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 gjerne takke medlemmene av Skok lab, spesielt Susannah Hewitt, for diskusjoner og kommentarer. Dette arbeidet er støttet av National Institute of Health tilskudd R01GM086852, RC1CA145746 (JAS). JAS er en Leukemi og lymfom Society forsker. JC er en Irvington Institute Fellow ved Cancer Research Institute. MM er støttet av National Science Foundation Grant Integrative Graduate Utdannings-og forskningsdepartementet Praksisplass (NSF IGERT 0333389).

1. DNA Probe Merking med fluoroforer: Nick Translation (~ 6 timer) <ol><li> Renhold BAC DNA (fremstilt ved maksi-prep) eller plasmider eller PCR-produkter, alle resuspendert i H 2 O, kan brukes til merking. Merk at for en robust fiskeekkoet bør prober spenner minst 10 kb. </li><li> Inkuber DNA i RNase A i 30 minutter ved 37 ° C (alle reagenser er oppført i Tabell 1). </li><li> Inkuber nick-translasjon reaksjonen i 2 timer ved 16 ° C (se tabell 2 og 3). ...

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Teknikkene beskrevet ovenfor ble brukt i vårt laboratorium for å analysere reguleringen av V (D) J-rekombinasjon av immunoglobulin og Tcra / d loci i utviklingen lymfocytter 30,31. Vi er sikre på at denne teknikken kan tilpasses for påvisning av ulike kjernefysiske proteiner, kjernefysiske avdelinger og loci, i ulike celle-typer. Modifikasjoner av protokollen kan være nødvendig, og i dette tilfellet de viktigste trinnene å fokusere på er følgende. Først, kan lengden permeabilizatio...

Epigenetic mekanismer utløser etablering og arv av utviklings-og celle-type spesifikke transkripsjonelle profiler. På ett nivå innebærer dette modulering av kromatin emballasje som definerer aktive eller stille genomisk regioner. I større målestokk, global 3D organisering av genomet og kjernefysisk arkitektur også spille en rolle i kontroll av transkripsjonelle mønstre. Dermed er disseksjon av disse epigenomic funksjoner avgjørende for en full forståelse av hvordan genene reguleres 6-11. Kombinert immunfluorescens og 3D DNA FISH gir en unik mulighet til å utfylle biokjemiske og molekylærbiologiske analyser ved å vurdere konkrete interaksjoner / foreninger av DNA-sekvenser og / eller proteiner i kjernen. Videre, mens genom-wide høy gjennomstrømning teknikker som kromatin immunoutfelling (chip-seq) eller kromosom fangst konformasjon kombinert med dyp sekvensering (4C-seq, 5C, Hi-C) gi global data på cellepopulasjoner 12, immunofluorescence / DNA FISK teknikker gjør analyser på enkelt celle-nivå. Her beskriver vi en protokoll for samtidig påvisning av histonmodifikasjonene ved immunfluorescens og DNA-sekvenser av DNA fisk etterfulgt av 3D mikroskopi og analyser (3D immuno-FISH). Fordelen med denne protokollen er den kombinerte visualisering av DNA og bevaring av proteinstrukturer. Vår erfaring på dette feltet har gitt oss mulighet til å forbedre og forenkle eksisterende protokoller. Selv om vi har brukt denne protokollen for å oppdage DNA dobbel-strandet pauser i lymfocytter gjennomgår rekombinasjon, kan denne metoden brukes på andre proteiner og andre celle-typer.

