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

Gostaríamos de agradecer aos membros do laboratório Skok, especialmente Susannah Hewitt, para discussões e comentários. Este trabalho é apoiado pelo Instituto Nacional de bolsas de Saúde R01GM086852, RC1CA145746 (JAS). JAS é um Leukemia &amp; Lymphoma Society estudioso. JC é um Irvington Fellow Instituto do Cancer Research Institute. MM é apoiado por um National Science Foundation Grant Integrativa Pós-Graduação Pesquisa e Estágio (NSF IGERT 0.333.389).

1. DNA Labeling Sonda com fluoróforos: Nick Tradução (~ 6 horas) <ol><li> Limpeza BAC DNA (preparado por maxi-prep) ou plasmídeos ou produtos de PCR, todos ressuspenso em H 2 O, podem ser utilizados para a rotulagem. Note-se que para um sinal de FISH robusto, as sondas devem abranger pelo menos 10 kb. </li><li> Incubar DNA em RNase A durante 30 min a 37 ° C (todos os reagentes estão listados na Tabela 1). </li><li> Incubar a reacção de tradução de nick durante 2 h...

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	<li>Chaumeil, J., Okamoto, I., Heard, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=X-chromosome+inactivation+in+mouse+embryonic+stem+cells:+analysis+of+histone+modifications+and+transcriptional+activity+using+immunofluorescence+and+FISH.">X-chromosome inactivation in mouse embryonic stem cells: analysis of histone modifications and transcriptional activity using immunofluorescence and FISH.</a> Methods in enzymology. , 376-405 (2004).</li><li>Cremer, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicolor+3D+fluorescence+in+situ+hybridization+for+imaging+interphase+chromosomes.">Multicolor 3D fluorescence in situ hybridization for imaging interphase chromosomes.</a> Methods Mol. Biol. 463, 205-239 (2008).</li><li>Chaumeil, J., Augui, S., Chow, J. C., Heard, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+immunofluorescence,+RNA+fluorescent+in+situ+hybridization,+and+DNA+fluorescent+in+situ+hybridization+to+study+chromatin+changes,+transcriptional+activity,+nuclear+organization,+and+X-chromosome+inactivation.">Combined immunofluorescence, RNA fluorescent in situ hybridization, and DNA fluorescent in situ hybridization to study chromatin changes, transcriptional activity, nuclear organization, and X-chromosome inactivation.</a> Methods Mol. Biol. 463, 297-308 (2008).</li><li>Solovei, I., Cremer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D-FISH+on+cultured+cells+combined+with+immunostaining.">3D-FISH on cultured cells combined with immunostaining.</a> Methods Mol. Biol. 659, 117-126 (2010).</li><li>Markaki, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+of+3D-FISH+and+super-resolution+structured+illumination+microscopy+for+studies+of+3D+nuclear+architecture:+3D+structured+illumination+microscopy+of+defined+chromosomal+structures+visualized+by+3D+(immuno)-FISH+opens+new+perspectives+for+studies+of+nuclear+architecture.">The potential of 3D-FISH and super-resolution structured illumination microscopy for studies of 3D nuclear architecture: 3D structured illumination microscopy of defined chromosomal structures visualized by 3D (immuno)-FISH opens new perspectives for studies of nuclear architecture.</a> BioEssays : news and reviews in molecular, cellular and developmental biology. 34, 412-426 (2012).</li><li>Heard, E., Bickmore, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ins+and+outs+of+gene+regulation+and+chromosome+territory+organisation.">The ins and outs of gene regulation and chromosome territory organisation.</a> Current opinion in cell biology. 19, 311-316 (2007).</li><li>Misteli, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+the+sequence:+cellular+organization+of+genome+function.">Beyond the sequence: cellular organization of genome function.</a> Cell. 128, 787-800 (1016).</li><li>Fraser, P., Bickmore, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+organization+of+the+genome+and+the+potential+for+gene+regulation.">Nuclear organization of the genome and the potential for gene regulation.</a> Nature. 447, 413-417 (2007).</li><li>Cremer, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+territories--a+functional+nuclear+landscape.">Chromosome territories--a functional nuclear landscape.</a> Current opinion in cell biology. 18, 307-316 (2006).</li><li>Mao, Y. 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As técnicas descritas acima foram usadas no nosso laboratório para analisar a regulação da recombinação V (D) J de imunoglobulina e Tcra / d loci no desenvolvimento de linfócitos 30,31. Estamos confiantes de que esta técnica pode ser adaptada para a detecção de várias proteínas nucleares, compartimentos nucleares e locos, em diferentes tipos de células. Modificações do protocolo pode ser necessário, e, neste caso, os passos principais para se concentrar são as seguintes. Em ...

