Nos gustaría agradecer las contribuciones de Dörthe Jülich, Ronda de Jennifer y Andrew Mara en el desarrollo de este protocolo. Apoyo a la investigación proporcionada por el NICHD, la Sociedad Americana del Cáncer y la Fundación March of Dimes.

1. FIJACIÓN <ol><li> Fix embriones noche a 4 ° C con 4% paraformaldehído (PFA) en PBS. </li><li> Quitar arreglar y lavar 2 x PBS, 5 minutos cada uno, a temperatura ambiente (RT). </li><li> Embriones manualmente dechorionate en PBS en una placa de vidrio depresión usando pinzas de relojero. Después de dechorionation, los embriones se transfieren por medio de un pulido al fuego pipeta Pasteur de vidrio, ya que puede adherirse a las pipetas de polipropileno. </li><li> La transferencia de embriones a través de una serie de 25%, 50% y 75% de metanol en PBS durante 5 minutos cada uno. </li><li> Sustituir el líquido con metanol al 100%, incubar 5 minutos y luego vuelva a colocar con metanol nuevo. </li><li> Embriones lugar a -20 ° C durante un mínimo de una hora. (Por lo general los embriones se incuban durante la noche. Estándar de hibridación in situ trabajará con embriones almacenados durante más de un año, pero que no han examinado si la alta resolución de la hibridación fluorescente in situ se pierde por un almacenamiento prolongado.) </li><li> Lavado de embriones de 5 minutos cada uno en el 75%, 50%, 25% de metanol en PBST a temperatura ambiente. Lavar dos veces durante 5 minutos cada uno en PBST a temperatura ambiente. </li><li> Fijar de nuevo durante 20 minutos en PFA al 4% en PBS a temperatura ambiente. </li><li> Lavar dos veces durante 5 minutos cada uno en PBST a temperatura ambiente. </li></ol> NOTA CON RESPECTO A PFA: Nosotros guardamos la PFA al 4% en alícuotas a -20 º C (ver tabla de reactivos). Para la fijación inicial, sólo usamos PFA que no haya sido previamente descongelado. Para las fijaciones posteriores, a menudo utilizamos PFA que ha sido previamente descongelado. La fijación inicial parece ser crítica para la tinción de éxito con este protocolo. 2. Proteinasas y postfijación <ol><li> Digerir con proteinasa K (5μg/ml en PBST) a temperatura ambiente durante 3 a 12 minutos para permeabilizar los embriones. (El tiempo de incubación depende de la edad de los embriones más jóvenes las etapas son más sensibles. También depende de los lotes de la enzima.) Para los embriones etapa somitogenesis, por lo general permeabilizar durante 3-4 minutos. Durante la incubación, ponemos el tubo de microcentrífuga de su lado. </li><li> Enjuague brevemente en PBST y lavar una vez por 5 minutos en PBST. </li><li> Fijar de nuevo durante 20 minutos en PFA al 4% en PBS a temperatura ambiente. </li><li> Lavar dos veces, durante 5 minutos cada uno, en PBST a temperatura ambiente. </li></ol> 3. Prehibridación <ol><li> Incubar los embriones durante 5 minutos a 65 º C en HYB. </li><li> Prehybridize a 65 º C durante al menos 1 hora en HYB +. </li></ol> 4. HIBRIDACIÓN <ol><li> Eliminar todos menos 50 l de la preHYB, pero asegúrese de mantener los embriones completamente sumergido. </li><li> Agregue 1-2 l de cada riboprobe (digoxigenina y fluoresceína riboprobes) de los embriones y mezclar agitando suavemente el tubo. La cantidad de sonda suele ser de 1 2μl de una reacción de síntesis de la sonda 20μl. </li><li> Dado que la fluoresceína es sensible a la luz, los tubos deben ser envueltos en papel de aluminio o de lo contrario expone a la luz mínima a partir de ahora. </li><li> Incubar los embriones durante la noche a 65 º C. </li></ol> 5. SONDA DE RETIRO Tenga en cuenta que las soluciones a partir de este punto de la falta de detergente adelante. Eliminación de detergente parece ayudar a las reacciones de tinción, pero hace que el embrión para convertirse en algo pegajoso. <ol><li> Retire la riboprobe. </li><li> Lavado de 2 x 30 minutos a 65 º C en el 50% formamide/2xSSC. </li><li> Lávese las manos por 15 minutos a 65 º C en 2 x SSC. </li><li> Lavar por 30 minutos a 65 º C en 0,2 x SSC. </li></ol> 6. Anti-fluoresceína ANTICUERPOS INCUBACIÓN <ol><li> Bloque de al menos 1 hora a temperatura ambiente en 500μl de una solución de 1x tampón ácido maleico más reactivo 2% de bloqueo (véase la tabla de los reactivos). </li><li> Añadir el anticuerpo anti-fluoresceína-POD, suministrado por Roche, en una dilución de 1:500 en la solución de bloqueo. </li><li> Incubar toda la noche a 4 º C. Durante la incubación, ponemos el tubo de microcentrífuga de su lado. </li><li> Lavar 4 x 20 minutos en tampón 1x ácido maleico. Lavar dos veces durante 5 minutos cada uno en PBS. </li></ol> 7. DETECCIÓN DE fluoresceína ETIQUETA DE SONDA <ol><li> Incubar 30-60 minutos en TSA más una solución de fluoresceína. (Decantar sustrato TSA antes de tomar la solución de tinción. Para la reacción, se diluye el reactivo tyramide 1:50 en Perkin Elmer diluyente tampón de amplificación.) Durante esta incubación, colocar el tubo de microcentrífuga de su lado. El tiempo de reacción debe ser determinada empíricamente para cada sonda. Por desgracia, la reacción de tinción no se puede controlar visualmente que el sustrato es fluorescente, y se verá fluorescencia verde en todas partes a lo largo de la reacción de tinción. </li><li> Lávese las manos por 10 minutos cada uno en el 30%, 50%, 75% y 100% de metanol en PBS. </li><li> Se incuban en una solución de 1% de H 2 