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

Nos gustaría agradecer a los miembros del laboratorio Skok, especialmente Susannah Hewitt, para los debates y comentarios. Este trabajo es apoyado por el Instituto Nacional de Salud subvenciones R01GM086852, RC1CA145746 (JAS). JAS es un erudito Leukemia &amp; Lymphoma Society. JC es un compañero Irvington Instituto de Investigación del Cáncer Institute. MM es apoyado por una Educación Nacional Science Foundation Graduate Beca Integral y Prácticas de Investigación (NSF IGERT 0.333.389).

1. Etiquetado de ADN Probe con fluoróforos: Nick Translation (~ 6 horas) <ol><li> Clean BAC ADN (preparado por maxi-prep) o plásmidos o productos de PCR, todos resuspendió en H 2 O, se puede utilizar para el etiquetado. Tenga en cuenta que para una señal de FISH robusto, sondas deberían abarcar al menos 10 kb. </li><li> Incubar ADN en RNasa A durante 30 min a 37 ° C (Todos los reactivos se enumeran en la Tabla 1). </li><li> Incubar la reacción de traducción nick dur...

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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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Las técnicas detalladas anteriormente se han utilizado en nuestro laboratorio para analizar la regulación de V (D) J recombinación de la inmunoglobulina y loci TCRA / d en el desarrollo de los linfocitos 30,31. Estamos seguros de que esta técnica se puede adaptar para la detección de varias proteínas nucleares, compartimentos nucleares y los loci, en diferentes tipos de células. Las modificaciones del protocolo puede ser necesaria, y en este caso los pasos principales de enfocar son l...

Epigenética establecimiento de mecanismos de disparo y la herencia de desarrollo y el tipo de células perfiles de transcripción específicos. Por un lado se trata de la modulación de la cromatina embalaje que define regiones genómicas activas o en silencio. En una escala mayor, la organización global de 3D del genoma nuclear y de la arquitectura también desempeñar un papel en el control de los patrones transcripcionales. Por lo tanto, la disección de estas características epigenomic es esencial para una comprensión completa de cómo se regulan los genes 6-11. FISH Combinado de ADN de inmunofluorescencia y 3D ofrecen una oportunidad única para complementar los análisis moleculares y bioquímicos mediante la evaluación de las interacciones específicas / asociaciones de secuencias de ADN y / o proteínas en el núcleo. Por otra parte, mientras que en todo el genoma técnicas de alto rendimiento, tales como cromatina immunoprecipitation (CHIP-seq) o conformación cromosoma captura junto con la secuenciación profunda (4C-seq, 5C, Hi-C) ofrecen dat mundialuna en 12 poblaciones celulares, inmunofluorescencia / ADN técnicas de FISH permitir el análisis a nivel de células individuales. Aquí se describe un protocolo para la detección simultánea de modificaciones de las histonas por secuencias de ADN de inmunofluorescencia y por FISH ADN seguido por microscopía y análisis 3D (3D inmuno-FISH). La ventaja de este protocolo es la visualización combinada de ADN y la preservación de las estructuras proteicas. Nuestra experiencia en este campo nos ha permitido mejorar y simplificar los protocolos existentes. Aunque hemos utilizado este protocolo para detectar ADN de doble cadena se rompe en los linfocitos sometidos a recombinación, este método se puede aplicar a otras proteínas y otros tipos de células.