Estabelecimento de mecanismos epigenéticos gatilho e herança de desenvolvimento e de células do tipo perfis específicos de transcrição. Em um nível, este envolve a modulação da embalagem da cromatina, que define as regiões genômicas ativas ou em silêncio. Numa escala maior, a organização 3D global do genoma e arquitectura nuclear também desempenhar um papel no controlo da transcrição de padrões. Assim, o esvaziamento desses recursos epigenômico é essencial para um entendimento completo de como os genes são regulados 6-11. FISH DNA combinado imunofluorescência e 3D fornecem uma oportunidade única para complementar análises moleculares e bioquímicos, avaliando interacções específicas / associações de sequências de ADN e / ou proteínas dentro do núcleo. Além disso, enquanto do genoma técnicas de alto rendimento, como cromatina imunoprecipitação (CHIP-seq) ou conformação cromossomo captura juntamente com o seqüenciamento de profundidade (4C-seq, 5C, Hi-C) fornecer dat mundialuma população de células em 12, técnicas de imunofluorescência / ADN FISH permitir análises ao nível da célula individual. Aqui nós descrevemos um protocolo para a detecção simultânea de modificações de histonas por seqüências de DNA por imunofluorescência e FISH DNA seguido por microscopia 3D e análises (3D imuno-FISH). A vantagem deste protocolo é a visualização conjunta de DNA e preservação de estruturas de proteínas. A nossa experiência neste campo nos permitiu melhorar e simplificar os protocolos existentes. Apesar de termos utilizados neste protocolo para a detecção de ADN de cadeia dupla em intervalos linfócitos submetidos a recombinação, este método pode ser aplicado a outras proteínas e de outros tipos de células.

<table border="1"><tbody><tr><td> Nome do reagente / Material </td><td> Companhia </td><td> Número de Catálogo </td><td> Comentários </td></tr><tr><td> H 2 O </td><td> Pescador </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 a C-11401 </td><td></td></tr><tr><td> Cy3 ou Cy5 dUTP </td><td> Pescador </td><td> N º 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 mM filtros </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 ADN de 1 mg / ml </td><td> Genética Aplicada </td><td> # MHB </td><td></td></tr><tr><td> Esperma de salmão </td><td> Sigma </td><td> # D1626 </td><td> pó a serem ressuspensas a 10 mg / ml em H2O </td></tr><tr><td> NaAc (Acetato de sódio, pH 5,2, solução tampão) </td><td> Sigma </td><td> # S7899 </td><td></td></tr><tr><td> Ficoll 400 (Mol. Biol grau) </td><td> Pescador </td><td> # 525 </td><td></td></tr><tr><td> Polivinilpirrolidona (Mol Biol grau) </td><td> Pescador </td><td> # BP431 </td><td></td></tr><tr><td> Dextran pó de sulfato </td><td> Sigma </td><td> # D8906 </td><td></td></tr><tr><td> SSPE (Solução salina-fosfato de sódio-EDTA) solução 20x </td&gt; <td> Pescador </td><td> # BP1328 </td><td></td></tr><tr><td> Formamida </td><td> Pescador </td><td> # BP227 </td><td></td></tr><tr><td> Lamelas </td><td> Pescador </td><td> º 12-548-B </td><td></td></tr><tr><td> Slides </td><td> Pescador </td><td> # 12-550 </td><td></td></tr><tr><td> Placas de 6 poços </td><td> Pescador </td><td> # 0720080 </td><td></td></tr><tr><td> PBS, 10x </td><td> Pescador </td><td> # MT-46-013-CM </td><td></td></tr><tr><td> Poli-L-lisina solução </td><td> Sigma </td><td> # P8920 </td><td></td></tr><tr><td> Paraformaldeído, grânulos, 95% </td><td> Sigma </td><td> # 441244 </td><td></td></tr><tr><td> Triton-X-100, Mol Biol grau </td><td> Sigma </td><td> # T8787 </td><td></td></tr><tr><td> BSA (Albumina de Soro Bovino) Fracção V </td><td> Pescador </td><td> # BP 1600 </td><td></td></tr><tr><td> Soro normal de cabra </td><td> Vector Labs </td><td> # S-1000 </td><td></td></tr><tr><td> Tween-20, Mol Biol grau </td><td> Sigma </td><td> # P9416 </td><td></td></tr><tr><td> SSC (citrato de sódio Saline) solução 20x </td><td> Pescador </td><td> # BP1325 </td><td></td></tr><tr><td> Prolongar reagente Antifade Ouro </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> Melhor teste de um cimento de borracha casaco </td><td> Abastecimento de lojas de arte ou de escritório </td><td></td><td></td></tr><tr><td></td><td></td><td></td><td> Tabela 1. Reagentes específicos e pequenos equipamentos. </td></tr></tbody></table>

