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 durante 2 horas a 16 ° C (véanse los cuadros 2 y 3). Los métodos alternativos de etiquetado directo puede ser utilizado (Ejemplo: Etiqueta FISH, Invitrogen). </li><li> Inactivar la reacción durante una hora a -80 ° C o durante la noche a -20 ° C. </li><li> Determinar el tamaño de la sonda en un 2% de gel de agarosa. La prueba debe ser de entre 100 y 1.000 pares de bases, con la mayoría en aproximadamente 300-500 pb (Tenga en cuenta que la mancha de Cy5 sondas no es visible). Si el ADN no se digiere suficiente, más ADN Pol I yDNasa I se puede añadir a la reacción y se incubaron durante dos horas más a 16 ° C. </li><li> Purificar sondas en filtros 0,025 mm en dos vasos de precipitados litro llenos de H 2 0 durante dos horas en la oscuridad. </li><li> Añadir factores de bloqueo para las sondas de la siguiente manera: 10 g de ADN Cot-1, 10 g de ADN Hybloc y 100 g de esperma de salmón de 10 g de sondas de ADN. Sondas de ADN Tienda a -20 ° C. </li></ol> 2. Probe Precipitación / desnaturalización / hibridación Pre-(hr 3-4) <ol><li> Entre 0,3 mg y 1 g de sonda de ADN se utiliza por cubreobjetos. Las sondas se mezclan con 0,1 volumen de NaAc 3M (pH 5,2) y 2,5 volúmenes de etanol al 100%, incubar durante una hora a -80 ° C o durante la noche a -20 ° C y centrifugar a 13.000 rpm a 4 ° C durante 30 min. Tenga en cuenta que la cantidad de sonda de ADN puede ser ajustada dependiendo de la calidad del ADN y la región nuclear de la hibridación. Los métodos alternativos de etiquetado directo puede permitir el uso de cantidades reducidas desonda. Para la detección de múltiples señales de ADN de FISH en una diapositiva, las diferentes sondas pueden precipitarse juntos (excepto sondas que no deberían estar pre-recocido, como secuencias de repetición). Las sondas para varios experimentos realizados al mismo tiempo también se puede precipitar juntos. Pellets debe ser ligeramente coloreado de acuerdo con los fluoróforos utilizados (en caso de pequeñas cantidades de sondas se precipitan, el precipitado puede no ser visible). </li><li> Pellets secos y resuspender ellos en 15 l de tampón de hibridación (véase la Tabla 4) por cubreobjetos con agitación (usando un termomezclador Eppendorf) en la oscuridad a 37-45 ° C (aproximadamente 20 min). </li><li> Sondas desnaturalizan durante 5 minutos a 75-95 ° C. </li><li> Pre-recocido de sondas a 37 º C durante 30-60 min. Longitud de pre-recocido puede ser ajustada dependiendo de la complejidad y la calidad de las sondas. </li><li> Aplicar las sondas a los cubreobjetos para la hibridación durante la noche (véase la Sección 3). </li></ol> 3. 3Tridimensional del ADN FISH - Primer día (~ 3 h) <ol><li> Las células no adherentes se concentran en un pequeño volumen de PBS 1x (~ 2x10 5 células en 5-10 l de PBS 1x por cubreobjeto) y se dejó caer sobre el centro de un cubreobjetos recubierto con poli-L-lisina. Las células adherentes se cultivaron en cubreobjetos durante al menos 24 horas (confluencia ~ 70%) (cubreobjetos se pueden recubrir con gelatina 0,1%). Cuadrados 18x18 a 24-24 mm cubreobjetos se colocan en placas de 6 pocillos, y durante todo el protocolo, dos ml de cada solución se utiliza por pocillo (alternativamente redondas 1,5 mm cubreobjetos se pueden colocar en placas 12/24-well). </li><li> Fijar las células en 2% de paraformaldehído / PBS 1x, pH 7-7.4, durante 10 min a TA (4% PFA existencias puede alícuotas, se almacenó a -20 ° C). El paraformaldehído se pueden comprar en solución (16%), o 4% soluciones madre se puede preparar a partir de polvo / gránulos. Paraformaldehído en polvo se disuelve en 1x PBS a&gt; 60 ° C (pH puede aumentarse a 8.9 para facilitar la disolución), enfriado en hielo, ajustared a pH 7-7.4, se filtró, alícuotas de halcones y almacenados durante semanas a -20 ° C. </li><li> Después de tres enjuagues en PBS 1x, permeabilizar las células en hielo enfriado con 0,4% Triton-X-100/1 x PBS durante 5 min en hielo. Longitud de permeabilización puede ajustarse dependiendo de la célula de tipo NB:. Si FISH de ADN se combina con la inmunofluorescencia, la inmunotinción se debe realizar en esta etapa (véase la Sección 4). </li><li> Después de tres enjuagues en 1x PBS, las células se incuban con 0,1 g / l de RNasa A en 1x PBS durante una hora a 37 ° C. Los cubreobjetos se colocan células hacia abajo sobre una gota de 100 l de solución en parafilm o un portaobjetos en una cámara húmeda. Los cubreobjetos son entonces cuidadosamente con fórceps. Si alguno se encuentra resistencia, los cubreobjetos se inundó con 1x PBS con el fin de evitar daños en las células. </li><li> Después de tres enjuagues en PBS 1x, permeabilizar las células en hielo enfriado con 0,7% Triton-X-100 HCl / 0,1 M durante 10 min en hielo. </li><li> Después de tres lavados in 1x PBS, las células desnaturalizan en 1,9 M HCl durante 30 min a TA. Alternativamente, las células pueden ser desnaturalizados en formamida / SSC 2x 50% a 75-80 ° C durante al menos 30 min. Cuenta que la longitud de hibridación (y la temperatura) puede ser optimizada en función del tipo de célula. </li><li> Después de tres enjuagues en hielo frío 1x PBS, las células se hibridan con sondas durante la noche a 37 ° C en una cámara oscura y húmeda. Los cubreobjetos se colocan celda hacia abajo en una caída de 15 l de la sonda en un portaobjetos, y se sella con cemento de goma. </li></ol> 4. 