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

Biz tartışmalar ve yorumlar için Skok laboratuar, özellikle Susannah Hewitt, üyelerine teşekkür etmek istiyorum. Bu çalışma Sağlık hibe Ulusal Enstitüsü R01GM086852, RC1CA145746 (JAS) tarafından desteklenmektedir. JAS Bir Lösemi ve Lenfoma Derneği bilgindir. JC Kanser Araştırma Enstitüsü Irvington Enstitüsü üyesidir. MM Ulusal Bilim Vakfı Hibe Bütünleştirici Lisansüstü Eğitim ve Araştırma Staj (NSF IGERT 0333389) tarafından desteklenmektedir.

1. Fluorophores DNA Prob Etiketleme: Nick Çeviri (~ 6 saat) <ol><li> Temizlik BAC DNA (Maxi-prep göre hazırlanmış) ve plazmit veya PCR ürünleri, H2O içinde tekrar süspanse edilir, tüm etiketleme için de kullanılabilir. Not sağlam bir FISH sinyal için, sondalar, en az 10 kb kapsar gerektiğini. </li><li> RNaz DNA inkübe bir 37 30 dakika ° C (Bütün çözeltiler Tablo 1&#39;de listelenmiştir). </li><li> 16. az 2 saat süreyle nick translation reaksiyon inkü...

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Yukarıda ayrıntılı teknikler lenfositler 30,31 geliştirme İmmunoglobulin ve Tcra / d loci V (D) J rekombinasyon düzenleme analiz etmek için, laboratuar kullanılmıştır. Biz bu tekniği farklı hücre tiplerinde çeşitli nükleer proteinler, nükleer bölmeleri ve lokus tespiti için adapte edilebilir eminiz. Protokol değişiklikler gerekli olabilir, ve bu durumda, odaklanmak için önemli adımlar aşağıdaki gibidir. İlk olarak, geçirgenliği uzunluğu hücre tipine bağlı ...

Epigenetik mekanizmalar tetik kuruluş ve gelişim ve hücre tipi özel transkripsiyonel profilleri kalıtım. Bir düzeyde bu aktif veya sessiz genomik bölgelerde tanımlar kromatin paketlenmesi modülasyonu içerir. Daha büyük ölçekte, genom ve nükleer mimarisinin küresel 3D organizasyonu da transkripsiyonel desen kontrolünde bir rol oynamaktadır. Böylece, bu epigenomic özelliklerin diseksiyonu genler 6-11 düzenlenir nasıl tam bir anlayış için önemlidir. Kombine immünofloresan ve 3D DNA FISH spesifik etkileşimler / çekirdeğin içinde DNA dizileri ve / veya protein dernekler değerlendirerek, biyokimyasal ve moleküler analizler tamamlamak için eşsiz bir fırsat sunmaktadır. Bunun yanında, bu tür kromatin immünopresipitasyon (ChIP-seq) veya derin dizileme ile birleştiğinde kromozom yakalama konformasyon olarak genom yüksek verimlilik teknikleri (4C-seq, 5C, Hi-C) küresel dat sağlamakhücre popülasyonlarının 12 üzerinde, immünofloresan / DNA FISH teknikleri tek hücre düzeyinde analiz sağlar. Burada 3D mikroskopi ve analizler (3D immuno-FISH) izledi DNA FISH immünofloresan ve DNA dizileri tarafından histon modifikasyonları eşzamanlı tespiti için bir protokol açıklar. Bu protokolün avantaj DNA ve protein yapılarının korunması kombine görselleştirme olduğunu. Bu alanda tecrübemiz mevcut protokoller geliştirmek ve kolaylaştırmak için bize sağladı. Bu rekombinasyon geçiren lenfosit çift-sarmallı DNA mola tespit etmek için bu protokol kullanılmıştır, ancak bu yöntem, diğer proteinlere ve diğer hücre türleri uygulanabilir.

