system consisted of a matrix of a variable concentration of gelatin, which was mixed with given concentrations of rat pancreas amylase and fixed according to various fixation protocols. With a relatively mild fixation, there was no clear proportionality between anti-amylase gold labeling and amylase concentration in ultra-thin cryosections. This was presumably due to uncontrolled loss of amylase from the sections. After stronger fixation with 2% glutaraldehyde for 4 hr, labeling density reflected the amylase concentration very well. We observed that matrix (gelatin) density influenced labeling density. A low gelatin concentration of 5% allowed penetration of immunoreagents into the cryosection, resulting in a high and variable labeling density. In gelatin concentrations of 10% and 20%, labeling density was lower but proportional to amylase concentration. To establish an equal (minimal) penetration of immunoreagents, we embedded model blocks with different matrix densities in polyacrylamide (PAA). In ultra-thin cryosections of these PAA-embedded blocks, anti-amylase labeling was proportional to amylase concentration even at a low (5%) gelatin concentration. Anti-amylase labeling in ultra-thin sections from Lowicryl K4M low temperature-embedded blocks was higher than in PAA sections, but the results were less consistent and depended to some extent on matrix density. These results, together with the earlier observation that acrylamide completely penetrates intracellular compartments (Slot JW, Geuze HJ: Biol Cell 44:325, 1982), demonstrate that it is possible to measure true intracellular concentrations of soluble proteins in situ using ultra-thin cryosections of PAA-embedded tissue.

Patterns of DNase I sensitivity in the holocentric chromosomes of the aphid Megoura viciae

Genome ◽  
1998 ◽  
Vol 41 (2) ◽  
pp. 169-172 ◽  
Author(s):  
Gian Carlo Manicardi ◽  
Mauro Mandrioli ◽  
Davide Bizzaro ◽  
Umberto Bianchi
Keyword(s):  
Dnase I ◽  
Holocentric Chromosomes ◽  
Telomeric Region ◽  
Nick Translation ◽  
In Situ Nick Translation ◽  
Embryo Cells ◽  
Dnase I Sensitivity ◽  
Megoura Viciae ◽  
Active Genes

Using the in situ nick translation technique, we looked for the presence of DNase I sensitive sites in Megoura viciae chromosomes, to study the distribution of active or potentially active genes in aphids, a group of insects possessing holocentric chromosomes. Cytological preparations obtained by the spreading of embryo cells were treated in situ with increasing concentrations (ranging from 5 to 200 ng/mL) of DNase I. At DNase I concentrations below 50 ng/mL, only one hypersensitive site was observed, and this was located on a telomeric region of the X chromosome that contains transcriptionally active nucleolar organizing regions, as assayed by silver staining. Interestingly, at intermediate concentrations of DNase, the incorporation of biotinylated nucleotide occurred uniformly throughout all chromosomes, whereas at concentrations above 100 ng/mL, a C-like banding pattern was produced. Our data differ from results obtained with mammalian, frog, and grasshopper chromosomes, where it was found that DNase I nicking is concentrated at the distal regions of all chromosomes.Key words: aphids, holocentric chromosomes, DNase I sensitivity, nick translation.

scholarly journals Catecholamine innervation of enkephalinergic neurons in guinea pig hypothalamus: demonstration by an in vitro autoradiographic technique combined with a post-embedding immunogold method.

1988 ◽  
Vol 36 (5) ◽  
pp. 533-542 ◽  
Author(s):  
V Mitchell ◽  
J C Beauvillain ◽  
P Poulain ◽  
M Mazzuca
Keyword(s):  
Guinea Pig ◽  
Ultrastructural Level ◽  
Microscopic Level ◽  
Electron Microscopic Level ◽  
Morphological Evidence ◽  
Osmic Acid ◽  
Electron Microscopic ◽  
Double Labeling ◽  
Thin Sections

To study the relationship between the catecholamine (CA) nerve endings and the enkephalinergic cell bodies in the magnocellular dorsal nucleus (MDN) of guinea pig hypothalamus, double-labeling experiments were performed on the same tissue section at the electron microscopic level. An in vitro autoradiographic (ARG) method for [3H]-norepinephrine (NE) or [3H]-dopamine (DA) was combined with a post-embedding immunogold cytochemical technique for Met-enkephalin (Met-enk) in colchicine-treated animals. Hypothalamic slices (450 micrograms) were perfused with [3H]-NE or [3H]-DA at the fluid-gas interface, then fixed by immersion with glutaraldehyde and osmic acid. Semi-thin sections processed from the thickness of the slices showed adequate penetration of the tracers to all parts of the tissue. Frontal sections permitted visualization of some CA-uptake structures distributed around the cells. At the ultrastructural level, preservation appeared good on about 60% of the thickness of slices, and [3H]-CA structures were easily distinguished. Ultra-thin sections were successively incubated with Met-enk and colloidal gold-labeled antisera, followed by ARG processing. At the electron microscopic level, the good integrity of the tissue made possible visualization of [3H]-CA nerve terminals making synaptic contacts with enkephalinergic perikarya. These results provide morphological evidence for direct catecholaminergic control of enkephalinergic neurons of the MDN.

scholarly journals A specific ultrastructural method to reveal DNA: the NAMA-Ur.

