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

Vi vil gerne takke medlemmerne af Skok lab, især Susannah Hewitt, for diskussioner og kommentarer. Dette arbejde er støttet af National Institute of Health tilskud R01GM086852, RC1CA145746 (JAS). JAS er en Leukemia &amp; Lymphoma Society lærd. JC er en Irvington Institute Fellow af Cancer Research Institute. MM er støttet af en National Science Foundation Grant Integrativ Graduate Uddannelse og Forskning praktik (NSF IGERT 0.333.389).

1. DNA Probe Mærkning med fluoroforer: Nick Translation (~ 6 timer) <ol><li> Ren BAC DNA (fremstillet ved maxi-prep) eller plasmider eller PCR-produkter, alle resuspenderet i H2O, kan anvendes til mærkning. Bemærk, at for en robust FISH signal, bør prober spænde mindst 10 kb. </li><li> Inkuber DNA i RNase A i 30 minutter ved 37 ° C (Alle reagenser er anført i tabel 1). </li><li> Inkubér nick-translation reaktion i 2 timer ved 16 ° C (se tabel 2 og 3).</stro...

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Teknikkerne beskrevet ovenfor blev anvendt i vores laboratorium til analyse reguleringen af V (D) J rekombination af immunoglobulinet og Tcra / d loci i udviklingen af lymfocytter 30,31. Vi tror på, at denne teknik kan tilpasses til påvisning af forskellige nukleare proteiner, nukleare rum og loci, i forskellige celletyper. Ændringer af protokollen kan være nødvendig, og i dette tilfælde de vigtigste trin til at fokusere på, er følgende. For det første kan længden af ​​permeabi...

Epigenetiske mekanismer udløser etablering og arv af udviklings-og celletype specifikke transkriptionelle profiler. På et niveau dette indebærer modulering af kromatin emballage, der definerer aktive eller stille genomiske regioner. I en større målestok, også global 3D organisering af genomet og nuklear struktur spiller en rolle i kontrollen af ​​transkriptionelle mønstre. Således dissektion af disse epigenomic funktioner er afgørende for en fuld forståelse af, hvordan generne reguleres 6-11. Kombineret immunofluorescens og 3D DNA FISH giver en unik mulighed for at supplere molekylære og biokemiske analyser ved at vurdere specifikke interaktioner / sammenslutninger af DNA-sekvenser og / eller proteiner i kernen. Og selv om genom-dækkende high throughput-teknikker, såsom chromatin immunoprecipitation (chip-seq) eller kromosom capture konformation kombineret med dyb sekventering (4C-seq, 5C, Hi-C) giver global daten på cellepopulationer 12, immunofluorescens / DNA FISH teknikker muliggøre analyser på enkelt celle niveau. Her beskriver vi en protokol til samtidig påvisning af histon ændringer ved immunofluorescens og DNA sekvenser ved DNA FISK efterfulgt af 3D mikroskopi og analyser (3D immuno-FISH). Fordelen ved denne protokol er den kombinerede visualisering af DNA og bevarelse af proteinstrukturer. Vores erfaring på dette område har gjort det muligt for os at forbedre og forenkle eksisterende protokoller. Selv om vi har anvendt denne protokol til påvisning af DNA dobbeltstrengede brud i lymfocytter undergår rekombination, kan denne metode anvendes på andre proteiner og andre celletyper.