0 2 en metanol durante 30 minutos para inactivar la peroxidasa en primer lugar. </li><li> Lavado de 10 minutos cada uno en el 75%, 50% y 30% de metanol en PBS. A continuación, lavar dos veces durante 10 minutos cada uno en PBS. Es importante que todos se reunieronhanol ser eliminado. </li></ol> NOTAS SOBRE EL amplificación de la señal tiramida: Hemos estado utilizando los kits de Perkin Elmer TSA. Nos encontramos con que los sustratos Alexa-tiramida tiñen bien con el Perkin Elmer amplificación buffer diluyente, pero no hemos tenido éxito en el uso de la tinción de amortiguación siempre con los kits de sondas Invitrogen / Molecular. Por último, hemos encontrado que Cy5 fluorescencia se elimina por posteriores metanol / H 2 O 2, mientras que el tratamiento con fluoresceína y Alexa 647-no se ven afectadas. Cy3 también puede verse afectado negativamente por el metanol / H 2 O 2 tratamiento, ya que está relacionada estructuralmente con Cy5. Por esta razón, Cy3 y Cy5 reacciones TSA sólo debe ser utilizado para la reacción de tinción segundo de una doble fluorescente in situ protocolo. 8. ANTICUERPOS ANTI-digoxigenina INCUBACIÓN <ol><li> Bloquear los embriones de nuevo por lo menos 1 hora a temperatura ambiente en una solución de 1x tampón ácido maleico más el 2% reactivo de bloqueo. </li><li> Añadir el anticuerpo anti-DIG POD suministrado por Roche en una dilución 1:1000 en solución de bloqueo por encima de </li><li> Incubar toda la noche a 4 º C. Durante la incubación, ponemos el tubo de microcentrífuga de su lado. </li><li> Lavar 4 x 20 minutos en tampón 1x ácido maleico. Lavar dos veces durante 5 minutos cada uno en PBS. </li></ol> 9. DETECCIÓN DE digoxigenina-ETIQUETA DE SONDA <ol><li> Incubar 30-60 minutos en TSA Además Cy5 solución (Decantar sustrato TSA antes de tomar la solución de tinción. Para la reacción, se diluye el reactivo tyramide 1:50 en tampón de amplificación diluyente) Durante esta incubación, colocar el tubo de microcentrífuga de su lado. El tiempo de reacción debe ser determinada empíricamente para cada sonda. </li><li> Lavar tres veces durante 10 minutos cada uno en PBST. </li></ol> 10. Tinción con yoduro de propidio <ol><li> Lavar dos veces durante 5 minutos cada uno en 2 x SSC. </li><li> Incubar los embriones durante 30 minutos a 37 ° C en 50 l SSCT 2 x con 10 l de RNasa (para una concentración final de 100μg/ml). </li><li> Lávese las seis veces durante 3 minutos cada uno en 2 x SSC a temperatura ambiente. </li><li> Tinción de embriones durante 8 minutos en una solución de yoduro de propidio 330μg/ml en 2 x SSC. </li><li> Lávese las seis veces durante 3 minutos cada uno en 2 x SSC a temperatura ambiente. </li><li> Fijar durante 20 minutos en PFA al 4% en PBS a temperatura ambiente. </li><li> Lavar dos veces, durante 5 minutos cada uno, en PBST a temperatura ambiente. </li></ol> 11. MONTAJE <ol><li> Incubar los embriones durante 10 minutos cada uno en el 25% y 50% de glicerol en PBST. Borrar toda la noche en el 75% de glicerol a 4 º C. </li><li> Nos diseccionar y deyolk los embriones y plano que se montan en un portaobjetos de microscopio. La yema puede producir fluorescencia de fondo importantes. La disección es mucho más fácil después de que los embriones hayan desaparecido ovenight en gylcerol. </li></ol> SOLUCIONES: <table border="1"><tbody><tr><td> PBST </td><td> PBS más 0,1% de Tween </td><td></td></tr><tr><td> SSCT </td><td> SSC más el 0,1% de Tween </td><td></td></tr><tr><td> HYB- </td><td> Formamida al 50% 5xSSC 0,1% de Tween-20 </td><td></td></tr><tr><td> HYB + </td><td> HYB- 5mg/ml torula (levadura) de ARN 50μg/ml heparina </td><td style="width: 300px;"> El ARN torula es preparado por digestión con proteinasa K de ARN, con la consiguiente fenol, fenol-cloroformo y cloroformo-extraction.The ARN se precipita y se disuelven en agua tratada con DEPC. </td></tr><tr><td> 1 x tampón ácido maleico </td><td> 150 mM de ácido maleico, 100 mM NaCl (pH 7,5) </td><td></td></tr></tbody></table><img src="/files/ftp_upload/1229/Brend_Holley_Fig1.png" alt="Figura 1" width="600" height="688" /> Figura 1. Los resultados representativos del monte se doble hibridación in situ fluorescente. (AC). Las imágenes muestran una sola sección confocal a través de la región posterior de un embrión de pez cebra en la etapa de diez somite. La vista es dorsal anterior a la cima. Núcleos teñidos con yoduro de propidio son de color azul. (A) mRNA deltaC se detectó mediante un riboprobe digoxigenina marcado y el reactivo de TSA-Cy5. (B) ARNm transcrito a partir del gen HER1 se detectó con un riboprobe marcado con fluoresceína y reactivo TSA-fluoresceína. (C) La fusión de los canales verde y rojo identifica de forma inequívoca las regiones de la expresión de genes distintos o superposición de los dos genes. (D, E) Detalle de HER1 expresión que demuestra la alta resolución de este procedimiento. Localización subcelular de ARNm se puede discernir con claridad. Puntas de flecha indican una célula exhibe transcripción activa, revelada por los puntos de la tinción en el núcleo. Las flechas indican la celda que muestra la localización citoplasmática del ARNm. (F, G) Doble tinción de HER1 y deltaC revela que las células son la transcripción de los genes (punta de flecha blanca), o cualquiera de los genes por separado (puntas de flecha de color rojo y verde).

