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

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.

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Genomic in situ hybridization to sectioned nuclei shows chromosome domains in grass hybrids

Journal of Cell Science ◽

10.1242/jcs.95.3.335 ◽

1990 ◽

Vol 95 (3) ◽

pp. 335-341

Author(s):

A.R. Leitch ◽

W. Mosgoller ◽

T. Schwarzacher ◽

M.D. Bennett ◽

J.S. Heslop-Harrison

Keyword(s):

In Situ Hybridization ◽

Genomic Dna ◽

Microscope Level ◽

Hordeum Chilense ◽

Root Tip ◽

Interphase Nuclei ◽

Light Microscope Level ◽

Thin Sections ◽

Electron Microscopes

In situ hybridization using biotinylated total genomic DNA and avidin detection systems was adapted for examination of thin-sectioned plant material in the light and electron microscopes. Root tip material was preserved prior to sectioning, so that the in vivo disposition of the chromatin was maintained. Use of total genomic DNA from Secale africanum as a probe enabled the chromatin from the two parental genomes in the grass hybrid Hordeum chilense × S. africanum to be distinguished. The biotinylated probe preferentially labelled the chromosomes of S. africanum origin. DNA-DNA hybrids were visualized at the light-microscope level by Texas Red fluorescence and at the electron-microscope level by the enzymic precipitation of DAB (diaminobenzidine) or by colloidal gold particles. The use of thin sections allowed the location of probe hybridization to be established unequivocally in both metaphase and interphase nuclei. Analysis of interphase nuclei showed that chromatin originating from the two parental genomes did not intermix but occupied distinct domains.

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Eukaryotic genome organization analyzed by electron microscope in situ hybridization

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100084508 ◽

1991 ◽

Vol 49 ◽

pp. 40-41

Author(s):

Barbara A. Hamkalo ◽

Sandya Narayanswami ◽

Nadja Dvorkin

Keyword(s):

In Situ Hybridization ◽

Electron Microscope ◽

Genome Organization ◽

Cell Biology ◽

Eukaryotic Genome ◽

Dna Denaturation ◽

Metaphase Chromosomes ◽

Rna Sequences ◽

Gold Particles

In situ hybridization is a powerful tool for the localization of DNA/RNA sequences in nuclei and chromosomes. The introduction of nonisotopic labelling methodologies in conjunction with fluorescent or enzyme-linked detection have resulted in a dramatic increase in the application of this technique at the light microscope (LM) level and has placed it in a pivotal role in cell biology, development and genetics. Development of equivalent mapping protocols at the EM level offers increased spatial resolution. We have combined the use of nonisotopic probes with invmunogold labelling to investigate eukaryotic genome organization at high resolution.Metaphase chromosomes released from mitotically-arrested cells are deposited on gold EM grids by centrifugation through a sucrose cushion. After fixation (0.1% glutaraldehyde, 20 min) and DNA denaturation, chromosomes are hybridized to cloned probes enzymatically labelled with biotin-dUTP, digoxigenin-dUTP, dinitrophenyl-dUTP or covalently coupled to N-acetoxyacetoaminofluorene. Hybrid sites typically are detected by a two-step antibody incubation and 1-30 nm colloidal gold particles.

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Gold Cluster Compounds are Useful Immunoprobes

Microscopy and Microanalysis ◽

10.1017/s1431927600034139 ◽

2000 ◽

Vol 6 (S2) ◽

pp. 328-329

Author(s):

J.M. Robinson ◽

T. Takizawa ◽

D.D. Vandré

Keyword(s):

Electron Microscope ◽

Gold Cluster ◽

Cluster Compounds ◽

Microscope Level ◽

Reporter System ◽

Electron Microscope Level ◽

Light Microscope Level ◽

Antibody Recognition ◽

Particulate Probes

Immunocytochemistry generally refers to methods directed toward obtaining information on the in situ distribution of antigens in cells and tissues. Immunocytochemical methods can be applied at either the light or electron microscope levels, or both in concert. The detection of antibody recognition of an antigen (i.e., localization of an antigen) relies upon a reporter system. At the light microscope level, enzymes (e.g., horseradish peroxidase) or fluorochromes are the most widely used reporters in immunocytochemistry. At the electron microscope level, particulate probes (e.g., colloidal gold) are the most widely used reporters. However, enzymes and even fluorochromes can be used at the EM level.In this abstract, we discuss our use of gold cluster immunoprobes as the reporter system in both light and electron microscope level immuncytochemistry. These gold cluster immunoprobes, are commercially known as NanogoldTM (NG). These probes are very small with a diameter of 1.4-nm.