<table border="1"><tbody><tr><td> Reaktif / Malzeme Adı </td><td> Şirket </td><td> Katalog Numarası </td><td> Yorumlar </td></tr><tr><td> H2O </td><td> Balıkçı </td><td> # BP2470 </td><td></td></tr><tr><td> RNaz 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 veya Cy5, dUTP </td><td> Balıkçı </td><td> # 45-001-xxx </td><td></td></tr><tr><td> DNaz 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 mikron filtre </td><td> MilliporE </td><td> # VSWP02500 </td><td></td></tr><tr><td> Manşon-1 DNA 1 mg / ml </td><td> Invitrogen </td><td> # 18440 </td><td></td></tr><tr><td> Hybloc DNA 1 mg / ml </td><td> Uygulamalı Genetik </td><td> # MHB </td><td></td></tr><tr><td> Somon sperm </td><td> Sigma </td><td> # D1626 </td><td> toz H2O içinde 10 mg / ml &#39;de tekrar süspanse edilmesi </td></tr><tr><td> NAAC (Sodyum Asetat, pH 5.2, tampon çözeltisi) </td><td> Sigma </td><td> # S7899 </td><td></td></tr><tr><td> Ficoll 400 (Mol Biol grade) </td><td> Balıkçı </td><td> # 525 </td><td></td></tr><tr><td> Polivinilpirolidon (Mol Biol grade) </td><td> Balıkçı </td><td> # BP431 </td><td></td></tr><tr><td> Dekstran sülfat tozu </td><td> Sigma </td><td> # D8906 </td><td></td></tr><tr><td> SSPE (Tuzlu-Sodyum Fosfat-EDTA) 20x çözümü </td&gt; <td> Balıkçı </td><td> # BP1328 </td><td></td></tr><tr><td> Formamid </td><td> Balıkçı </td><td> # BP227 </td><td></td></tr><tr><td> Lameller </td><td> Balıkçı </td><td> # 12-548-B </td><td></td></tr><tr><td> Slaytlar </td><td> Balıkçı </td><td> # 12-550 </td><td></td></tr><tr><td> 6-well plate </td><td> Balıkçı </td><td> # 0720080 </td><td></td></tr><tr><td> 10x PBS, </td><td> Balıkçı </td><td> # MT-46-013-CM </td><td></td></tr><tr><td> Poli-L-lisin çözelti </td><td> Sigma </td><td> # P8920 </td><td></td></tr><tr><td> Paraformaldehyde, zerreciklerinin,% 95 </td><td> Sigma </td><td> # 441244 </td><td></td></tr><tr><td> Triton-X-100, Mol Biol dereceli </td><td> Sigma </td><td> # T8787 </td><td></td></tr><tr><td> BSA (Bovine Serum Albumin) Fraksiyon V </td><td> Balıkçı </td><td> # BP 1600 </td><td></td></tr><tr><td> Normal keçi serumu </td><td> Vektör Labs </td><td> # S-1000 </td><td></td></tr><tr><td> Tween-20, Mol Biol dereceli </td><td> Sigma </td><td> # P9416 </td><td></td></tr><tr><td> SSC (Salin Sodyum Sitrat) 20x çözümü </td><td> Balıkçı </td><td> # BP1325 </td><td></td></tr><tr><td> Altın antifade reaktifi uzatmak </td><td> Invitrogen </td><td> # P36930 </td><td></td></tr><tr><td> DAPI (4 &#39;,6-diamidino-2-fenilindol) </td><td> Sigma </td><td> # D9542 </td><td></td></tr><tr><td> En testi tek kat kauçuk çimento </td><td> Sanat veya ofis malzemeleri mağazaları </td><td></td><td></td></tr><tr><td></td><td></td><td></td><td> Tablo 1. Özgül reaktifleri ve küçük ekipman. </td></tr></tbody></table>

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

3D konfokal mikroskopi ardından 3 boyutlu korunmuş çekirdekleri üzerinde DNA problar kullanılarak floresan in situ hibridizasyon (3D DNA FISH) bireysel hücrelerin alt bölgeler kromozomal gen lokuslarının yeri veya tüm ülkelerle görselleştirmek için en doğru yolu gösterir. Bu tip analiz çekirdeğin küresel mimari gibi nükleer boşluk içinde belirli lokus ve genomik bölgenin davranışının fikir verir. Immunofluorescence, diğer taraftan, nükleer proteinlerin tespiti (modifiye edilmiş histon, histon türevleri ve değiştiriciler, transkripsiyon faktörleri ve makine, nükleer alt-bölme, vb) izin verir. Immünofloresan ve 3B DNA FISH birleştirme içinde önemli bir sorun çekirdeğin 3D mimarisi yanı sıra antikor tarafından tespit edilen epitopu korumak için bir taraftan, ve diğer taraftan, DNA prob nüfuz tespit etmesi için gen lokus veya kromozom bölgeleri 1-5. Burada 3D korunmuş çekirdeklerinde genomik lokus ile kromatin modifikasyonları görselleştirme birleştiren bir protokol sağlar.

DNA ve immüno-FISH B ve T lenfosit gelişimi sırasında antijen reseptörünün bir lokus, V (D) J rekombinasyon işlemi ile bağlantılı olarak nükleer organizasyon değişiklikleri çalışması için Skok laboratuvar kullanılmaktadır. Yukarıda ayrıntılı teknikleri) bize i odağının iki ucu (kasılma) arasında) mesafeleri ölçmek için ii) allel veya lokus (eşleştirme) arasındaki mesafeleri ölçmek, iii), lokus içindeki iv meydana DNA hasarı analiz etkinleştirmek allellerin yerini değerlendirmek ...

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

Burada 3D mikroskopi ve analizler (3D immuno-DNA FISH) izledi DNA FISH immünofloresan ve DNA dizileri tarafından histon modifikasyonları eşzamanlı tespiti için bir protokol açıklar.

3D Nükleer teşkilatında değişiklikler Eğitim için 3D-korunmuş Tane Çekirdekle ilgili Kombine İmmünofloresan ve DNA FISH

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

Kertenkeleler Hareket Hız ve Yön Visual Hassasiyet Test

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 tabanlı Balık Türleri Tanımlama Protokolü

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

Doğrudan Etiketli Problar kullanarak Sağlam 3D DNA BALIK

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

Bıldırcın-ördek Chimeras kullanma Kraniyofasiyal Kalkınma için Türlere özgü Katılımlar değerlendirilmesi

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

Kombine DNA-RNA Floresan<em&gt; In situ</em&gt; Hibridizasyon (FISH) Farklılaştırılmış Kadın Fare Embriyonik Kök Hücreleri X kromozomu inaktivasyonu Eğitim için

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

Kronik sonra fare dalak DNA hasar ve Onarım Ölçme<em&gt; In Vivo</em&gt; Beta ve gamma-Radyasyonun Çok Düşük Doz Maruz Kalma

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

Deneysel Yaklaşımlar Mitokondriyal Yerelleşme ve Nükleer Hücre Döngüsü Kinaz, Cdk1 Fonksiyonu Eğitim için

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

Belirli Genomik Bölgeler ve enChIP tarafından İlişkili Moleküllerin Tanımlanması İzolasyonu

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

Yöntemleri ile Caenorhabditis elegans çağda doğal Protein toplama çalışma değişikliklere

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

Cryosec, zebrafish embriyo üzerinde sıralı Immünofluorescence ve Immünhistokimya

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