DP es receptor de Ruth Premio Kirschstein DA021977 1F32-01A1 del Instituto Nacional sobre Abuso de Drogas. Este trabajo fue apoyado por la Fundación Margaret M. Alkek a RHF.

<html> I. Esquema general: <img src="/files/ftp_upload/old/1027_2009_1_22_2/Figure1.png" alt="Figura 1" width="460" height="384" /> Este video muestra las diferentes etapas de ensayo para la inducción neural en el embrión de pollo. En primer lugar, el embrión de acogida es explantados en la cultura de Nuevo [NI1]. Luego, el embrión es donnor explantados en solución salina y el nodo de Hensen (el &quot;organizador&quot; de pollo) está marcada con fluorescente DII colorante [NI2]. Nodo de Hensen se extirpa el embrión a partir de donnor [NI3] y se trasplantan en el embrión de acogida [NI4, NI5]. El embrión de acogida luego se cultiva durante un máximo de 22 horas, después de los cuales el tejido donnor ha inducido a un eje ectópico en la zona de acogida opaca [NI6]. A raíz de la cultura, el embrión de acogida se fija y se procesaron para fotooxidación en presencia de DAB: En esta reacción química, las células donnor derivados, que aparecen de color rojo con fluorescencia FITC antes de la fotooxidación [NI7], se convierten en reacción marrón siguiente con DAB [NI8]. El embrión es luego procesada por hibridación in situ con un marcador neural [NI8]; tejido del huésped derivados se ha formado el tejido nervioso en presencia del injerto donnor. Este tejido se ve azul. 1: Preparar el embrión de acogida para la cultura de Nueva <ol><li> Este protocolo de inducción neural ensayo comienza con los huevos que se incuban en Hamburger y Hamilton etapa 3 + (o HH3 +). Estos huevos han sido incubados establecidas a su favor en una incubadora húmeda durante 13 horas. </li><li> Preparar el embrión de acogida para la cultura de Nueva (medidas 3.1. A 5.5 9). </li><li> Cubra la superficie del embrión anfitrión con 200 l de solución salina. Esto facilitará la posterior traslado y colocación del injerto donnor. </li><li> Por último, cubrir el conjunto con un invertido Falcon 35 mm placa de cultivo. </li></ol> 2: explantar el embrión donnor en solución salina <ol><li> Abrir el huevo tocando en la cubierta con unas pinzas y la eliminación de piezas de la cáscara, de todo el shell. Retire la parte superior de la concha y el descarte. </li><li> Eliminar la albúmina de espesor con pinzas, y la inclinación del saco vitelino con pinzas gruesas para que el embrión hacia arriba. </li><li> Con la tijera fina, cortar un cuadrado de alrededor del saco vitelino del embrión. Extraer el embrión de la yema de huevo con una cuchara, y colocar en un recipiente lleno de PBS. </li><li> Con unas pinzas, separar el embrión de la zona pelúcida donnor. </li><li> La transferencia de embriones donnor a un plato cubierto Sylgard contiene PBS. </li></ol> 3: El etiquetado, la escisión y el trasplante del injerto donnor <ol><li> El uso de un extractor de microelectrodos, tire 50 pipetas de vidrio microcapilar l. Bajo el microscopio, cortar la punta de la micropipeta con unas pinzas finas. Micropipeta lugar en una placa de Petri llenas de una cinta de plasteline. </li><li> Prepare la solución de colorante que se utiliza para etiquetar el injerto antes del trasplante: carbocyanine tinte DII (1,1 &#39;-dioctadecil-3, 3,3&#39;, 3&#39;-tetramethylindocarbocyanine perclorato): En primer lugar, preparar un inventario de DII en 1 ml etanol absoluto en un 0,5%. Este archivo puede ser almacenado a-20C en la oscuridad. En un baño de agua a 42 ° C, mezclar 40 ml precalentado acciones DII a 360μl 0,3 M de sacarosa. La temperatura del agua es necesaria para prevenir la precipitación de DII y la formación de cristales insolubles. En pocas palabras girar la solución en un spectrafuge mini (5-10 segundos). La solución DII ya está listo para su uso. </li><li> El uso de clavos de insectos, seguro que el embrión donnor en la parte inferior del plato Sylgard cubiertos. </li><li> Volver a llenar la micropipeta con DII. Aplicando una presión suave con un conjunto de tubo aspirador, aplicar un pequeño bolo de DII en el nodo de Hensen. Asegúrese de que todo el nodo de Hensen está etiquetado. </li><li> Con una pipeta microcapilar o un cuchillo microdissecting, corte nodo de Hensen (&quot;organizador&quot;, injerto donnor). </li><li> Marque la parte ventral y el extremo posterior del injerto con carmín. Esto asegurará que el injerto se coloca con una adecuada orientación dorso-ventral en el paso 4.3. </li><li> Usando una pipeta Gilson 200μl, la transferencia del injerto al embrión de acogida. </li></ol> 4: Asegurar el injerto y el cultivo de los embriones de acogida <ol><li> Quitar salina de embrión de acogida, asegurándose de que el injerto se mantiene posicionado en las proximidades de un sitio de acogida. </li><li> Con una pipeta microcapilar o un cuchillo microdissecting, hacer una pequeña incisión (80-100μm) en la zona pelúcida / área región de la frontera opaca, a nivel del nodo de Hensen del embrión huésped. </li><li> Con una pipeta microcapilar o posición de la cuchilla microdissecting, el injerto de modo que la parte ventral está orientada hacia usted y el extremo posterior es paralela a la región equivalente en el embrión de acogida. </li><li> Quitar salina restantes, y el proceso de acogida para el embrión nueva cultura de 81-22 h (6,1 a 6,6 pasos 9). </li><li> El embrión de