We thank Hongjae Sunwoo for helpful advice for oligonucleotide design in RNA FISH. We also thank Serenity Curtis for editing the manuscript. N.Y. was supported by a Postdoctoral Fellowship for Research Abroad of the Japan Society for the Promotion of Science (JSPS). This work was supported by the NIH (RO1-GM102184), the March of Dimes Research Foundation (#6-FY12-337), and the Developmental Fund and Trustee Grant at Cincinnati Children's Hospital Medical Center to Y.O.

1. Preparación de la sonda <ol><li> Obtener múltiples oligonucleótidos únicos en Xist (oligonucleótidos 20-30 nucleótidos de longitud, 63-65 ºC de temperatura de fusión, Tabla 1) con una modificación 5&#39;-amino y suspender en agua. </li><li> Piscina cantidades equimolares de los oligonucleótidos con 5&#39;-amino modificación (en total 4,5 g) y la etiqueta con colorante fluorescente-amina reactiva siguiendo las instrucciones del fabricante. <ol><li> Disolver los oligonucleótidos agrupados en últimos 5 l de agua libre de nucleasa y añadir 3 l de 1 M de bicarbonato de sodio a la solución de oligonucleótidos. </li><li> Añadir 2 l DMSO al vial de tinte y de vórtice-amina reactiva brevemente. </li><li> Mezclar la solución de oligonucleótido con el colorante reactivo en DMSO y se incuba la mezcla a temperatura ambiente durante 1 h en la oscuridad. </li></ol></li><li> Purificar las sondas de oligonucleótidos marcados con fluorescencia por la columna G-25 seguido por precipitación con etanol. <ol><li&gt; Centrifugar la columna G-25 a 750 xg durante 1 min para eliminar el exceso de tampón de almacenamiento. </li><li> Añadir 40 l de agua libre de nucleasa a la mezcla de etiquetado producido en la Etapa 1.2.3 y aplicar a la columna G-25. Centrifugar a 750 xg durante 2 min. </li><li> Añadir 50 l de agua libre de nucleasa, 10 l 3 M acetato de sodio (pH 5,2) y 250 l de etanol a la sonda purificada de la etapa 1.3.2. Conservar a -80 ° C durante 30 minutos y se centrifuga a 21.000 xg durante 15 min a 4 ° C. </li><li> Lavar los oligonucleótidos precipitados una vez con 1 ml de etanol al 70%. </li><li> Vuelva a suspender en 50 l de agua libre de nucleasa o TE (10 mM Tris-HCl, pH 7,5; EDTA 1 mM) para una concentración final de aproximadamente 10 mM. </li></ol></li><li> Diluir las sondas de oligonucleótidos marcados con fluorescencia a una concentración final de 250 nM con tampón de hibridación (10% formamida; 2x SSC [NaCl 300 mM; citrato de sodio 30 mM]; 2 mg / ml BSA; sulfato de dextrano al 10%). NOTA: Con este protocolo FISH rápida, oligonucleótido pbatas en Xist fueron diseñados y utilizados a una concentración mayor que el protocolo normal de FISH usando sondas de oligonucleótidos con el fin de acortar el tiempo de incubación para la hibridación. </li></ol> 2. Preparación de los portaobjetos NOTA: Los portaobjetos se puede preparar para la inmuno-FISH utilizando madre de ratón embrionarias (ES) o el cuerpo embrioide (EB) diferenciación de las células 20, 21 ya sea por crecimiento de ellos directamente en un portaobjetos tratados con poli-lisina o utilizando cytospin con suspensión celular. Aquí, se muestra la preparación de diapositivas cytospin. <ol><li> Aspirar el medio de cultivo en una placa de 6 pocillos. Lavar con PBS (temperatura ambiente), y aspirar a fondo. </li><li> Añadir 0,5 ml de tripsina al plato de 6 pocillos, se incuba durante 5-10 minutos a 37 ° C. </li><li> Añadir 5 ml de medio y dispersar las células pipeteando suavemente hacia arriba y abajo varias veces. </li><li> Transferir las células a un tubo de 15 ml, centrifugar durante 5 min a 200 xg a temperatura ambiente. </li><li> Deseche el medio de unad suspender suavemente las células en 2 ml de PBS. </li><li> Contar las células usando un hemocitómetro, y se diluye la suspensión celular en 4 x 10 5 células / ml de PBS. </li><li> Montar una diapositiva, una tarjeta de filtro y una cámara con un clip y colocarlos en las ranuras correspondientes en el rotor de la cytospin. Añadir 250 l de cada muestra preparada en el paso 2.6 en cada cámara de las unidades ensambladas en el cytospin. Centrifugar a 1500 rpm durante 10 min a temperatura ambiente. </li><li> Llenar 3 frascos de Coplin en hielo con 40 ml cada uno de tampón PBS, CSK enfriado con hielo (tubos 10 mM, pH 6,8; NaCl 100 mM; sacarosa 300 mM; 3 mM de MgCl 2), y de amortiguamiento CSKT (tampón CSK con 0,5% Triton