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

We willen de leden van de Skok lab, in het bijzonder Susannah Hewitt, bedanken voor discussies en commentaar. Dit werk wordt ondersteund door de National Institute of Health subsidies R01GM086852, RC1CA145746 (JAS). JAS is een Leukemia &amp; Lymphoma Society geleerde. JC is een Irvington Instituut Fellow van het Cancer Research Institute. MM wordt ondersteund door een National Science Foundation Grant Integratieve Graduate Onderwijs en Onderzoek Traineeship (NSF IGERT 0333389).

1. DNA Probe Labeling met fluoroforen: Nick Vertaling (~ 6 uur) <ol><li> Schoon BAC DNA (bereid door maxi-prep) of plasmiden of PCR producten alle geresuspendeerd in H 2 O, kunnen worden gebruikt voor labeling. Merk op dat een robuust FISH signaal probes ten minste 10 kb omvatten. </li><li> Incubeer DNA in RNase A gedurende 30 min bij 37 ° C (alle reagentia zijn vermeld in tabel 1). </li><li> Incubeer de nicktranslatie reactie gedurende 2 uur bij 16 ° C (zie tabel...

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De hierboven beschreven technieken werden in ons laboratorium van de regulering van V (D) J recombinatie van het immunoglobuline en Tcra / d loci in ontwikkeling lymfocyten 30,31 analyseren. We zijn ervan overtuigd dat deze techniek kan worden aangepast voor detectie van verschillende nucleaire eiwitten, nucleaire compartimenten en loci, in verschillende celtypen. Wijzigingen van het protocol noodzakelijk zijn, en in dit geval de belangrijke stappen te concentreren op de volgende. Ten eerste...

Epigenetische mechanismen trekker vestiging en overerving van ontwikkelings-en cel-type specifieke transcriptionele profielen. Op een bepaald niveau gaat het om de modulatie van chromatine verpakking dat de actieve of stille genomische regio&#39;s definieert. Op grotere schaal, algemene 3D organisatie van het genoom en nucleaire architectuur een rol spelen in de controle van transcriptionele patronen. Zo dissectie van deze epigenomisch functies is van essentieel belang voor een goed begrip van de manier waarop genen worden gereguleerd 6-11. Gecombineerd immunofluorescentie en 3D DNA FISH bieden een unieke gelegenheid om moleculaire en biochemische analyses aan te vullen met het beoordelen van specifieke interacties / verenigingen van DNA-sequenties en / of eiwitten in de kern. Bovendien, terwijl genoom-brede high throughput technieken zoals chromatine immunoprecipitatie (ChIP-seq) of chromosoom capture conformatie in combinatie met diepe sequencing (4C-seq, 5C, Hi-C) bieden wereldwijde thateen op celpopulaties 12, immunofluorescentie / DNA FISH technieken maken van de analyses op de enkele cel niveau. Hier beschrijven we een protocol voor gelijktijdige detectie van histonmodificaties door immunofluorescentie en DNA-sequenties van DNA FISH gevolgd door 3D microscopie en analyses (3D-immuno FISH). Het voordeel van dit protocol is de gecombineerde visualisatie van DNA en behoud van eiwitstructuren. Onze ervaring op dit gebied heeft ons in staat te verbeteren en vereenvoudiging van de bestaande protocollen. Hoewel we hebben dit protocol DNA dubbelstrengs breuken gedetecteerd in lymfocyten ondergaan recómbinatie, kan deze werkwijze worden toegepast op andere eiwitten en andere celtypen.