3-dimensional Immuno-FISH - First Day (~ 6-7 h) <ol><li> Para combinar inmunofluorescencia / ADN FISH, inmuno-tinción debe realizarse después de la permeabilización en el 0,4% de Triton-X-100 (Paso 3,3). </li><li> Se incuban las células en solución de bloqueo durante 30 min a TA (2,5% albúmina de suero bovino (BSA), 10% de suero de cabra normal, 0,1% de Tween-20). La solución de bloqueo se filtra, se dividió en alícuotas en Eppendorfs 1,5 ml y se almacenó para meses a -20 ° C. </li><li> Se incuban las células con el anticuerpo primario contra la proteína o la modificación de las histonas de interés diluido en solución de bloqueo, durante una hora a temperatura ambiente en una cámara húmeda. Aquí se muestra el ejemplo de un anticuerpo contra fosforilados serina-139 de H2AX (γ-H2AX, Millipore, Ver Tabla 5 para más detalles). Los cubreobjetos se colocan células hacia abajo sobre una gota de 100 l de solución en parafilm o un portaobjetos en una cámara húmeda. Los cubreobjetos son entonces cuidadosamente con fórceps (inundado con 1x PBS si es necesario). Nota que la dilución y la duración de la incubación puede ser ajustado dependiendo de la calidad de los anticuerpos y de células tipos. </li><li> Lavar las células en 0,2% BSA / 0,1% Tween-20 / 1x PBS, tres veces 5 min a TA con agitación. </li><li> Se incuban las células con el apropiado fluoróforo conjugado con anticuerpo secundario diluido en solución de bloqueo durante una hora a RT en una cámara oscura y húmeda. Aquí se muestra un ejemplo de detección con unsecundario de cabra anti-ratón de anticuerpos (Alexa Fluor 488 o 555, Invitrogen; Ver Tabla 5 para más detalles). Hapteno conjugado con anticuerpos secundarios (biotina, digoxigenina, etc) también podría ser utilizado. En este caso, un paso adicional para la detección apropiado (estreptavidina, anti-dig, etc) se puede realizar ya sea antes o después de ADN-FISH hibridación durante la noche. </li><li> Enjuagar las células en 0,1% de Tween-20 / 1x PBS, tres veces 5 min a TA con agitación en la oscuridad. </li><li> Post-fix células en 2% de paraformaldehído / PBS 1x durante 10 min a TA en la oscuridad. </li><li> Continuar FISH ADN como anteriormente a partir de la incubación en RNasa A (Paso 3,4). </li></ol> 5. 3-dimensional de ADN FISH / Immuno-FISH - Segundo día (~ 2 h) <ol><li> Retire con cuidado el cemento de goma, cubreobjetos de inundación con 2x SSC, retire con cuidado con unas pinzas y colóquelos en los pocillos con solución de lavado. </li><li> Lavar las células en 2x SSC durante 30 minutos a 37 ° C en la oscuridad con agitación.</li><li> Lavar las células en 2x SSC durante 30 min a TA en la oscuridad con agitación. </li><li> Lavar las células en 1x SSC durante 30 min a TA en la oscuridad con agitación Tenga en cuenta que en caso de desnaturalización con formamida, lavados debe ser sustituido por el siguiente:. 3 lavados en lavados de 50% de formamida / SSC 2x y 3 en 2x SSC, 5 minutos cada uno, a 37 º C. </li><li> Montar las células en ProLong oro medio de montaje que contiene 1,5 mg / ml DAPI (4,6-diamidino-2-fenilindol) para la contratinción de ADN total. Los cubreobjetos se colocan células hacia abajo sobre una gota de 10-15 l de medio de montaje en un portaobjetos y se sellaron con esmalte de uñas. Las diapositivas se mantuvo durante meses a -20 ° C. </li></ol> 6. Microscopia y Análisis de 3D <ol><li> Imágenes 3D pueden ser adquiridos con los sistemas de microscopios. En nuestros experimentos, las secciones ópticas separadas por 0,3 micras se recogen en un sistema Leica SP5 AOBS confocal (acústico-óptico divisor de haz). Tenga en cuenta que la separación entre Optical planos se pueden ajustar dependiendo del tipo de análisis. </li><li> Pilas de imágenes 3D se pueden analizar usando software diferente y herramientas. Usamos el software Image J para evaluar las mediciones de distancia entre loci de interés y analizar diferentes tipos de asociación de los lugares de interés con los sub-compartimientos nucleares o proteínas. </li><li> Todos los análisis estadísticos se realizaron para comparar dos (o más) diferentes condiciones biológicas con pairwise evaluación (s) de significación: Comparación entre diferentes tipos de células o fases, la comparación entre los diferentes loci de interés. En todas las pruebas estadísticas, los valores de p ≤ 5.00e-2 (a = 0,05) se considera que son significativos (1,00 E-2 ≤ P ≤ 5.00e-2 * significativo; 1,00 E-3 ≤ P ≤ 1,00 E-2 * * muy significativa, p &lt;1,00 E-3 *** altamente significativo). </li></ol> El análisis estadístico de las distribuciones de enteros de las distancias entre los dos alelos. Hemos primera construct acumulativos curvas de distribución de frecuencias en toda la gama de distancias medidas entre el alleles.To NAEP la significación de las diferencias en las distribuciones empíricas de inter-alélicas distancias que usamos la prueba no paramétrica de dos muestras de Kolmogorov-Smirnov (KS) prueba de 13,14. El análisis estadístico de una estrecha asociación (es decir, el emparejamiento) de dos alelos con una distancia óptima de corte. Identificar el rango de la mayoría de las diferencias robustas en las distancias entre dos alelos o loci, utilizamos una serie de 15 dos colas prueba exacta de Fisher s. Es decir, en cada distancia medida se prueba si una condición es significativamente más representadas en distancias más cortas entre las dos muestras. El mínimo en la distribución de los valores P indica la gama de distancias que deben ser considerados como un límite para cerrar asociación (es decir, emparejamiento) de los dos alelos. Posteriormente, la prueba de dos colas exacta de Fisher se utiliza to evaluar la importancia de emparejamiento de dos alelos en el identificada valor de corte 15. Correcciones de prueba múltiples se aplica a la cuenta para el número total de pruebas de Fisher realizó 16. El análisis estadístico de la asociación del locus de interés con sub-compartimentos nucleares o proteínas. La prueba de dos colas de Fisher-exacta se utiliza para analizar la significación de la asociación del locus de interés con diferentes compartimentos o proteínas 15.