X-100), respectivamente. </li><li> Después de que se terminó la cytospin, transferir rápidamente el portaobjetos en PBS enfriado con hielo y se incuba durante 5 min. </li><li> Transferir el portaobjetos en tampón de CSK enfriado con hielo y se incuba durante 1 min. </li><li> Transferir el portaobjetos en tampón de CSKT enfriado con hielo y se incuba durante 5 min. </li><li> Transferir el portaobjetos de nuevo en helada CSK buffer e incubar durante 1 min. </li><li> Transferir el portaobjetos en 4% de paraformaldehído en PBS y se incuba durante 10 min a temperatura ambiente. </li></ol> 3. La inmunofluorescencia <ol><li> Colocar los portaobjetos en un tarro Coplin con PBST (1x PBS con 0,1% de Tween-20) durante varios segundos. </li><li> Colocar los portaobjetos en una inclinación que permite buffer restante a fluir por el tobogán. Limpie cuidadosamente el exceso de líquido fuera de la diapositiva y colocarla en una caja de pipeta de punta vacío con agua. Superposición de la diapositiva con 40 l de solución de bloqueo (1x PBS, 1% de BSA, 0,1% de Tween-20), colocar suavemente un cubreobjetos, y se incuba durante 10 min a temperatura ambiente. </li><li> Retire el cubreobjetos, eliminar la solución de bloqueo, y añadir 40 l de anticuerpo primario diluido H3K27me3 histona a una concentración de 1: 500 en solución de bloqueo. Coloque el cubreobjetos de nuevo en el porta y se incuba durante 30 min a temperatura ambiente. NOTA: Un inhibidor de RNasa en la solución de bloqueo podría ser útil para preservar ARN en este ARN FISProtocolo H cuando se utilizan anticuerpos policlonales. </li><li> Lave deslice 2 veces en frascos Coplin llenos de PBST, durante 5 minutos cada uno, agitando suavemente durante el lavado. </li><li> Colocar los portaobjetos en una inclinación que permite buffer restante a fluir por el tobogán. Limpie cuidadosamente el exceso de líquido y coloque el portaobjetos de nuevo en la caja de propina. Superposición con 40 l de anticuerpo secundario (1: 500) diluido en solución de bloqueo, colocar un cubreobjetos, y se incuba durante 15 min en la oscuridad. Para los pasos siguientes, mantenga la diapositiva en la oscuridad en todo momento. </li><li> Lavar 2 veces en Coplin frascos con PBST como en el paso 3.4. </li></ol> 4. ARN FISH con sondas de oligonucleótidos <ol><li> Pre-calentar un cuadro de pipeta de punta vacío con agua en la parte inferior para evitar que las diapositivas de secado a 37 ° C para el ARN FISH en el paso 4.3. </li><li> Colocar los portaobjetos en una inclinación que permite buffer restante a fluir por el tobogán. Limpie cuidadosamente el exceso de líquido fuera de la diapositiva desde el paso 3.6 y colocar la placa en la caja de pipeta de punta con water en la parte inferior. Añadir 500 l de tampón de lavado FISH (10% de formamida en 2x SSC) en la diapositiva y se incuba durante 5 min a temperatura ambiente. </li><li> Retire la solución de lavado FISH, añadir 8 l de sondas de oligonucleótidos en la diapositiva, coloque una hoja de la cubierta, y la transferencia de la diapositiva a una caja de pipeta de punta pre calentado con agua en el fondo; incubar a 37 ° C durante 1-2 horas. </li><li> Coloque 3 frascos pre-calentado Coplin en un baño de agua a 37 ° C con tampón de lavado FISH, FISH tampón de lavado con DAPI, y 2x SSC. </li><li> Después de 1-2 horas de incubación de ARN FISH, poner la diapositiva en el tampón de lavado FISH precalentado. </li><li> Incubar el portaobjetos durante unos minutos hasta que el cubreobjetos se cae de la diapositiva. </li><li> Colocar el portaobjetos de nuevo en el tampón de lavado, y continuar la incubación durante 5 min. </li><li> Transferir la diapositiva a otra jarra Coplin con tampón de lavado pre-calentado FISH con 0,5 ng / ml DAPI y se incuba durante 5 min. </li><li> Transferir la diapositiva a otra jarra Coplin con pre-calentado 2x SSC y se incuba durante 5 millasn. </li><li> Colocar los portaobjetos en una inclinación que permite buffer restante a fluir por el tobogán. Limpie cuidadosamente el exceso de líquido y coloque el portaobjetos de nuevo en una caja de punta seca. Añadir 4 l de antifade con DAPI sobre el portaobjetos y colocar un cubreobjetos. </li><li> Sellar el cubreobjetos con esmalte de uñas y observar con un microscopio. La corredera se puede almacenar a 4 ° C en la oscuridad durante unos pocos meses. </li></ol>