<table border="1"><tbody><tr><td> Nombre del reactivo / material </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> H 2 O </td><td> Pescador </td><td> # BP2470 </td><td></td></tr><tr><td> RNasa 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 o Cy5 dUTP </td><td> Pescador </td><td> º 45-001-xxx </td><td></td></tr><tr><td> DNasa I </td><td> Roche </td><td> # 04536282001 </td><td></td></tr><tr><td> ADN Pol I </td><td> Biolabs </td><td> # M0209 </td><td></td></tr><tr><td> 0,025 micras 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 1 mg / ml </td><td> Genética Aplicada </td><td> # MHB </td><td></td></tr><tr><td> De esperma de salmón </td><td> Sigma </td><td> # D1626 </td><td> polvo que se volvieron a suspender a 10 mg / ml en H 2 O </td></tr><tr><td> NaAc (acetato de sodio, pH 5,2, solución tampón) </td><td> Sigma </td><td> # S7899 </td><td></td></tr><tr><td> Ficoll 400 (Mol Biol. grado) </td><td> Pescador </td><td> # 525 </td><td></td></tr><tr><td> Polivinilpirrolidona (Mol Biol. grado) </td><td> Pescador </td><td> # BP431 </td><td></td></tr><tr><td> Polvo de sulfato de dextrano </td><td> Sigma </td><td> # D8906 </td><td></td></tr><tr><td> SSPE (solución salina fosfato de sodio-EDTA) 20x solución </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> Cubreobjetos </td><td> Pescador </td><td> # 12-548-B </td><td></td></tr><tr><td> Diapositivas </td><td> Pescador </td><td> # 12-550 </td><td></td></tr><tr><td> 6-y las placas </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 </td><td> Sigma </td><td> # P8920 </td><td></td></tr><tr><td> Paraformaldehído, gránulos, 95% </td><td> Sigma </td><td> # 441244 </td><td></td></tr><tr><td> Triton-X-100, Mol Biol. grado </td><td> Sigma </td><td> # T8787 </td><td></td></tr><tr><td> BSA (albúmina de suero bovino) fracción V </td><td> Pescador </td><td> # BP 1600 </td><td></td></tr><tr><td> Suero normal de cabra </td><td> Vector Labs </td><td> # S-1000 </td><td></td></tr><tr><td> Tween-20, Mol Biol. grado </td><td> Sigma </td><td> # P9416 </td><td></td></tr><tr><td> SSC (citrato sódico salino) 20x solución </td><td> Pescador </td><td> # BP1325 </td><td></td></tr><tr><td> Prolongar reactivo Antifade Oro </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> Mejor prueba de una capa de cemento de goma </td><td> Arte o tiendas de suministros de oficina </td><td></td><td></td></tr><tr><td></td><td></td><td></td><td> Tabla 1. Reactivos específicos y pequeños equipos. </td></tr></tbody></table>

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

La hibridación fluorescente in situ con sondas de ADN sobre el 3-dimensional núcleos conservan seguido por microscopía confocal 3D (3D FISH AND) representa la forma más directa para visualizar la localización de loci de genes, cromosomas sub-regiones o territorios enteros en celdas individuales. Este tipo de análisis permite conocer la arquitectura global del núcleo, así como el comportamiento de los loci genómicos y regiones específicas dentro del espacio nuclear. Inmunofluorescencia, por otro lado, permite la detección de proteínas nucleares (histonas modificadas, variantes de histonas y modificadores, maquinaria de transcripción y factores nucleares, sub-compartimientos, etc.) El principal reto en la combinación de ADN FISH inmunofluorescencia y 3D es, por un lado, para preservar el epítopo detectados por el anticuerpo, así como la arquitectura 3D del núcleo, y por otra parte, para permitir la penetración de la sonda de ADN para detectar loci de genes o cromosomas territorios 1-5. A continuación presentamos un protocolo que combina la visualización de las modificaciones de la cromatina con loci genómicos en los núcleos 3D conserva.

ADN y la inmuno-FISH se utilizan en el laboratorio Skok para estudiar los cambios en la organización nuclear asociados con el proceso de V (D) J recombinación de loci receptores de antígenos durante el desarrollo de linfocitos B y T. Las técnicas detalladas anteriormente nos permiten i) medir distancias entre los dos extremos de un locus (contracción) ii) medir distancias entre los alelos o loci (emparejamiento), iii) analizar el daño del ADN que ocurre dentro de loci, iv) evaluar la ubicación de los alelos y en ...

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

Aquí se describe un protocolo para la detección simultánea de las modificaciones de histonas por secuencias de ADN de inmunofluorescencia y por FISH de ADN seguido por microscopía 3D y análisis (3D inmuno-ADN FISH).

Inmunofluorescencia y FISH Combinado de ADN en 3D conservados núcleos en interfase para estudiar los cambios en la organización 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

Prueba de sensibilidad visual a la velocidad y dirección del movimiento en lagartos

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

Investigación

Biología

DNA-based Fish Species Identification Protocol

Basada en el ADN de especies de peces Protocolo de identificación

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

Robusto PESCADO ADN 3D utilizando sondas marcadas directamente

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

Evaluar Aportes específicos de la especie para el desarrollo craneofacial Utilizando quimeras Quail-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

ADN-ARN combinado fluorescente<em&gt; In situ</em&gt; La hibridación (FISH) para el Estudio de inactivación del cromosoma X en las células madre embrionarias de ratón Diferenciada Mujer

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

Medición de daños y reparación del ADN en esplenocitos de ratón Después crónica<em&gt; En Vivo</em&gt; La exposición a dosis muy bajas de beta y gamma Radiación

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

Los enfoques experimentales para estudiar mitocondrial localización y función de un ciclo celular quinasa nuclear, 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

El aislamiento de regiones genómicas específicas e identificación de moléculas asociadas por 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 estudio cambios de agregación inherente de proteína con la edad en 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

Inmunofluorescencia secuencial e inmunohistoquímica en embriones de pez cebra crioseccionados

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