<ol>
	<li>Schulte-Merker, S., Ho, R. K., Herrmann, B. G., N&#252;sslein-Volhard, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+protein+product+of+the+zebrafish+homologue+of+the+mouse+T-gene+is+expressed+in+nuclie+of+the+germ+ring+and+the+notochord+of+the+early+embryo.">The protein product of the zebrafish homologue of the mouse T-gene is expressed in nuclie of the germ ring and the notochord of the early embryo.</a> Development. 116, 1021-1027 (1992).</li><li>Jowett, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double+in+situ+hybridization+techniques+in+zebrafish.">Double in situ hybridization techniques in zebrafish.</a> Methods. 23, 345-358 (2001).</li><li>J&#252;lich, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=beamter/deltaC+and+the+role+of+Notch+ligands+in+the+zebrafish+somite+segmentation,+hindbrain+neurogenesis+and+hypochord+differentiation.">beamter/deltaC and the role of Notch ligands in the zebrafish somite segmentation, hindbrain neurogenesis and hypochord differentiation.</a> Dev Biol. 286, 391-404 (2005).</li><li>Mara, A., Schroeder, J., Chalouni, C., Holley, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Priming,+Initiation+and+Synchronization+of+the+Segmentation+Clock+by+deltaD+and+deltaC.">Priming, Initiation and Synchronization of the Segmentation Clock by deltaD and deltaC.</a> Nat Cell Biol. 9, 523-530 (2007).</li><li>Lecuyer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+analysis+of+mRNA+localization+reveals+a+prominent+role+in+organizing+cellular+architecture+and+function.">Global analysis of mRNA localization reveals a prominent role in organizing cellular architecture and function.</a> Cell. 131, 174-187 (2007).</li></ol>