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No conflicts of interest declared.

En este trabajo, hemos presentado un protocolo de inmuno-FISH rápida que tarda menos de 5 horas para completar la preparación de diapositivas, Xist ARN FISH, y la inmunofluorescencia para H3K27me3. En comparación con el enfoque general de inmuno-FISH para Xist ARN de detección, que por lo general tiene una noche de incubación de ARN FISH, este protocolo no sólo reduce significativamente el tiempo pasado sino que también mejora la sensibilidad inmune-FISH usando sondas de oligonucleótidos. <p class="jove_cont...

Mamíferos inactivación del cromosoma X (XCI) es una estrategia para compensar el desequilibrio en la dosificación del gen ligado al cromosoma X entre XX y XY, en el que uno de los dos cromosomas X en las mujeres es transcripcionalmente inactiva 1. Inactivación del cromosoma X es un gran modelo para el sistema a largo ARN no codificante (lncRNA) investigación. Inactivación del cromosoma X está regulado por múltiples lncRNAs, y ha sido ampliamente estudiado en los últimos decenios para descubrir los mecanismos de diafonía entre lncRNAs, transcripción, estructura de la cromatina y la organización nuclear 2, 3. El centro de la inactivación X (XIC), ubicado en el cromosoma X es un locus genético complejo compuesto de un número de genes que producen ARN no codificantes 4. -X inactivo transcripción específico (Xist) lncRNA en los mamíferos euterios es uno de esos lncRNA que desempeña un papel crucial en la inactivación del cromosoma X 5,6. Transcripciones Xist rodean la ubicación de la futura Xi para iniciar inactivación del cromosoma X, y aparece como una nube cuando se visualizan utilizando ARN FISH; esta formación se denomina como el &quot;Xist nube&quot; 7. Desde Xist ARN interactúa con varias enzimas de modificación de la cromatina, se observa co-localización de las nubes Xist con diferentes modificaciones epigenéticas de la cromatina y la transcripción en silencio durante represivo X-inactivación del cromosoma 8. Por ejemplo, Xist ARN interactúa con polycomb complejo represivo 2 (PRC2) que es responsable de H3K27me3 e induce un estado represivo cromatina 9. La aparición de la nube Xist en el Xi y su co-localización con la modificación intensiva H3K27me3 representa un paisaje heterocromatina facultativa de la Xi 10,11. Técnicas citogenéticas, como el ADN / ARN FISH han recorrido un largo camino desde el método tradicional utilizando sondas radiomarcado 12 a las técnicas más recientes y avanzados con mayor sensibilidad yimágenes fluorescentes utilizando múltiples sondas de oligonucleótidos 13,14. ADN / ARN FISH junto con inmunofluorescencia se ha utilizado de forma rutinaria como una herramienta citológico de entender la organización espacio-temporal nuclear, la localización del ARN, la estructura de la cromatina y modificaciones. La preparación de la sonda estándar de la mayoría de ARN FISH implica el uso de plásmido o cromosoma artificial (BAC) clones bacterianos y su posterior etiquetado ya sea con la traducción nick o cebado aleatorio 15. Sin embargo, casi el 70% de los genes en ratones y 40% de los genes en los seres humanos muestran una superposición de sentido y antisentido transcripciones 16, por lo que requieren un método FISH específica de hebra a fin de distinguir sentido y antisentido transcripciones. In vitro sondas de ARN transcritos (ribosonda ) se utilizan a menudo para el capítulo específico de ARN FISH 17,18; sin