1991 ◽  
Vol 39 (10) ◽  
pp. 1427-1438 ◽  
Author(s):  
P S Testillano ◽  
M A Sanchez-Pina ◽  
A Olmedilla ◽  
M A Ollacarizqueta ◽  
C J Tandler ◽  
...  
Keyword(s):  
Ultrastructural Level ◽  
Microscopic Level ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
En Bloc ◽  
Easy Method ◽  
Transmission Electron ◽  
Pre Treatment ◽  
Alkali Hydrolysis

We have developed a new, simple, and reproducible cytochemical method to specifically stain DNA at the electron microscopic level: the NAMA-Ur. It is based on the extraction of RNA and phosphate groups from phosphoproteins by a weak alkali hydrolysis (NA) which does not affect DNA, followed by blockage of the amino and carboxyl groups by methylation and acetylation (MA). Finally, sections are stained by uranyl (Ur), which can bind only to DNA. The efficiency of the pre-treatment (NA and MA) was demonstrated by X-ray microanalysis at the transmission electron microscopic level. The NAMA-Ur method has been successfully performed en bloc and on Lowicryl sections in mammalian and plant cells. A specific contrast is observed in the DNA-containing structures after this method, whose sensitivity allows visualization of electron-dense chromatin fibers of 10-12 nm composed of 3-nm DNA fibrils. This staining method has been combined with anti-DNA antibodies, providing complementary information to detect DNA in situ. We propose the NAMA-Ur as an easy method to investigate the chromatin organization in situ at the ultrastructural level.

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.

A New Technique for the Light and Electron Microscopic Study of Individual Cerebro-Spinal Fluid Cells in a Mona Plane Layer

1976 ◽  
Vol 34 ◽  
pp. 362-363
Author(s):  
L.A. Dell
Keyword(s):  
Microscopic Study ◽  
Electron Microscopic Study ◽  
Plane Layer ◽  
Ultrastructural Level ◽  
Spinal Fluid ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
Easy Method ◽  
Cell Pellet ◽  
A New Technique

A new method has been developed which readily offers the microscopist a possibility for both light and electron microscopic study of selected cells from the cerebrospinal fluid. Previous attempts to examine these cells in the spinal fluid at the ultrastructural level were based on modifications of cell pellet techniques developed for peripheral blood. These earlier methods were limited in application by the number of cells in spinal fluid required to obtain a sufficient size pellet and by the lack of an easy method of cellular identification between the light and electron microscopic level. The newly developed method routinely employs microscope slides coated with Siliclad and tungsten oxide for duplicate cytocentrifuge preparations of diagnostic spinal fluid specimens. Work done by Kushida and Suzuki provided a basis for our use of the metal oxide.

X-Ray Microanalysis of Nickel and Cobalt Intensified Peroxidase- Antiperoxidase For Verification of Reaction Sites

1985 ◽  
Vol 43 ◽  
pp. 468-469
Author(s):  
A. Angel ◽  
K. Miller ◽  
V. Seybold ◽  
R. Kriebel
Keyword(s):  
Reaction Mechanisms ◽  
Visual Discrimination ◽  
Osmium Tetroxide ◽  
Ultrastructural Level ◽  
Microscopic Level ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
X Ray ◽  
Insoluble Polymer ◽  
Cytochemical Reaction

Localization of specific substances at the ultrastructural level is dependent on the introduction of chemicals which will complex and impart an electron density at specific reaction sites. Peroxidase-antiperoxidase(PAP) methods have been successfully applied at the electron microscopic level. The PAP complex is localized by addition of its substrate, hydrogen peroxide and an electron donor, usually diaminobenzidine(DAB). On oxidation, DAB forms an insoluble polymer which is able to chelate with osmium tetroxide becoming electron dense. Since verification of reactivity is visual, discrimination of reaction product from osmiophillic structures may be difficult. Recently, x-ray microanalysis has been applied to examine cytochemical reaction precipitates, their distribution in tissues, and to study cytochemical reaction mechanisms. For example, immunoreactive sites labelled with gold have been ascertained by means of x-ray microanalysis.

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