<table border="1"><tbody><tr><td> Navn på reagens / Material </td><td> Selskapet </td><td> Katalognummer </td><td> Kommentarer </td></tr><tr><td> H 2 O </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 mikrometer 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> Applied Genetics </td><td> # MHB </td><td></td></tr><tr><td> Laksesperm </td><td> Sigma </td><td> # D1626 </td><td> pulver som skal resuspendert ved 10 mg / ml i H 2 O </td></tr><tr><td> NaAc (natriumacetat, pH 5,2, bufferoppløsning) </td><td> Sigma </td><td> # S7899 </td><td></td></tr><tr><td> Ficoll 400 (Mol Biol grade) </td><td> Fisher </td><td> # 525 </td><td></td></tr><tr><td> Polyvinylpyrrolidon (Mol Biol grade) </td><td> Fisher </td><td> # BP431 </td><td></td></tr><tr><td> Dekstransulfat pulver </td><td> Sigma </td><td> # D8906 </td><td></td></tr><tr><td> SSPE (Saline-natriumfosfat-EDTA) 20x løsning </td&gt; <td> Fisher </td><td> # BP1328 </td><td></td></tr><tr><td> Formamide </td><td> Fisher </td><td> # BP227 </td><td></td></tr><tr><td> Dekkglass </td><td> Fisher </td><td> # 12-548-B </td><td></td></tr><tr><td> Lysbilder </td><td> Fisher </td><td> # 12-550 </td><td></td></tr><tr><td> 6-brønners plater </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-lysin løsning </td><td> Sigma </td><td> # P8920 </td><td></td></tr><tr><td> Paraformaldehyd, priller, 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 (bovint serum albumin) fraksjon V </td><td> Fisher </td><td> # BP 1600 </td><td></td></tr><tr><td> Normal geit serum </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 løsning </td><td> Fisher </td><td> # BP1325 </td><td></td></tr><tr><td> Forlenge Gold antifade reagens </td><td> Invitrogen </td><td> # P36930 </td><td></td></tr><tr><td> DAPI (4 &#39;,6-diamidino-2-phenylindole) </td><td> Sigma </td><td> # D9542 </td><td></td></tr><tr><td> Best test ett strøk gummi sement </td><td> Kunst eller på kontoret forsyne butikkene </td><td></td><td></td></tr><tr><td></td><td></td><td></td><td> Tabell 1. Spesifikke reagenser og små utstyr. </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-dimensjonalt bevart kjerner etterfulgt av 3D konfokalmikroskopi (3D DNA FISH) representerer den mest direkte måten å visualisere plasseringen av genloci, kromosomal subregioner eller hele områder i enkeltceller. Denne typen analyse gir et innblikk i global arkitektur av kjernen så vel som virkemåten av spesifikke genomisk loci og regioner innenfor kjernefysiske plass. Immunfluorescens, på den annen side, tillater påvisning av kjernefysiske proteiner (modifiserte histoner, histone varianter og modifikatorer, transkripsjon maskiner og faktorer, kjernefysiske sub-avdelinger, etc). Den største utfordringen i å kombinere immunfluorescens og 3D DNA FISH er, på den ene side for å bevare epitopen detektert av antistoffet samt 3D arkitekturen av kjernen, og på den annen side, for å tillate inntrengning av DNA probe for å påvise genloci eller kromosom territorier 1-5. Her tilbyr vi en protokoll som kombinerer visualisering av kromatin modifikasjoner med genomisk loci i 3D bevart kjerner.

DNA og immuno-fisk brukes i Skok lab til studere endringene i kjernefysisk organisasjon knyttet til prosessen av V (D) J-rekombinasjon av antigen-reseptor loci under B og T-lymfocytt utvikling. Teknikkene beskrevet ovenfor gjør oss i stand til å i) måle avstander mellom de to endene av et locus (sammentrekning) ii) måle avstander mellom alleler eller loci (sammenkobling), iii) analysere DNA skade som oppstår i løpet av loci, iv) vurdere plasseringen av alleler og loci i forhold til kjernekraft sub-avdelinger (unde...

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 protokoll for samtidig påvisning av histonmodifikasjonene ved immunfluorescens og DNA-sekvenser av DNA fisk etterfulgt av 3D mikroskopi og analyser (3D immuno-DNA FISH).

Kombinert Immunofluorescence og DNA FISH på 3D bevarte Interphase Nuclei å studere endringer 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 Bruke Direkte merkede 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 artsspesifikke bidrag til Kraniofacial Development Bruke Quail-duck 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

Kombinert DNA-RNA Fluorescent<em&gt; In situ</em&gt; Hybridisering (FISH) for å studere X kromosom inaktivering i Differensiert Kvinne Mouse 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

Måle DNA skade og reparer i musesplenocyter Etter Kronisk<em&gt; I Vivo</em&gt; Eksponering for svært lave doser av 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 tilnærminger til å studere mitokondrie lokalisering og funksjon for en kjernefysisk 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 av Spesifikke Genomiske Regioner og Identifisering av Associated Molekyler av 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 for å studere endringer i iboende Protein samling 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

Sekvensiell Immunofluorescence og immunhistokjemi på Cryosectioned sebrafisk embryo

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