embargo, esto implica la preparación de un clon de plásmido o producto de PCR con T7, SP6, o el promotor T3 y sintetizar ribosondas. Además, ribosondas derivado de genomaic ADN o ADNc a menudo contienen regiones no específicas y elementos repetitivos, que resultan en alto ruido de fondo. Otra cuestión es que ribosondas, que son unos pocos cientos de nucleótidos de longitud y contienen múltiples fluoróforos, no pueden penetrar de manera eficiente en el núcleo. Para evitar esto, múltiples sondas de oligonucleótidos más cortos marcados con un solo fluoróforo en el extremo han sido desarrollados que tienen una buena sensibilidad, potencia de la señal uniforme, y la facilidad de purificación y el manejo de 14. Además, los oligonucleótidos de ADN son generalmente más estable que el ARN. Se aplicó una estrategia similar de utilizar oligonucleótidos en Xist ARN FISH 19 con inmunofluorescencia para comprender la dinámica epigenéticas de la Xi inducidos por Xist lncRNA durante el proceso de inactivación del cromosoma X. Este protocolo describe la creación de sondas de oligonucleótidos y la preparación apropiada de las células, así como la utilización de inmunofluorescencia y ARN FISH. Xist ARN FISH utilizando múltiples oligonucleOtides es enfoque rentable en el largo plazo si Xist ARN FISH se realiza de forma rutinaria en el laboratorio de uno. Esta técnica se puede utilizar para identificar lncRNAs en las células mientras que la cartografía simultáneamente su co-localización con modificaciones o factores epigenéticos. Una ventaja importante del protocolo es la capacidad de modificar fácilmente para adaptarse a los propios intereses de investigación.

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		<tr height="21">
			<td height="21" style="height:21px;width:267px;">oligonucleotides with 5&#8217;-amine modification</td>
			<td style="width:187px;">IDT</td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Alexa Fluor 488 Reactive Dye</td>
			<td style="width:187px;">Life Technologies</td>
			<td style="width:119px;">A32750</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">MicroSpin G-25 columns</td>
			<td style="width:187px;">GE Healthcare</td>
			<td style="width:119px;">27-5325-01</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Cytospin 2</td>
			<td style="width:187px;">Shandon</td>
			<td align="right" style="width:119px;">59900102</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Bovine Serum Albumin, Molecular Biology Grade (for hybridization buffer)</td>
			<td style="width:187px;">Roche</td>
			<td style="width:119px;">10715859103</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Histone H3K27me3 antibody</td>
			<td style="width:187px;">Active Motif</td>
			<td style="width:119px;">61017</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Accutase</td>
			<td style="width:187px;">Innovative Cell Technologies</td>
			<td style="width:119px;">AT104</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Alexa Fluor 555 Goat Anti-Mouse, highly cross-adsorbed</td>
			<td style="width:187px;">Life Technologies</td>
			<td style="width:119px;">A21424</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">ProLong Gold antifade reagent</td>
			<td style="width:187px;">Life Technologies</td>
			<td style="width:119px;">P36931</td>
		</tr>
	</tbody>
</table>

quick fluorescent in situ hybridization protocol for xist rna combined with immunofluorescence of histone modification in x-chromosome inactivation