El protocolo presentado aquí funciona bien con las sondas que dar una señal fuerte limpia después de la tinción de 30-45 minutos en una fosfatasa alcalina típica mediada por la reacción. Antes de realizar la fluorescencia en el protocolo de hibridación in situ, siempre prueba de nuestras sondas utilizando el estándar de protocolo no fluorescente (siempre en el material complementario junto con el protocolo de síntesis de la sonda). Hemos tenido menos éxito con los fluorescentes en el protocolo de hibridación ...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Paraformaldehyde 16% solution, EM Grade</td>
<td>&nbsp;</td>
<td>Electron Microscopy Services</td>
<td>15710</td>
<td>Dilute to 4% in 1.33x PBS (final 1x). Store in 2ml aliquots at &#x2013;20&deg;C</td>
</tr>
<tr>
<td>Proteinase K, recombinant PCR grade</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>03115879001</td>
<td>Make a 20mg/ml stock solution in water. Store in 1ml aliquots at &#x2013;20&deg;C</td>
</tr>
<tr>
<td>Blocking Reagent</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11096176001</td>
<td>Make a 10% stock solution in 1x maleic acid buffer. Store in 50ml aliquots at &#x2013;20&deg;C. Stable over multiple freeze/thaws.</td>
</tr>
<tr>
<td>Anti-Fluorescein-POD, Fab fragments</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11426346910</td>
<td>Aliquot and store at -20&deg;C. Keep one working aliquot at 4&deg;C.</td>
</tr>
<tr>
<td>Anti-Digoxigenin-POD, Fab fragments</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11207739910</td>
<td>As above.</td>
</tr>
<tr>
<td>TSA Plus Fluorescein system</td>
<td>&nbsp;</td>
<td>Perkin Elmer</td>
<td>NEL741001KT</td>
<td>Prepare each vial of TSA reagent only when needed. Dissolve in 60&mu;l DMSO. Store at 4&deg;C.</td>
</tr>
<tr>
<td>TSA Plus Cyanine5 system</td>
<td>&nbsp;</td>
<td>Perkin Elmer</td>
<td>NEL745001KT</td>
<td>As above</td>
</tr>
<tr>
<td>TSA Plus Cyanine3 system</td>
<td>&nbsp;</td>
<td>Perkin Elmer</td>
<td>NEL744001KT</td>
<td>As above</td>
</tr>
<tr>
<td>TSA Kit #16 AlexaFluor647 tyramide (plus HRP-goat-anti-rabbit IgG)</td>
<td>&nbsp;</td>
<td>Molecular Probes /Invitrogen</td>
<td>T20926</td>
<td>Dissolve tyramide reagent in 150&mu;l DMSO, aliquot and store at &#x2013;20&deg;C.</td>
</tr>
<tr>
<td>RNase, DNase free</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11119915001</td>
<td>Supplied as 500&mu;g/ml solution</td>
</tr>
<tr>
<td>Propidium Iodide</td>
<td>&nbsp;</td>
<td>Molecular Probes /Invitrogen</td>
<td>P-3566</td>
<td>Supplied as 1mg/ml solution in water.</td>
</tr>		</tbody>
		</table>
		</div>