<table border="1"><tbody><tr><td> Naam van de Reagens / Materiaal </td><td> Vennootschap </td><td> Catalogus nummer </td><td> Reacties </td></tr><tr><td> H 2 O </td><td> Visser </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 naar C-11401 </td><td></td></tr><tr><td> Cy3 of Cy5 dUTP </td><td> Visser </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 micrometer filters </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> Toegepaste Genetica </td><td> # MHB </td><td></td></tr><tr><td> Zalmsperma </td><td> Sigma </td><td> # D1626 </td><td> poeder worden gesuspendeerd bij 10 mg / ml in H 2 O </td></tr><tr><td> NaAc (natriumacetaat, pH 5,2, buffer oplossing) </td><td> Sigma </td><td> # S7899 </td><td></td></tr><tr><td> Ficoll 400 (Mol Biol grade) </td><td> Visser </td><td> # 525 </td><td></td></tr><tr><td> Polyvinylpyrrolidone (Mol Biol grade) </td><td> Visser </td><td> # BP431 </td><td></td></tr><tr><td> Dextraansulfaat poeder </td><td> Sigma </td><td> # D8906 </td><td></td></tr><tr><td> SSPE (Saline-Sodium Phosphate-EDTA) 20x-oplossing </td&gt; <td> Visser </td><td> # BP1328 </td><td></td></tr><tr><td> Formamide </td><td> Visser </td><td> # BP227 </td><td></td></tr><tr><td> Dekglaasjes </td><td> Visser </td><td> Nr. 12-548-B </td><td></td></tr><tr><td> Slides </td><td> Visser </td><td> # 12 tot 550 </td><td></td></tr><tr><td> 6-well platen </td><td> Visser </td><td> # 0720080 </td><td></td></tr><tr><td> PBS, 10x </td><td> Visser </td><td> # MT-46-013-CM </td><td></td></tr><tr><td> Poly-L-lysine oplossing </td><td> Sigma </td><td> # P8920 </td><td></td></tr><tr><td> Paraformaldehyde, prils, 95% </td><td> Sigma </td><td> # 441244 </td><td></td></tr><tr><td> Triton-X-100, Mol Biol graad </td><td> Sigma </td><td> # T8787 </td><td></td></tr><tr><td> BSA (Bovine Serum Albumine) Fraction V </td><td> Visser </td><td> # BP 1600 </td><td></td></tr><tr><td> Normaal geitenserum </td><td> Vector Labs </td><td> # S-1000 </td><td></td></tr><tr><td> Tween-20, Mol Biol graad </td><td> Sigma </td><td> # P9416 </td><td></td></tr><tr><td> SSC (Saline natriumcitraat) 20x oplossing </td><td> Visser </td><td> # BP1325 </td><td></td></tr><tr><td> ProLong Gold antifade reagens </td><td> Invitrogen </td><td> # P36930 </td><td></td></tr><tr><td> DAPI (4 &#39;,6-diamidino-2-fenylindool) </td><td> Sigma </td><td> # D9542 </td><td></td></tr><tr><td> Beste test een laag rubber cement </td><td> Art of kantoor aanbod winkels </td><td></td><td></td></tr><tr><td></td><td></td><td></td><td> Tabel 1. Specifieke reagentia en klein materiaal. </td></tr></tbody></table>

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

Fluorescente in situ hybridisatie met DNA probes on 3-dimensionaal bewaard kernen gevolgd door 3D confocale microscopie (3D DNA FISH) is de meest directe manier om de locatie van genloci, chromosomale deelgebieden of gehele grondgebied in individuele cellen te visualiseren. Dit type analyse geeft inzicht in de globale architectuur van de kern en het gedrag van specifieke genomische loci en gebieden binnen de nucleaire ruimte. Immunofluorescentie, anderzijds, voor het vaststellen van nucleaire eiwitten (gemodificeerde histonen, histon varianten en modifiers, transcriptiemachinerie en factoren, nucleaire subcompartimenten, etc). De grote uitdaging in combinatie immunofluorescentie en 3D DNA FISH is enerzijds aan de epitoop herkend door het antilichaam en de 3D structuur van de kern te behouden en anderzijds, zodat de penetratie van de DNA probe te detecteren genloci of chromosoom gebieden 1 tot 5. Hier bieden wij een protocol dat visualisatie van chromatine modificaties met genomische loci in 3D bewaarde kernen combineert.

DNA en immuno-FISH worden gebruikt in de Skok lab de veranderingen in nucleaire organisatie bij het proces van V (D) J recombinatie van antigen receptor loci tijdens B en T lymfocyten ontwikkeling te bestuderen. De technieken hierboven beschreven kunnen we i) afstanden meten tussen de twee uiteinden van een locus (contractie) ii) afstanden meten tussen allelen of loci (pairing), iii) het analyseren van de DNA-schade die binnen loci, iv) een beoordeling van de locatie van allelen en loci ten opzichte van nucleaire sub-co...

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 beschrijven we een protocol voor gelijktijdige detectie van histonmodificaties door immunofluorescentie en DNA-sequenties van DNA FISH gevolgd door 3D microscopie en analyses (3D-immuno DNA FISH).

Gecombineerd Immunofluorescentie en DNA FISH op 3D-bewaarde interfase kernen aan veranderingen in 3D Nuclear Organisatie Studie

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

Research

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Biology

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

Het testen van visuele gevoeligheid voor de snelheid en richting van de beweging in Lizards

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

DNA-based Fish Species Identification Protocol

DNA-gebaseerde Vissoorten 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

Robuuste 3D DNA FISH behulp direct gemerkt Probes

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

Het beoordelen Soortspecifieke Bijdragen aan craniofaciale ontwikkeling met behulp van Quail-eend Chimeras

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

Gecombineerde DNA-RNA-TL<em&gt; In situ</em&gt; Hybridisatie (FISH) aan X-chromosoom inactivatie studeren in Gedifferentieerde vrouwelijke muis embryonale stamcellen

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

Het meten van DNA-schade en herstel in Mouse Splenocyten Na Chronische<em&gt; In Vivo</em&gt; Blootstelling aan zeer lage doses van bèta en gamma-straling

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

Experimentele benaderingen om te studeren mitochondriale lokalisatie en functie van een nucleair celcyclus 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

Isolatie van specifieke genomische regio en identificatie van geassocieerde moleculen door 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 om wijzigingen van de studie in inherente aggregatie van eiwitten met de leeftijd 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

Sequentiële immunofluorescentie en immunohistochemie op Gecryode zebravis-embryo's

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