La combinación de ARN hibridación in situ fluorescente (FISH) con inmunofluorescencia (inmuno-FISH) crea una técnica que se puede emplear en el nivel de células individuales para detectar la dinámica espacial de la localización del ARN con la penetración simultánea en la localización de las proteínas, las modificaciones epigenéticas y otros detalles que puede ser resaltado mediante inmunofluorescencia. Inactivación del cromosoma X es un paradigma para largo ARN no codificante (lncRNA) mediada por silenciamiento génico. Transcripción específico (Xist) la acumulación lncRNA-X inactivo (llamado una nube de Xist) sobre uno de los dos cromosomas X en las hembras de mamíferos es un paso crítico para iniciar X-inactivación del cromosoma. ARN Xist interactúa directamente o indirectamente con diversas enzimas de la cromatina modificar y presenta distintos paisajes epigenéticos al cromosoma X inactivo (Xi). Una conocida seña de identidad de la epigenética Xi es la histona H3 trimetil-lisina 27 (H3K27me3) modificación. A continuación, describimos una forma sencilla y rápida inmuno-FISHProtocolo para la detección de Xist ARN utilizando ARN FISH con múltiples sondas de oligonucleótidos junto con inmunofluorescencia de H3K27me3 a examinar la localización de Xist ARN y modificaciones epigenéticas asociadas. El uso de sondas de oligonucleótidos resultados en un tiempo de incubación más corto y la detección más sensible de Xist ARN en comparación con el transcrito in vitro sondas de ARN (ribosondas). Este protocolo proporciona una poderosa herramienta para la comprensión de la dinámica de lncRNAs y su modificación epigenética asociada, la estructura de la cromatina, organización nuclear y la regulación transcripcional.

Imágenes representativas de rápida inmuno-FISH se muestran en la Figura 1A. Se detectó co-localización de la nube y H3K27me3 señal de Xist ARN en el Xi en la diferenciación de las células femeninas. Al día 12 de la diferenciación, más del 90% de las células EB tenía una nube Xist (Figura 1B). Sondas de oligonucleótidos corto penetraron de manera eficiente en los núcleos, que conduce a una visualización de casi todas las señales H3K27me3 co-localizada con Xist ARN <strong...

Watch this Scientific Journal Video about Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation at JoVE.co

Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation

We developed an easily customized strand-specific fluorescent in situ hybridization (FISH) protocol combined with immunofluorescence. This allows for a detailed examination of RNA dynamics with simultaneous insight into the chromatin structure, nuclear organization, and transcriptional regulation at the single cell level.

Fluorescente Rápida<em&gt; In Situ</em&gt; Protocolo de hibridación para Xist ARN combinado con inmunofluorescencia de histona Modificación de inactivación del cromosoma X.

Combining RNA fluorescent in situ hybridization (FISH) with immunofluorescence (immuno-FISH) creates a technique that can be employed at the single cell ...

quick-fluorescent-situ-hybridization-protocol-for-xist-rna-combined

Research

JoVE Journal

Biology

Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation

13.8K vistas.  Cincinnati Children's Hospital Medical Center. We developed an easily customized strand-specific fluorescent in situ hybridization (FISH) protocol combined with immunofluorescence. This allows for a detailed examination of RNA dynamics with simultaneous insight into the chromatin structure, nuclear organization, and transcriptional regulation at the single cell level. 