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

Hibridização fluorescente in situ utilizando sondas de DNA em 3-dimensionalmente núcleos preservados seguido por microscopia confocal 3D (3D FISH DNA) representa a forma mais direta de visualizar a localização de locos gênicos, cromossômica sub-regiões ou territórios inteiros em células individuais. Este tipo de análise fornece insights sobre a arquitetura global do núcleo, bem como o comportamento de loci genômicos e regiões específicas dentro do espaço nuclear. Imunofluorescência, por outro lado, permite a detecção de proteínas nucleares (histonas modificados, variantes de histonas e modificadores, maquinaria de transcrição e factores nucleares, sub-compartimentos, etc.) O principal desafio na combinação FISH DNA imunofluorescência e 3D é, por um lado, para preservar o epitopo detectada pelo anticorpo, bem como a arquitectura 3D do núcleo, e, por outro lado, para permitir a penetração da sonda de ADN para detectar locos gênicos ou territórios cromossômicos 1-5. Aqui nós fornecemos um protocolo que combina visualização de modificações da cromatina com loci genômicos em núcleos preservados 3D.

DNA e imuno-FISH são utilizados no laboratório Skok para estudar as alterações na organização nuclear associadas com o processo de recombinação V (D) J de receptor de antigénio, durante loci B e T o desenvolvimento de linfócitos. As técnicas acima descritas nos permitir i) medir distâncias entre as duas extremidades de um locus (contração) ii) medir distâncias entre alelos ou loci (emparelhamento), iii) analisar os danos no DNA que ocorrem dentro loci, iv) avaliar a localização de alelos e locos em rela...

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

Aqui nós descrevemos um protocolo para a detecção simultânea de modificações de histonas por imunofluorescência e seqüências de DNA por FISH DNA seguido por microscopia 3D e análises (3D FISH imuno-DNA).

Imunofluorescência combinados e FISH DNA em 3D preservado núcleos interfásicos para estudar mudanças na organização nuclear 3D

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

Teste de sensibilidade visual para a velocidade e direção do movimento em Lagartos

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

Biologia

DNA-based Fish Species Identification Protocol

Baseados em DNA Peixe Protocolo Identificação de Espécies

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 FISH DNA 3D utilizando sondas Diretamente rotuladas

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

Avaliando Contribuições Espécies específicas Para o Desenvolvimento Craniofacial Utilizando Quimeras Codorniz-pato

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

Hibridização in situ fluorescente (FISH) combinada de DNA-RNA para estudar a inativação do cromossomo X em células-tronco embrionárias diferenciadas de camundongos fêmeas

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

Medindo danos DNA e reparar no mouse Esplenócitos Depois crônica<em&gt; In Vivo</em&gt; A exposição a doses muito baixas de beta e gama-radiação

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

Abordagens experimentais para estudar mitocondrial Localização e função de um ciclo celular Nuclear 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

O isolamento de regiões específicas genômico e identificação de moléculas pela Associated 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

Métodos de estudo mudanças na agregação de proteínas inerente com a idade em 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

Imunofluorescência sequencial e imuno-histoquímica em embriões de zebrafish Crioseccionados

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