zebrafish whole mount high-resolution double fluorescent in situ hybridization

Todo el montaje<em&gt; In situ</emHibridación&gt; es una de las técnicas más utilizadas en la biología del desarrollo. Aquí se presenta una alta resolución de doble fluorescente<em&gt; In situ</em&gt; Hibridación protocolo para el análisis del patrón de expresión precisa de un solo gen y para determinar la superposición de los dominios de expresión de dos genes. El protocolo es una versión modificada de la norma<em&gt; In situ</em&gt; Hibridación con fosfatasa alcalina y los sustratos tales como NBT / BCIP y Fast Red<sup&gt; 1,2</sup&gt;. Este protocolo utiliza sondas estándar digoxigenina y fluoresceína junto a la amplificación de la señal tyramide (TSA)<sup&gt; 3</sup&gt;. Los kits disponibles en el mercado TSA permite un diseño flexible experimental como la emisión de fluorescencia de color verde a rojo lejano se puede utilizar en combinación con diferentes manchas nucleares, como el yoduro de propidio, o inmunohistoquímica de fluorescencia de las proteínas. TSA produce un sustrato reactivo fluorescente que rápidamente se une covalentemente a restos, los residuos de tirosina general, en las inmediaciones de la riboprobe antisentido marcado. Los patrones de tinción resultantes son de alta resolución en que la localización subcelular de los ARNm se puede observar con microscopía confocal de barrido láser<sup&gt; 3,4</sup&gt;. Se puede observar transcripciones naciente en los loci cromosómicos, distinguir la tinción nuclear y citoplasmática y visualizar los patrones de otros, tales como la localización cortical del ARNm. Los estudios realizados en<em&gt; Drosophila</em&gt; Indican que aproximadamente el 70% de los ARNm muestran patrones específicos de la localización subcelular de que con frecuencia se correlacionan con la función de la proteína codificada<sup&gt; 5</sup&gt;. Cuando se combina con la reconstrucción por ordenador de conjuntos de datos 3D confocal, el protocolo permite el análisis detallado de la distribución del ARNm con resolución subcelular en embriones de los vertebrados conjunto.

Watch this Scientific Journal Video about Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization at JoVE.com

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Todo el montaje hibridación in situ es una de las técnicas más utilizadas en la biología del desarrollo. Aquí se presenta una alta resolución de doble fluorescente en el protocolo de hibridación in situ para analizar el patrón de expresión precisa de un solo gen y para determinar la superposición de los dominios de expresión de dos genes. Se incluye un yoduro de propidio nuclear tinción de contraste para destacar la organización del tejido.

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 ...

zebrafish-whole-mount-high-resolution-double-fluorescent-situ

Research

JoVE Journal

Biology

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

23.1K vistas.  Yale University. Todo el montaje hibridación in situ es una de las técnicas más utilizadas en la biología del desarrollo. Aquí se presenta una alta resolución de doble fluorescente en el protocolo de hibridación in situ para analizar el patrón de expresión precisa de un solo gen y para determinar la superposición de los dominios de expresión de dos genes. Se incluye un yoduro de propidio nuclear tinción de contraste para destacar la organización del tejido.

Video: Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

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

Method for Whole Mount Antibody Staining in Chick

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 antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

Método para la tinción de anticuerpos enteros en el monte del polluelo

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of specific mRNA transcripts within whole mount specimen. This technique (adapted from Albertson and Yelick, 2005) was used in an upper level undergraduate Comparative Vertebrate Biology laboratory classroom at Syracuse University. The first two thirds of the Comparative Vertebrate Biology lab course gave students the opportunity to study the embryology and gross anatomy of several organisms representing various chordate taxa primarily via traditional dissections and the use of models. The final portion of the course involved an innovative approach to teaching anatomy through observation of vertebrate development employing molecular techniques in which WISH was performed on zebrafish embryos. A heterozygous fibroblast growth factor 8 a (fgf8a) mutant line, ace, was used. Due to Mendelian inheritance, ace intercrosses produced wild type, heterozygous, and homozygous ace/fgf8a mutants in a 1:2:1 ratio. RNA probes with known expression patterns in the midline and in developing anatomical structures such as the heart, somites, tailbud, myotome, and brain were used. WISH was performed using zebrafish at the 13 somite and prim-6 stages, with students performing the staining reaction in class. The study of zebrafish embryos at different stages of development gave students the ability to observe how these anatomical structures changed over ontogeny. In addition, some ace/fgf8a mutants displayed improper heart looping, and defects in somite and brain development. The students in this lab observed the normal development of various organ systems using both external anatomy as well as gene expression patterns. They also identified and described embryos displaying improper anatomical development and gene expression (i.e., putative mutants).
For instructors at institutions that do not already own the necessary equipment or where funds for lab and curricular innovation are limited, the financial cost of the reagents and apparatus may be a factor to consider, as will the time and effort required on the part of the instructor regardless of the setting. Nevertheless, we contend that the use of WISH in this type of classroom laboratory setting can provide an important link between developmental genetics and anatomy. As technology advances and the ability to study organismal development at the molecular level becomes easier, cheaper, and increasingly popular, many evolutionary biologists, ecologists, and physiologists are turning to research strategies in the field of molecular biology. Using WISH in a Comparative Vertebrate Biology laboratory classroom is one example of how molecules and anatomy can converge within a single course. This gives upper level college students the opportunity to practice modern biological research techniques, leading to a more diversified education and the promotion of future interdisciplinary scientific research.