<ol>
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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multifunctional+element+in+the+mouse+Igkappa+locus+that+specifies+repertoire+and+Ig+loci+subnuclear+location.">A multifunctional element in the mouse Igkappa locus that specifies repertoire and Ig loci subnuclear location.</a> Journal of Immunology. 186, 5356-5366 (2011).</li><li>Hewitt, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RAG-1+and+ATM+coordinate+monoallelic+recombination+and+nuclear+positioning+of+immunoglobulin+loci.">RAG-1 and ATM coordinate monoallelic recombination and nuclear positioning of immunoglobulin loci.</a> Nature immunology. 10, 655-664 (2009).</li><li>Deriano, L., 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=The+RAG2+C+terminus+suppresses+genomic+instability+and+lymphomagenesis.">The RAG2 C terminus suppresses genomic instability and lymphomagenesis.</a> Nature. 471, 119-123 (2011).</li><li>Brown, K. E., Baxter, J., Graf, D., Merkenschlager, M., Fisher, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+repositioning+of+genes+in+the+nucleus+of+lymphocytes+preparing+for+cell+division.">Dynamic repositioning of genes in the nucleus of lymphocytes preparing for cell division.</a> Molecular cell. 3, 207-217 (1999).</li><li>Fernandez-Capetillo, O., Lee, A., Nussenzweig, M., Nussenzweig, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=H2AX:+the+histone+guardian+of+the+genome.">H2AX: the histone guardian of the genome.</a> DNA repair. 3, 959-967 (2004).</li><li>Croft, J. A., 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=Differences+in+the+localization+and+morphology+of+chromosomes+in+the+human+nucleus.">Differences in the localization and morphology of chromosomes in the human nucleus.</a> The Journal of cell biology. 145, 1119-1131 (1999).</li><li>Chaumeil, J., Le Baccon, P., Wutz, A., 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=A+novel+role+for+Xist+RNA+in+the+formation+of+a+repressive+nuclear+compartment+into+which+genes+are+recruited+when+silenced.">A novel role for Xist RNA in the formation of a repressive nuclear compartment into which genes are recruited when silenced.</a> Genes Dev. 20, 2223-2237 (2006).</li><li>Walter, J., 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=Towards+many+colors+in+FISH+on+3D-preserved+interphase+nuclei.">Towards many colors in FISH on 3D-preserved interphase nuclei.</a> Cytogenetic and genome research. 114, 367-378 (2006).</li><li>Toomre, D., Bewersdorf, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+wave+of+cellular+imaging.">A new wave of cellular imaging.</a> Annual review of cell and developmental biology. 26, 285-314 (2010).</li><li>Schermelleh, L., Heintzmann, R., Leonhardt, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+super-resolution+fluorescence+microscopy.">A guide to super-resolution fluorescence microscopy.</a> The Journal of cell biology. 190, 165-175 (2010).</li><li>Dobbie, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OMX:+a+new+platform+for+multimodal,+multichannel+wide-field+imaging.">OMX: a new platform for multimodal, multichannel wide-field imaging.</a> Cold Spring Harbor protocols. , 899-909 (2011).</li><li>Boyle, S., Rodesch, M. J., Halvensleben, H. A., Jeddeloh, J. A., Bickmore, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+in+situ+hybridization+with+high-complexity+repeat-free+oligonucleotide+probes+generated+by+massively+parallel+synthesis.">Fluorescence in situ hybridization with high-complexity repeat-free oligonucleotide probes generated by massively parallel synthesis.</a> Chromosome research : an international journal on the molecular, supramolecular and evolutionary aspects of chromosome biology. 19, 901-909 (2011).</li></ol>