Video: Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

Monte doble entero hibridación in situ de embriones de pollo temprano

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Investigación

Biología

Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species

The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of tissue specific expression is limited because of problems in dissecting individual tissues. Expression data needs to be confirmed and complemented with expression patterns using e.g. in situ hybridization, a technique used to localize cell specific mRNA expression. The in situ hybridization method is laborious, time-consuming and often requires extensive optimization depending on species and tissue. In situ experiments are relatively more difficult to perform in woody species such as the conifer Norway spruce (Picea abies). Here we present a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including P. abies. With just a few adjustments, including altered RNase treatment and proteinase K concentration, we could use the protocol to study tissue specific expression of homologous genes in male reproductive organs of one gymnosperm and two angiosperm species; P. abies, Arabidopsis thaliana and Brassica napus. The protocol worked equally well for the species and genes studied. AtAP3 and BnAP3 were observed in second and third whorl floral organs in A. thaliana and B. napus and DAL13 in microsporophylls of male cones from P. abies. For P. abies the proteinase K concentration, used to permeablize the tissues, had to be increased to 3 g/ml instead of 1 g/ml, possibly due to more compact tissues and higher levels of phenolics and polysaccharides. For all species the RNase treatment was removed due to reduced signal strength without a corresponding increase in specificity. By comparing tissue specific expression patterns of homologous genes from both flowering plants and a coniferous tree we demonstrate that the DIG in situ protocol presented here, with only minute adjustments, can be applied to a wide range of plant species. Hence, the protocol avoids both extensive species specific optimization and the laborious use of radioactively labeled probes in favor of DIG labeled probes. We have chosen to illustrate the technically demanding steps of the protocol in our film.
Anna Karlgren and Jenny Carlsson contributed equally to this study.
Corresponding authors: Anna Karlgren at Anna.Karlgren@ebc.uu.se and Jens F. Sundstr&#246;m at Jens.Sundstrom@vbsg.slu.se

Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species

We describe a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including Norway spruce. With just a few adjustments, including altered RNase treatment and proteinase K concentration, the protocol may be used in studies of different tissues and species.

No radiactivo In situ La hibridación aplicable para el abeto rojo protocolo y una serie de especies de plantas

The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of ...

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

Pez cebra todo el montaje de alta resolución doble fluorescente In Situ La hibridación

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic process in the parents. In human males, these errors can lead to the production of spermatozoa with numerical chromosome abnormalities which represent an increased risk of transmitting these anomalies to the offspring.
For this reason, the technique of fluorescence in situ hybridization (FISH) on sperm nuclei has become a protocol widely incorporated in the context of clinical diagnosis. This practice provides an estimate of the frequencies of numerical chromosome abnormalities in the gametes of the patients that seek for genetic reproductive advice.
To date, the chromosomes most frequently included in sperm FISH analysis are chromosomes X, Y, 13, 18 and 21.
This video-article describes, step by step, how to process and fix a human semen sample, how to decondense and denature the sperm chromatin, how to proceed to obtain sperm FISH preparations, and how to visualize the results at the microscope. Special remarks of the most relevant steps are given to achieve the best results.

Fluorescence in situ hybridization FISH Protocol in Human Sperm

This video-article describes, step by step, how to process a semen sample to achieve good-quality fluorescence in situ hybridization on human spermatozoa. Preparations obtained can be used for aneuploidy screening in the context of clinical diagnosis.

Fluorescencia In situ</emHibridación> (FISH) Protocolo en el esperma humano

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic ...