<table border="1"><tbody><tr><td> Navn på reagens / Material </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer </td></tr><tr><td> H2O </td><td> Fisher </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 til C-11401 </td><td></td></tr><tr><td> Cy3 eller Cy5 dUTP </td><td> Fisher </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 filtre </td><td> Millipore </td><td> # VSWP02500 </td><td></td></tr><tr><td> Cot-1-DNA 1 mg / ml </td><td> Invitrogen </td><td> # 18.440 </td><td></td></tr><tr><td> Hybloc DNA 1 mg / ml </td><td> Applied Genetics </td><td> # MHB </td><td></td></tr><tr><td> Laksesperma </td><td> Sigma </td><td> # D1626 </td><td> pulver, der skal resuspenderes ved 10 mg / ml i H2O </td></tr><tr><td> NaAc (natriumacetat, pH 5,2, pufferopløsning) </td><td> Sigma </td><td> # S7899 </td><td></td></tr><tr><td> Ficoll 400 (Mol Biol kvalitet) </td><td> Fisher </td><td> # 525 </td><td></td></tr><tr><td> Polyvinylpyrrolidon (Mol Biol kvalitet) </td><td> Fisher </td><td> # BP431 </td><td></td></tr><tr><td> Dextransulfat pulver </td><td> Sigma </td><td> # D8906 </td><td></td></tr><tr><td> SSPE (saltvand-natriumphosphat-EDTA) 20x opløsning </td&gt; <td> Fisher </td><td> # BP1328 </td><td></td></tr><tr><td> Formamid </td><td> Fisher </td><td> # BP227 </td><td></td></tr><tr><td> Dækglas </td><td> Fisher </td><td> # 12-548-B </td><td></td></tr><tr><td> Slides </td><td> Fisher </td><td> # 12-550 </td><td></td></tr><tr><td> 6-brøndsplader </td><td> Fisher </td><td> # 0720080 </td><td></td></tr><tr><td> PBS, 10x </td><td> Fisher </td><td> # MT-46-013-CM </td><td></td></tr><tr><td> Poly-L-lysinopløsningen </td><td> Sigma </td><td> # P8920 </td><td></td></tr><tr><td> Paraformaldehyd, prills, 95% </td><td> Sigma </td><td> # 441.244 </td><td></td></tr><tr><td> Triton-X-100, Mol Biol kvalitet </td><td> Sigma </td><td> # T8787 </td><td></td></tr><tr><td> BSA (bovint serumalbumin) fraktion V </td><td> Fisher </td><td> # BP 1600 </td><td></td></tr><tr><td> Normalt gedeserum </td><td> Vector Labs </td><td> # S-1000 </td><td></td></tr><tr><td> Tween-20, Mol Biol kvalitet </td><td> Sigma </td><td> # P9416 </td><td></td></tr><tr><td> SSC (Saline natriumcitrat) 20x opløsning </td><td> Fisher </td><td> # BP1325 </td><td></td></tr><tr><td> Forlæng Guld antiblegemiddel reagens </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> Best test ét ​​lag gummi cement </td><td> Kunst eller på kontoret levering butikker </td><td></td><td></td></tr><tr><td></td><td></td><td></td><td> Tabel 1. Specifikke reagenser og mindre udstyr. </td></tr></tbody></table>

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

Fluorescerende in situ hybridisering med DNA-prober på 3-dimensionelt bevarede kerner efterfulgt af 3D konfokal mikroskopi (3D DNA FISH) repræsenterer den mest direkte måde at visualisere placeringen af gen loci, kromosomale subregioner eller hele områder i de enkelte celler. Denne type analyse giver indblik i den globale arkitektur af kernen samt opførsel af specifikke genomiske loci og regioner i det nukleare område. Immunofluorescens på den anden side tillader påvisning af kerneproteiner (modificerede histoner, histon varianter og modifikatorer, transkriptionsmekanisme og faktorer, nukleare delrum osv.). Den største udfordring i at kombinere immunofluorescens og 3D DNA FISH er, på den ene side at bevare epitopen detekteres af antistoffet såvel som 3D arkitektur af kernen, og på den anden side, for at tillade gennemtrængning af DNA-probe til påvisning gen loci eller kromosom territorier 1-5. Her giver vi en protokol, der kombinerer visualisering af kromatin modifikationer med genomiske loci i 3D konserveret kerner.

DNA og immuno-FISH anvendes i Skok laboratoriet at studere ændringer i nuklear organisering i forbindelse med fremgangsmåden ifølge V (D) J rekombination af antigen-receptor loci i B-og T-lymfocyt-udvikling. De teknikker beskrevet ovenfor giver os mulighed for i) måle afstande mellem de to ender af et locus (sammentrækning) ii) måle afstande mellem alleler eller loci (parring), iii) at analysere DNA-skader forekommer inden loci, iv) vurdere placeringen af ​​alleler og loci i forhold til nukleare delrum (repres...

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

Her beskriver vi en protokol til samtidig påvisning af histon ændringer ved immunofluorescens og DNA sekvenser ved DNA FISK efterfulgt af 3D mikroskopi og analyser (3D immuno-DNA FISH).

Kombineret Immunofluorescens og DNA FISH på 3D-bevaret Interfasen Kerner til Uddannelse Ændringer i 3D Nuclear Organization

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

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Biology

Testing Visual Sensitivity to the Speed and Direction of Motion 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

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 Brug Direkte mærkede prober

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

Vurdere artsspecifikke bidrag til kraniofaciale Development Brug Quail-ænder kimærer

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

Kombineret DNA-RNA Fluorescent<em&gt; In situ</em&gt; Hybridisering (FISH) til at studere X-kromosom inaktivering differentieret hunmus embryonale stamceller

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

Måling DNA Skader og reparation i musesplenocytter Efter Kronisk<em&gt; In vivo</em&gt; Udsættelse for meget lave doser af beta og gamma-stråling

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

Eksperimentelle tilgange til at studere mitokondrie Lokalisering og funktion af en nuklear Cell Cycle 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

Isolering af specifikke genomiske regioner og Identifikation af associerede molekyler ved 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

Metoder til undersøgelse ændringer i iboende Protein sammenlægning med alder i 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

Sekventiel immunofluorescens og Immunhistokemi på Kryosecerede Zebrafish-embryoner

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