Los autores declaran que no tienen intereses en conflicto financieros.

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 ...

combined-immunofluorescence-dna-fish-on-3d-preserved-interphase

Research

JoVE Journal

Biology

18.2K vistas.  New York University School of Medicine. 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).

Video: Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization

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 the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

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

We have developed a fast, simple, and accurate DNA-based screening method to identify the fish species present in fresh and processed seafood samples. This versatile method employs PCR amplification of genomic DNA extracted from fish samples, followed by restriction fragment length polymorphism (RFLP) analysis to generate fragment patterns that can be resolved on the Agilent 2100 Bioanalyzer and matched to the correct species using RFLP pattern matching software.
The fish identification method uses a simple, reliable, spin column- based protocol to isolate DNA from fish samples. The samples are treated with proteinase K to release the nucleic acids into solution. DNA is then isolated by suspending the sample in binding buffer and loading onto a micro- spin cup containing a silica- based fiber matrix. The nucleic acids in the sample bind to the fiber matrix. The immobilized nucleic acids are washed to remove contaminants, and total DNA is recovered in a final volume of 100 &mu;l. The isolated DNA is ready for PCR amplification with the provided primers that bind to sequences found in all fish genomes. The PCR products are then digested with three different restriction enzymes and resolved on the Agilent 2100 Bioanalyzer. The fragment lengths produced in the digestion reactions can be used to determine the species of fish from which the DNA sample was prepared, using the RFLP pattern matching software containing a database of experimentally- derived RFLP patterns from commercially relevant fish species.

This publication describes how to use the Agilent Fish Species Identification System to identify the species of a fish by extracting DNA and performing PCR and RFLP analysis.