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Whole mount in situ hybridization (WISH) was used in an upper level undergraduate Comparative Vertebrate Biology course in addition to vertebrate dissections. This gave students the opportunity to study gene expression patterns as well as gross anatomy, linking the study of molecular and organismal biology within one course.

Todo el montaje utilizando In situ La hibridación de Enlace de Organismos y Biología Molecular

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of ...

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures are lengthy and technically demanding with multiple important steps that collectively contribute to the quality of the final result. This protocol describes in detail several key quality control steps for optimizing probe labeling and performance. Overall, our protocol provides a detailed description of the critical steps necessary to reproducibly obtain high quality results. First, we describe the generation of digoxygenin (DIG) labeled RNA probes via in vitro transcription of DNA templates generated by PCR. We describe three critical quality control assays to determine the amount, integrity and specific activity of the DIG-labeled probes. These steps are important for generating a probe of sufficient sensitivity to detect endogenous mRNAs in a whole mouse embryo. In addition, we describe methods for the fixation and storage of E8.5-E11.5 day old mouse embryos for in situ hybridization. Then, we describe detailed methods for limited proteinase K digestion of the rehydrated embryos followed by the details of the hybridization conditions, post-hybridization washes and RNase treatment to remove non-specific probe hybridization. An AP-conjugated antibody is used to visualize the labeled probe and reveal the expression pattern of the endogenous transcript. Representative results are shown from successful experiments and typical suboptimal experiments.

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

This whole mount in situ hybridization protocol discusses critical steps that ensure reproducible high quality results for gene expression studies in E8.5-E11.5 day old mouse embryos.

Todo el monte In Situ La hibridación de E8.5 a E11.5 embriones de ratón

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures ...

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 ...

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression both in terms of tissue distribution and developmental stage. Protocols are described for the use of WISH of zebrafish embryos using antisense RNA probes labeled with digoxigenin. Probes are generated by incorporating digoxigenin-linked nucleotides through in vitro transcription of gene templates that have been cloned and linearized. The chorions of embryos harvested at defined developmental stages are removed before incubation with specific probes. Following a washing procedure to remove excess probe, embryos are incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase. By employing a chromogenic substrate for alkaline phosphatase, specific gene expression can be assessed. Depending on the level of gene expression the entire procedure can be completed within 2-3 days.

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

The zebrafish, a small tropical fish, has become a popular model for studying gene function during vertebrate development and disease. The temporal and spatial expression of target genes can be determined by in situ hybridization. Our improved protocol allows for the detection of low abundant transcripts with low non-specific background signal.

Alta resolución todo el montaje de<em&gt; In Situ</em&gt; La hibridación en embriones de pez cebra para estudiar la expresión génica y la función

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression ...

Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. The whole-mount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning and/or colabeling is ultimately required to pinpoint the exact cell types undergoing apoptosis. The whole-mount Casp3 assay is optimized for analysis of fixed embryos between the 4-cell stage and 32 hr-post-fertilization and is useful for a number of applications, including analysis of zebrafish mutants and morphants, overexpression of mutant and wild-type mRNAs, and exposure to chemicals. Compared to acridine orange staining, which can identify apoptotic cells in live embryos in a matter of hours, Casp3 and TUNEL assays take considerably longer to complete (2-4 days). However, because of the dynamic nature of apoptotic cell formation and clearance, analysis of fixed embryos ensures accurate comparison of apoptotic cells across multiple samples at specific time points. We have also found the Casp3 assay to be superior to analysis of apoptotic cells by the whole-mount TUNEL assay in regard to cost and reliability. Overall, the Casp3 assay represents a robust, highly reproducible assay in which to analyze apoptotic cells in early zebrafish embryos.

Certain genetic perturbations or exposure to toxins can disrupt normal developmental processes leading to death of specific cell types. The analysis of activated Caspase 3 by whole-mount immunofluorescence in zebrafish embryos reveals stage- and tissue-specific localization of cells specifically undergoing apoptosis.

Análisis de la apoptosis en embriones de pez cebra en Todo el montaje de inmunofluorescencia para detectar la caspasa 3 activada

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis ...