Name: combined immunofluorescence and dna fish on 3d-preserved interphase nuclei to study changes in 3d nuclear organization
Uploaded: 2013-02-03T14:00:00
Description: Watch this Scientific Journal Video about Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization at JoVE.com

Wir möchten die Mitglieder des Skok Labor, vor allem Susannah Hewitt, für Diskussionen und Kommentare. Diese Arbeit wird durch das National Institute of Health Zuschüsse R01GM086852, RC1CA145746 (JAS) unterstützt. JAS ist ein Leukemia &amp; Lymphoma Society Gelehrter. JC ist ein Irvington Institute Fellow des Cancer Research Institute. MM wird von der National Science Foundation Grant Integrative Graduate Bildung und Forschung Traineeship (NSF IGERT 0.333.389) unterstützt.

Ein. DNA Probe Labeling mit Fluorophoren: Nick Translation (~ 6 h) <ol><li> Saubere BAC DNA (hergestellt von Maxi-Prep) oder Plasmiden oder PCR-Produkte, die alle in H 2 O resuspendiert, kann für die Etikettierung verwendet werden. Beachten Sie, dass für eine robuste FISH Signal, Sonden sollten mindestens 10 kb überspannen. </li><li> Inkubieren DNA in RNase A während 30 min bei 37 ° C (alle Reagenzien sind in Tabelle 1 aufgeführt). </li><li> Inkubieren Sie die Nick-Tr...

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Die Techniken detailliert wurden oben in unserem Labor verwendet werden, um die Regulation der V (D) J-Rekombination der Immunglobulin und TCRA / d loci in Entwicklungsländern Lymphozyten 30,31 analysieren. Wir sind sicher, dass diese Technik für die Detektion verschiedener Kernproteinen, nukleare Abteile und Loci, in verschiedenen Zelltypen angepasst werden kann. Modifikationen des Protokolls notwendig sein kann, und in diesem Fall die Hauptschritte zu konzentrieren sind die folgenden. Er...

Epigenetische Mechanismen auslösen Aufbau und Vererbung von Entwicklungs-und Zelltyp-spezifische Transkriptions-Profilen. Auf einer Ebene Dies beinhaltet Modulation der Chromatinstruktur Verpackungen, die aktive oder stille genomischen Regionen definiert. In einem größeren Maßstab, globale 3D Organisation des Genoms und Zellkernarchitektur spielen ebenfalls eine Rolle bei der Kontrolle der Transkriptions-Mustern. So ist Dissektion dieser epigenomischen wesentliche Merkmale für ein volles Verständnis, wie Gene 6-11 geregelt. Kombiniert Immunfluoreszenz-und 3D-DNA FISH bieten eine einzigartige Möglichkeit, molekulare und biochemische Analysen Beurteilung spezifischer Interaktionen / Verbände von DNA-Sequenzen und / oder Proteinen im Zellkern zu ergänzen. Hinzu kommt, dass genomweite Hochdurchsatztechniken wie Chromatin-Immunopräzipitation (ChIP-seq) oder Chromosom capture Konformation mit deep sequencing gekoppelt (4C-seq, 5C, Hallo-C) liefern globale data auf Zellpopulationen 12 ermöglichen Immunfluoreszenz / DNA FISH-Techniken Analysen der einzelnen Zelle Ebene. Hier beschreiben wir ein Protokoll zur gleichzeitigen Detektion von Histonmodifikationen durch Immunfluoreszenz und DNA-Sequenzen von DNA-FISH durch 3D-Mikroskopie und-Analysen (3D Immuno-FISH) gefolgt. Der Vorteil dieses Protokolls ist die kombinierte Visualisierung von DNA und Konservierung von Proteinstrukturen. Unsere Erfahrung in diesem Bereich hat es uns ermöglicht, zu verbessern und zu vereinfachen bestehende Protokolle. Obwohl wir dieses Protokoll verwendet wurden, um DNA-Doppelstrang-Pausen in Lymphozyten unterziehen Rekombination detektieren kann dieses Verfahren an andere Proteine ​​und andere Zelltypen verwendet werden.