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

3D DNA FISH has become a major tool for analyzing three-dimensional organization of the nucleus, and several variations of the technique have been published. In this article we describe a protocol which has been optimized for robustness, reproducibility, and ease of use. Brightly fluorescent directly labeled probes are generated by nick-translation with amino-allyldUTP followed by chemical coupling of the dye. 3D DNA FISH is performed using a freeze-thaw step for cell permeabilization and a heating step for simultaneous denaturation of probe and nuclear DNA. The protocol is applicable to a range of cell types and a variety of probes (BACs, plasmids, fosmids, or Whole Chromosome Paints) and allows for high-throughput automated imaging. With this method we routinely investigate nuclear localization of up to three chromosomal regions.

We describe a robust and versatile protocol for analyzing nuclear architecture by 3D DNA FISH using directly labeled fluorescent 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

The generation of chimeric embryos is a widespread and powerful approach to study cell fates, tissue interactions, and species-specific contributions to the histological and morphological development of vertebrate embryos. In particular, the use of chimeric embryos has established the importance of neural crest in directing the species-specific morphology of the craniofacial complex. The method described herein utilizes two avian species, duck and quail, with remarkably different craniofacial morphology. This method greatly facilitates the investigation of molecular and cellular regulation of species-specific pattern in the craniofacial complex.&#160;Experiments in quail and duck chimeric embryos have already revealed neural crest-mediated tissue interactions and cell-autonomous behaviors that regulate species-specific pattern in the craniofacial skeleton, musculature, and integument. The great diversity of neural crest derivatives suggests significant potential for future applications of the quail-duck chimeric system to understanding vertebrate development, disease, and evolution.

This article describes a method to generate chimeric embryos that are&#160;designed to test the species-specific contributions of neural crest and/or other tissues to craniofacial development.

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

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in cytogenetics and cancer diagnostics, and can detect aberrations of the genome, which often has important clinical implications. RNA FISH can be used to detect RNA molecules in cells and has provided important insights in regulation of gene expression. Combining DNA and RNA FISH within the same cell is technically challenging, as conditions suitable for DNA FISH might be too harsh for fragile, single stranded RNA molecules. We here present an easily applicable protocol which enables the combined, simultaneous detection of Xist RNA and DNA encoded by the X chromosomes. This combined DNA-RNA FISH protocol can likely be applied to other systems where both RNA and DNA need to be detected.

Combined DNA-RNA Fluorescent In situ Hybridization FISH to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) allows the detection of nucleic acids in their native environment within cells. We here describe a protocol for the combined, simultaneous detection of RNA and DNA by means of FISH, which can be used to study X chromosome inactivation in 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

Low dose radiation exposure may produce a variety of biological effects that are different in quantity and quality from the effects produced by high radiation doses. Addressing questions related to environmental, occupational and public health safety in a proper and scientifically justified manner heavily relies on the ability to accurately measure the biological effects of low dose pollutants, such as ionizing radiation and chemical substances. DNA damage and repair are the most important early indicators of health risks due to their potential long term consequences, such as cancer. Here we describe a protocol to study the effect of chronic in vivo exposure to low doses of &#947;- and &#946;-radiation on DNA damage and repair in mouse spleen cells. Using a commonly accepted marker of DNA double-strand breaks, phosphorylated histone H2AX called &#947;H2AX, we demonstrate how it can be used to evaluate not only the levels of DNA damage, but also changes in the DNA repair capacity potentially produced by low dose in vivo exposures. Flow cytometry allows fast, accurate and reliable measurement of immunofluorescently labeled &#947;H2AX in a large number of samples. DNA double-strand break repair can be evaluated by exposing extracted splenocytes to a challenging dose of 2 Gy to produce a sufficient number of DNA breaks to trigger repair and by measuring the induced (1 hr post-irradiation) and residual DNA damage (24 hrs post-irradiation). Residual DNA damage would be indicative of incomplete repair and the risk of long-term genomic instability and cancer. Combined with other assays and end-points that can easily be measured in such in vivo studies (e.g., chromosomal aberrations, micronuclei frequencies in bone marrow reticulocytes, gene expression, etc.), this approach allows an accurate and contextual evaluation of the biological effects of low level stressors.

Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

A protocol to evaluate changes in DNA damage levels and DNA repair capacity that may be induced by chronic in vivo low dose irradiation in mouse spleen lymphocytes, by measuring phosphorylated histone H2AX, a marker of DNA double-strand breaks, using flow cytometry is presented.