In Situ Hybridization for the Precise Localization of Transcripts in Plants

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene expression. Microarray and next generation sequencing approaches are being used to investigate developmental, physiological and stress response processes, dissect epigenetic and small RNA pathways, and build large gene regulatory networks1-3. While these techniques facilitate the simultaneous analysis of large gene sets, they typically provide a very limited spatiotemporal resolution of gene expression changes. This limitation can be partially overcome by using either profiling method in conjunction with lasermicrodissection or fluorescence-activated cell sorting4-7. However, to fully understand the biological role of a gene, knowledge of its spatiotemporal pattern of expression at a cellular resolution is essential. Particularly, when studying development or the effects of environmental stimuli and mutants can the detailed analysis of a gene's expression pattern become essential. For instance, subtle quantitative differences in the expression levels of key regulatory genes can lead to dramatic phenotypes when associated with the loss or gain of expression in specific cell types. 
Several methods are routinely used for the detailed examination of gene expression patterns. One is through analysis of transgenic reporter lines. Such analysis can, however, become time-consuming when analyzing multiple genes or working in plants recalcitrant to transformation. Moreover, an independent validation to ensure that the transgene expression pattern mimics that of the endogenous gene is typically required. Immunohistochemical protein localization or mRNA in situ hybridization present relatively fast alternatives for the direct visualization of gene expression within cells and tissues. The latter has the distinct advantage that it can be readily used on any gene of interest. In situ hybridization allows detection of target mRNAs in cells by hybridization with a labeled anti-sense RNA probe obtained by in vitro transcription of the gene of interest. 
Here we outline a protocol for the in situ localization of gene expression in plants that is highly sensitivity and specific. It is optimized for use with paraformaldehyde fixed, paraffin-embedded sections, which give excellent preservation of histology, and DIG-labeled probes that are visualized by immuno-detection and alkaline-phosphatase colorimetric reaction. This protocol has been successfully applied to a number of tissues from a wide range of plant species, and can be used to analyze expression of mRNAs as well as small RNAs8-14.

In Situ Hybridization for the Precise Localization of Transcripts in Plants

The in situ hybridization protocol described here allows a direct localization of mRNA and small RNA expression at the cellular level with high sensitivity and specificity. The procedure is optimized for paraffin-embedded plant tissue sections, is applicable to a wide range of plants and tissues, and can be completed within ten days.

Hibridación in situ para la localización precisa de las transcripciones de las plantas

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

La detección de ARN viral por fluorescencia<em&gt; In situ</em&gt; La hibridación (FISH)

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes.
The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE1.
Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System2 (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription and regulation of a variety of genes expressed during the different stages of the P. falciparum life cycle and is adaptable to other malaria parasite species and other organisms and cell types.

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH

Fluorescent in situ hybridization (FISH) to identify mRNA transcripts in individual cells allows analysis of polygenic activity such as the simultaneous transcription of more than one member of the var multigene family in Plasmodium falciparum infected erythrocytes 1. The technique is adaptable and can be used on different types of genes, cells and organisms.

Análisis de la transcripción de genes de una sola célula de ARN fluorescente<em&gt; In Situ</em&gt; La hibridación (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 ...

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The specification of replication timing is thought to be a dynamic process regulated by tissue-specific and developmental cues that are responsive to epigenetic modifications. However, the mechanisms regulating where and when DNA replication initiates along chromosomes remains poorly understood. Homologous chromosomes usually replicate synchronously, however there are notable exceptions to this rule. For example, in female mammalian cells one of the two X chromosomes becomes late replicating through a process known as X inactivation1. Along with this delay in replication timing, estimated to be 2-3 hr, the majority of genes become transcriptionally silenced on one X chromosome. In addition, a discrete cis-acting locus, known as the X inactivation center, regulates this X inactivation process, including the induction of delayed replication timing on the entire inactive X chromosome. In addition, certain chromosome rearrangements found in cancer cells and in cells exposed to ionizing radiation display a significant delay in replication timing of &gt;3 hours that affects the entire chromosome2,3. Recent work from our lab indicates that disruption of discrete cis-acting autosomal loci result in an extremely late replicating phenotype that affects the entire chromosome4. Additional 'chromosome engineering' studies indicate that certain chromosome rearrangements affecting many different chromosomes result in this abnormal replication-timing phenotype, suggesting that all mammalian chromosomes contain discrete cis-acting loci that control proper replication timing of individual chromosomes5.
Here, we present a method for the quantitative analysis of chromosome replication timing combined with fluorescent in situ hybridization. This method allows for a direct comparison of replication timing between homologous chromosomes within the same cell, and was adapted from6. In addition, this method allows for the unambiguous identification of chromosomal rearrangements that correlate with changes in replication timing that affect the entire chromosome. This method has advantages over recently developed high throughput micro-array or sequencing protocols that cannot distinguish between homologous alleles present on rearranged and un-rearranged chromosomes. In addition, because the method described here evaluates single cells, it can detect changes in chromosome replication timing on chromosomal rearrangements that are present in only a fraction of the cells in a population.