<table border="1"><tbody><tr><td> Name des Reagenz / Material </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> H 2 O </td><td> Fischer </td><td> # BP2470 </td><td></td></tr><tr><td> RNase A </td><td> Sigma </td><td> # R4642 </td><td></td></tr><tr><td> dNTP </td><td> Sigma </td><td> # DNTP100 </td><td></td></tr><tr><td> Alexa dUTP </td><td> Invitrogen </td><td> # C11397-C-11401 </td><td></td></tr><tr><td> Cy3 oder Cy5 dUTP </td><td> Fischer </td><td> # 45-001-xxx </td><td></td></tr><tr><td> DNase I </td><td> Roche </td><td> # 04536282001 </td><td></td></tr><tr><td> DNA Pol I </td><td> Biolabs </td><td> # M0209 </td><td></td></tr><tr><td> 0,025 um Filter </td><td> Millipore </td><td> # VSWP02500 </td><td></td></tr><tr><td> Cot-1-DNA 1 mg / ml </td><td> Invitrogen </td><td> # 18440 </td><td></td></tr><tr><td> Hybloc DNA 1 mg / ml </td><td> Angewandte Genetik </td><td> # MHB </td><td></td></tr><tr><td> Lachssperma </td><td> Sigma </td><td> # D1626 </td><td> Pulver bei 10 mg / ml in H 2 O resuspendiert werden </td></tr><tr><td> NaAc (Natriumacetat, pH 5,2, Puffer-Lösung) </td><td> Sigma </td><td> # S7899 </td><td></td></tr><tr><td> Ficoll 400 (Mol Biol grade) </td><td> Fischer </td><td> # 525 </td><td></td></tr><tr><td> Polyvinylpyrrolidon (Mol Biol grade) </td><td> Fischer </td><td> # BP431 </td><td></td></tr><tr><td> Dextransulfat Pulver </td><td> Sigma </td><td> # D8906 </td><td></td></tr><tr><td> SSPE (Saline-Sodium Phosphate-EDTA) 20x-Lösung </td&gt; <td> Fischer </td><td> # BP1328 </td><td></td></tr><tr><td> Formamid </td><td> Fischer </td><td> # BP227 </td><td></td></tr><tr><td> Deckgläser </td><td> Fischer </td><td> # 12-548-B </td><td></td></tr><tr><td> Slides </td><td> Fischer </td><td> # 12-550 </td><td></td></tr><tr><td> 6-Well-Platten </td><td> Fischer </td><td> # 0720080 </td><td></td></tr><tr><td> PBS, 10x </td><td> Fischer </td><td> # MT-46-013-CM </td><td></td></tr><tr><td> Poly-L-Lysin-Lösung </td><td> Sigma </td><td> # P8920 </td><td></td></tr><tr><td> Paraformaldehyd, Prills, 95% </td><td> Sigma </td><td> # 441244 </td><td></td></tr><tr><td> Triton-X-100, Mol Biol Grade </td><td> Sigma </td><td> # T8787 </td><td></td></tr><tr><td> BSA (Bovine Serum Albumin) Fraktion V </td><td> Fischer </td><td> # BP 1600 </td><td></td></tr><tr><td> Normalem Ziegenserum </td><td> Vector Labs </td><td> # S-1000 </td><td></td></tr><tr><td> Tween-20, Mol Biol Grade </td><td> Sigma </td><td> # P9416 </td><td></td></tr><tr><td> SSC (Saline Sodium Citrate) 20x-Lösung </td><td> Fischer </td><td> # BP1325 </td><td></td></tr><tr><td> ProLong Gold-antifade Reagenz </td><td> Invitrogen </td><td> # P36930 </td><td></td></tr><tr><td> DAPI (4 &#39;,6-Diamidino-2-Phenylindol) </td><td> Sigma </td><td> # D9542 </td><td></td></tr><tr><td> Bester Test eine Schicht Gummi-Zement </td><td> Kunst oder Büromärkten </td><td></td><td></td></tr><tr><td></td><td></td><td></td><td> Tabelle 1. Spezifische Reagenzien und Kleingeräten. </td></tr></tbody></table>

combined immunofluorescence and dna fish on 3d-preserved interphase nuclei to study changes in 3d nuclear organization

Fluoreszenz-in situ-Hybridisierung mit DNA-Sonden auf 3-dimensional erhalten Kerne durch 3D-konfokale Mikroskopie verfolgt (3D DNA FISH) stellt die direkteste Weg, um die Lage der Gen-Loci, chromosomalen Subregionen oder ganze Gebiete in einzelnen Zellen zu visualisieren. Diese Art der Analyse gibt einen Einblick in die globale Architektur des Zellkerns als auch das Verhalten der spezifischen genomischen Loci und Regionen innerhalb der nuklearen Raum. Immunfluoreszenz, auf der anderen Seite erlaubt die Detektion von nuklearen Proteinen (modifizierte Histone, Histon-Varianten und Modifikatoren, Transkription Maschinen und Faktoren, Atom-U-Fächer, etc). Die große Herausforderung in der Kombination Immunfluoreszenz und 3D DNA FISH ist, einerseits um das Epitop von dem Antikörper sowie die 3D-Architektur des Kerns detektiert erhalten und andererseits, damit das Eindringen der DNA-Sonde zu detektieren Genloci oder Chromosom Gebieten 1-5. Hier bieten wir ein Protokoll, das die Visualisierung von Chromatin Modifikationen mit genomischen Loci in 3D erhalten Kernen kombiniert.

DNA und Immuno-FISH sind im Laborbetrieb Skok um die Änderungen in Atom-Organisation mit dem Verfahren der V (D) J-Rekombination von Antigenrezeptors Loci während der B-und T-Lymphozyten-Entwicklung verbunden studieren. Die Techniken zuvor dargelegten uns ermöglichen, i) Messung Abstände zwischen den beiden Enden eines Locus (Kontraktion) ii) messen Abstände zwischen Allelen oder Loci (Paarung), iii) eine Analyse der DNA-Schädigung, die innerhalb Loci, iv) Bewertung der Standort der Allele und loci Bezug auf Atom-...

Watch this Scientific Journal Video about Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization at JoVE.com

Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization

Hier beschreiben wir ein Protokoll zur gleichzeitigen Detektion von Histonmodifikationen durch Immunfluoreszenz und DNA-Sequenzen von DNA-FISH durch 3D-Mikroskopie und-Analysen (3D Immuno-DNA FISH) gefolgt.

Kombiniert Immunfluoreszenz und DNA FISH auf 3D erhaltene Interphase Nuclei, um Änderungen in 3D Nuclear Organization Study

Fluorescent in situ hybridization using DNA probes  on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA  FISH) represents the ...

Research

JoVE Journal

Biology

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testen visueller Sensibilität für die Geschwindigkeit und Richtung der Bewegung in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Forschung

Biologie

DNA-based Fish Species Identification Protocol

DNA-basierte Fischarten Identification Protocol

We have developed a fast, simple, and accurate DNA-based screening method to identify the fish species present in fresh and processed seafood samples. ...

Robust 3D DNA FISH Using Directly Labeled Probes

Robust 3D DNA FISH Mit Direkt markierten Sonden

3D DNA FISH has become a major tool for  analyzing three-dimensional organization of the nucleus, and several variations  of the technique have been ...

Assessing Species-specific Contributions To Craniofacial Development Using Quail-duck Chimeras

Die Beurteilung artspezifische Um Beiträge Craniofacial Entwicklung mit Wachtel-Ente Chimären

The generation of chimeric embryos is a widespread and powerful approach to study cell fates, tissue interactions, and species-specific contributions to ...

Combined DNA-RNA Fluorescent In situ Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Combined DNA-RNA Fluorescent In situ Hybridization FISH to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Kombinierte DNA-RNA-Leuchtstoff<em&gt; In situ</em&gt; Hybridisierung (FISH) zu X-Chromosom-Inaktivierung in Differenzierte Weiblich embryonalen Stammzellen der Maus Studieren

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in ...

Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

Mess DNA-Schädigung und -Reparatur in Maus-Splenozyten nach chronischer<em&gt; In Vivo</em&gt; Die Exposition gegenüber sehr niedrigen Dosen von Beta- und Gamma-Strahlung

Low dose radiation exposure may produce a variety of biological effects that are different in quantity and quality from the effects produced by high ...

Experimental Approaches to Study Mitochondrial Localization and Function of a Nuclear Cell Cycle Kinase, Cdk1

Experimentelle Ansätze zur Untersuchung Mitochondriale Lokalisation und Funktion eines Kernzellzyklus-Kinase, Cdk1

Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

Isolierung von spezifischen genomischen Regionen und Identifizierung von assoziierten Moleküle durch enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

Methoden zur Untersuchung Veränderungen inhärenten Proteinaggregation mit dem Alter in Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...

Sequential Immunofluorescence and Immunohistochemistry on Cryosectioned Zebrafish Embryos

Sequenzielle Immunfluoreszenz und Immunhistochemie an kryosektionierten Zebrafisch-Embryonen

Investigation of intercellular interactions often requires discrete labeling of specific cell populations and precise protein localization. The zebrafish ...