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 ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

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

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These age-associated disorders are characterized by the appearance of protein aggregates with fibrillary structure in the brains of these patients. Exactly why normally soluble proteins undergo an aggregation process remains poorly understood. The discovery that protein aggregation is not limited to disease processes and instead part of the normal aging process enables the study of the molecular and cellular mechanisms that regulate protein aggregation, without using ectopically expressed human disease-associated proteins. Here we describe methodologies to examine inherent protein aggregation in Caenorhabditis elegans through complementary approaches. First, we examine how to grow large numbers of age-synchronized C. elegans to obtain aged animals and we present the biochemical procedures to isolate highly-insoluble-large aggregates. In combination with a targeted genetic knockdown, it is possible to dissect the role of a gene of interest in promoting or preventing age-dependent protein aggregation by using either a comprehensive analysis with quantitative mass spectrometry or a candidate-based analysis with antibodies. These findings are then confirmed by in vivo analysis with transgenic animals expressing fluorescent-tagged aggregation-prone proteins. These methods should help clarify why certain proteins are prone to aggregate with age and ultimately how to keep these proteins fully functional.

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

The goal of the method presented here is to explore protein aggregation during normal aging in the model organism C. elegans. The protocol represents a powerful tool to study the highly insoluble large aggregates that form with age and to determine how changes in proteostasis impact protein aggregation.

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

Investigation of intercellular interactions often requires discrete labeling of specific cell populations and precise protein localization. The zebrafish embryo is an excellent tool for examining such interactions with an in vivo model. Whole-mount immunohistochemical and immunofluorescence assays are frequently applied in zebrafish embryos to assess protein expression. However, it can be difficult to achieve accurate mapping of co-localized proteins in three-dimensional space. In addition, some studies may require the use of two antibodies that are not compatible with the same technique (e.g., antibody 1 is only suitable for immunohistochemistry and antibody 2 is only suitable for immunofluorescence). The purpose of the method described herein is to perform sequential immunofluorescence and/or immunohistochemistry on individual cryosections derived from early-stage zebrafish embryos. Here we describe the use of sequential rounds of immunofluorescence, imaging, immunohistochemistry, imaging for a single cryosection in order to achieve precise identification of protein expression at the single-cell level. This methodology is suitable for any study in early-stage zebrafish embryos that requires accurate identification of multiple protein targets in individual cells.

This protocol demonstrates sequential immunofluorescence and immunohistochemistry on cryosections from early-stage zebrafish embryos enabling precise colocalization analyses in specific cell populations.

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 ...

Visualization of DNA Repair Proteins Interaction by Immunofluorescence

Mammalian cells are constantly exposed to chemicals, radiations, and naturally occurring metabolic by-products, which create specific types of DNA insults. Genotoxic agents can damage the DNA backbone, break it, or modify the chemical nature of individual bases. Following DNA insult, DNA damage response (DDR) pathways are activated and proteins involved in the repair are recruited. A plethora of factors are involved in sensing the type of damage and activating the appropriate repair response. Failure to correctly activate and recruit DDR factors can lead to genomic instability, which underlies many human pathologies including cancer. Studies of DDR proteins can provide insights into genotoxic drug response and cellular mechanisms of drug resistance.
There are two major ways of visualizing&#160;proteins in vivo: direct observation, by tagging the protein of interest with a fluorescent protein and following it by live imaging, or indirect immunofluorescence on fixed samples. While visualization of fluorescently tagged proteins allows precise monitoring over time, direct tagging in N- or C-terminus can interfere with the protein localization or function. Observation of proteins in their unmodified, endogenous version is preferred. When DNA repair proteins are recruited to the DNA insult, their concentration increases locally and they form groups, or &#8220;foci&#8221;, that can be visualized by indirect immunofluorescence using specific antibodies.
Although detection of protein foci does not provide a definitive proof of direct interaction, co-localization of proteins in cells indicates that they regroup to the site of damage and can inform of the sequence of events required for complex formation. Careful analysis of foci spatial overlap in cells expressing wild type or mutant versions of a protein can provide precious clues on functional domains important for DNA repair function. Last, co-localization of proteins indicates possible direct interactions that can be verified by co-immunoprecipitation in cells, or direct pulldown using purified proteins.

Following DNA damage, human cells activate essential repair pathways to restore the integrity of their genome. Here, we describe the method of indirect immunofluorescence as a means to detect DNA repair proteins, analyze their spatial and temporal recruitment, and help interrogate protein-protein interaction at the sites of DNA damage.