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

A quantitative method for the analysis of chromosome replication timing is described. The method utilizes BrdU incorporation in combination with fluorescent in situ hybridization (FISH) to assess replication timing of mammalian chromosomes. This technique allows for the direct comparison of rearranged and un-rearranged chromosomes within the same cell.

Replicación del cromosoma Timing Combinado con fluorescente<em&gt; In situ</em&gt; La hibridación

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The ...

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels 1. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest 2. As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens 3. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources 4-11. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure 12. Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.

Todo el monte ARN fluorescente<em&gt; In situ</em&gt; La hibridación de<em&gt; Drosophila</em&gt; Los embriones

Assessing  the expression pattern of a gene, as well as the subcellular localization  properties of its transcribed RNA, are key features for ...

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

Marine elasmobranchs are valued animal models for biomedical and genomic studies as they are the most primitive vertebrates to have adaptive immunity and have unique mechanisms for osmoregulation 1-3. As the most primitive living jawed-vertebrates with paired appendages, elasmobranchs are an evolutionarily important model, especially for studies in evolution and development. Marine elasmobranchs have also been used to study aquatic toxicology and stress physiology in relationship to climate change 4. Thus, development and adaptation of methodologies is needed to facilitate and expand the use of these primitive vertebrates to multiple biological disciplines. Here I present the successful adaptation of RNA whole mount in situ hybridization and histological techniques to study gene expression and cell histology in elasmobranchs.
Monitoring gene expression is a hallmark tool of developmental biologists, and is widely used to investigate developmental processes 5. RNA whole mount in situ hybridization allows for the visualization and localization of specific gene transcripts in tissues of the developing embryo. The expression pattern of a gene's message can provide insight into what developmental processes and cell fate decisions a gene may control. By comparing the expression pattern of a gene at different developmental stages, insight can be gained into how the role of a gene changes during development.
While whole mount in situ's provides a means to localize gene expression to tissue, histological techniques allow for the identification of differentiated cell types and tissues. Histological stains have varied functions. General stains are used to highlight cell morphology, for example hematoxylin and eosin for general staining of nuclei and cytoplasm, respectively. Other stains can highlight specific cell types. For example, the alcian blue stain reported in this paper is a widely used cationic stain to identify mucosaccharides. Staining of the digestive tract with alcian blue can identify the distribution of goblet cells that produce mucosaccharides. Variations in mucosaccharide constituents on short peptides distinguish goblet cells by function within the digestive tract 6. By using RNA whole mount in situ's and histochemical methods concurrently, cell fate decisions can be linked to gene-specific expression.
Although RNA in situ's and histochemistry are widely used by researchers, their adaptation and use in marine elasmobranchs have met limited and varied success. Here I present protocols developed for elasmobranchs and used on a regular basis in my laboratory. Although further modification of the RNA in situ's hybridization method may be needed to adapt to different species, the protocols described here provide a strong starting point for researchers wanting to adapt the use of marine elasmobranchs to their scientific inquiries.

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

By combining methods for RNA whole mount in situ hybridization and histology, gene expression can be linked with cell fate decisions in the developing embryo. These methods have been adapted to marine elasmobranchs and facilitate the use of these animals as model organisms for biomedical, toxicology and comparative studies.

ARN<em&gt; In situ</emHibridación&gt; Los embriones en todo el montaje y la histología de células Adaptado para elasmobranquios marinos

Marine  elasmobranchs are valued animal models for biomedical and genomic studies as  they are the most primitive vertebrates to have adaptive immunity ...