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LOCALIZATION OF U1, U2 snRNAs AND POLY(A) RNA BY IN SITU HYBRIDIZATION AT THE ELECTRON MICROSCOPE LEVEL. CHARACTERIZATON OF A NOVEL NUCLEAR DOMAIN CONTAINING U1 but not U2 snRNA AND PRESENCE OF POLY(A) RNA IN INTERCHROMATIN GRANULES

Biology of the Cell ◽

10.1016/0248-4900(93)90279-n ◽

1993 ◽

Vol 79 (1) ◽

pp. 85-85

Author(s):

PUVION Edmond ◽

VISA Neus ◽

PUVION-DUTILLEUL Francine ◽

HARPER Francis ◽

BACHELLERIE Jean-Pierre

Keyword(s):

In Situ Hybridization ◽

Electron Microscope ◽

Microscope Level ◽

Electron Microscope Level ◽

U2 Snrna ◽

Nuclear Domain ◽

Interchromatin Granules

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Notes on the flagellar apparatus and taxonomy of Pavlova mesolychnon Van der Veer, and on the status of Pavlova Butcher and related genera within the Haptophyceae

Journal of the Marine Biological Association of the United Kingdom ◽

10.1017/s0025315400020671 ◽

1976 ◽

Vol 56 (3) ◽

pp. 595-602 ◽

Cited By ~ 25

Author(s):

J. C. Green

Keyword(s):

Electron Microscope ◽

Light Microscope ◽

Type Species ◽

Flagellar Apparatus ◽

Microscope Level ◽

New Genera ◽

Light Microscope Level ◽

The Status ◽

Freshwater Habitats

INTRODUCTIONPavlova gyrans Butcher, the type species of the genus, was described at the light microscope level in 1952 (Butcher, 1952). Since then this species has been re-investigated with the electron microscope (Green & Manton, 1970), a number of other species described from marine, brackish and freshwater habitats (for literature, see p. 600) and at least two new genera erected which appear to be closely related to Pavlova (Corcontochrysis Kalina, 1970;Exanthemachrysis Lepailleur, 1970).

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Detection of hepatopancreatic parvovirus (HPV) of penaeid shrimp by in situ hybridization at the electron microscope level

Diseases of Aquatic Organisms ◽

10.3354/dao044087 ◽

2001 ◽

Vol 44 ◽

pp. 87-96 ◽

Cited By ~ 12

Author(s):

CR Pantoja ◽

DV Lightner

Keyword(s):

In Situ Hybridization ◽

Electron Microscope ◽

Penaeid Shrimp ◽

Microscope Level ◽

Electron Microscope Level ◽

Hepatopancreatic Parvovirus

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DNA sequence mapping in interphase and metaphase chromosomes by fluorescence in situ hybridization

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100122885 ◽

1992 ◽

Vol 50 (1) ◽

pp. 496-497

Author(s):

Barbara Trask ◽

Susan Allen ◽

Anne Bergmann ◽

Mari Christensen ◽

Anne Fertitta ◽

...

Keyword(s):

In Situ Hybridization ◽

Dna Sequence ◽

Dna Sequences ◽

Dual Band ◽

Nick Translation ◽

Metaphase Chromosomes ◽

Band Pass ◽

Texas Red ◽

Fluorescent Spot

Using fluorescence in situ hybridization (FISH), the positions of DNA sequences can be discretely marked with a fluorescent spot. The efficiency of marking DNA sequences of the size cloned in cosmids is 90-95%, and the fluorescent spots produced after FISH are ≈0.3 μm in diameter. Sites of two sequences can be distinguished using two-color FISH. Different reporter molecules, such as biotin or digoxigenin, are incorporated into DNA sequence probes by nick translation. These reporter molecules are labeled after hybridization with different fluorochromes, e.g., FITC and Texas Red. The development of dual band pass filters (Chromatechnology) allows these fluorochromes to be photographed simultaneously without registration shift.

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Lateral Elements Inside Synaptonemal Complex-Like Polycomplexes in ndt80 Mutants of Yeast Bind DNA

Genetics ◽

10.1093/genetics/163.2.539 ◽

2003 ◽

Vol 163 (2) ◽

pp. 539-544 ◽

Cited By ~ 1

Author(s):

Hasanuzzaman Bhuiyan ◽

Gunilla Dahlfors ◽

Karin Schmekel

Keyword(s):

In Situ Hybridization ◽

Electron Microscope ◽

Synaptonemal Complex ◽

Immunoelectron Microscopy ◽

Meiotic Prophase ◽

Lateral Element ◽

Homologous Chromosomes ◽

Meiotic Prophase I ◽

Dna Antibodies

Abstract The synaptonemal complex (SC) keeps the synapsed homologous chromosomes together during pachytene in meiotic prophase I. Structures that resemble stacks of SCs, polycomplexes, are sometimes found before or after pachytene. We have investigated ndt80 mutants of yeast, which arrest in pachytene. SCs appear normal in spread chromosome preparations, but are only occasionally found in intact nuclei examined in the electron microscope. Instead, large polycomplexes occur in almost every ndt80 mutant nucleus. Immunoelectron microscopy using DNA antibodies show strong preferential labeling to the lateral element parts of the polycomplexes. In situ hybridization using chromosome-specific probes confirms that the chromosomes in ndt80 mutants are paired and attached to the SCs. Our results suggest that polycomplexes can be involved in binding of chromosomes and possibly also in synapsis.

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