acogida se fija la noche a 4 ° C (7,1 a 7,7 pasos 9). </li></ol>e &quot;&gt; 5: Photooxidise el injerto en la fluorescencia <ol><li> Bajo campana extractora de humos, preparar una solución de trabajo DAB en tampón Tris: Disolver sustrato DAB (3,3 &#39;-diaminobenzidina tetrahidrocloruro) en tampón Tris (100 mM Tris-HCl, pH 7,4) en 500μg/ml; mantener la solución en la oscuridad, en el hielo. </li><li> Lavar el embrión dos veces en PBS durante 5 minutos cada uno y una vez en tampón Tris de 5 minutos. </li><li> Transferencia del embrión a la cavidad de una diapositiva de vidrio que contiene 1 ml de tampón Tris. </li><li> Vuelva a colocar la solución tampón Tris con 1,5 ml de solución de DAB. Disponer de Eppendorf punta en un cubo que contiene una solución de lejía al 10% con el fin de descontaminar el DAB. Coloque un cubreobjetos en la parte superior de la cavidad de diapositivas. </li><li> Bajo FITC (isotiocianato de fluoresceína) de fluorescencia, el enfoque del objetivo del microscopio en el injerto (células donnor aparecerá de color rojo fluorescente) y exponer a la fluorescencia FITC hasta que todas las células teñidas de rojo fluorescente se han convertido en marrón (esto ocurre como resultado de la DII fluorocromo se photoconverted insoluble para cristales de color marrón, por la exposición a la longitud de onda de excitación FITC (488 nm) en presencia de DAB. Este proceso tarda entre 20 minutos y 2 horas. </li><li> Tras la finalización de la reacción de fotooxidación, quitar el cubreobjetos con unas pinzas finas. La transferencia del embrión a un vial de centelleo de vidrio de 20 ml que contiene PBTw (o PBS con 0,1% de Tween-20). </li><li> Desactivar DAB de cubreobjetos y portaobjetos de vidrio mediante la incubación de la cavidad en la solución de lejía al 10%. </li></ol> 6: Proceso de embrión de hibridación in situ con la fotografía, y la sección <ol><li> Proceder a la hibridación in situ (3,7 a 6,5 pasos 9), la incubación del embrión sólo DIG sonda marcada. </li><li> Proceder con fotografía (paso 8.2 9) y la sección (medidas 8.3 y 9 8.4). </li></ol> Resultados representante En el siguiente ejemplo de ensayo de inducción neural, el injerto se muestra donnor inducir la expresión del marcador neuronal sin cola a partir de tejido del huésped derivados. En este ejemplo particular, se evaluó la adquisición de la capacidad de inducir neural de las regiones de embriones que normalmente no tienen &quot;organizador&quot; habilidades: el injerto donnor se origina a partir de un embrión en el que ha sido el &quot;organizador&quot; región de la extirpación quirúrgica del embrión en la etapa de donnor 3 +. Después de la ablación, el embrión donnor se le permite desarrollarse en la cultura nueva, hasta que el agujero se ha curado y una estructura parecida a nodo de Hensen se ha formado. Esta estructura se denominan entonces con DII, eliminado del embrión donnor y se injerta a la zona pelúcida / zona de la frontera opaca de un embrión de acogida. A raíz de la nueva cultura, un eje en miniatura se forma adyacente a la sede de embriones: este eje en miniatura consiste en una varilla de glóbulos rojos (donnor derivados) y una estructura similar a la cabeza (host derivados): En (A), donnor derivados DII etiquetado las células se muestran antes de la fijación bajo microscopía de fluorescencia FITC. Estas células donnor derivados se han multiplicado para formar una miniatura notocorda, que aparece como barra de glóbulos rojos. (B) fotoconversión Después de DII, estas células donnor derivados de un color marrón. El injerto donnor ha inducido la formación de tejido nervioso desde el host: este tejido se compone de los precursores para el cerebro, según lo revelado por su expresión para los siguientes marcadores sin cola de hibridación in situ [reproducido a partir de 6]. <img src="/files/ftp_upload/old/1027_2009_1_22_3/Figure 2.png" alt="la figura 2" width="230" height="154" /> Nota: En este procedimiento, se han utilizado células DII etiquetados con el fin de diferenciar los tejidos induciendo neural donnor (rojo / marrón) del host derivados &quot;neural&quot; del tejido (azul). En lugar de utilizar una chica derivados, injerto DII etiquetados donnor, algunos autores también utilizan tejidos donnor derivadas de embriones de codorniz en vez de pollo [por ejemplo, 6]. Este método alternativo también permite diferenciar donnor de tejido del huésped. En este caso, el injerto (tejido donnor) se origina a partir de huevos de codorniz en vez de huevos de gallina. Siguiendo el paso 4.5, el anfitrión es procesadas para inmunohistoquímica montar todo con un anticuerpo específico para el tejido de codorniz (QCP1) y los pasos 3,1 a 3,4 y 5,1 a 5,7 se omiten en el protocolo. Un ejemplo de este procedimiento alternativo es el siguiente: En este caso, se utilizó el mismo procedimiento experimental como en A, B, pero usamos la codorniz tejido derivado donnor (como se muestra en la expresión de anticuerpos QCPN [negro]): La regeneración nodo derivadas de embriones de codorniz ha inducido la expresión de la pan-neuronal marcador Sox2 en el host [reproducido a partir de 6]: <img src="/files/ftp_upload/old/1027_2009_1_22_4/Figure 3.png" alt="Figura 3" width="154" height="230" />

<ol>
	<li>Waddington, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experiments+on+the+development+of+chick+and+duck+embryos,+cultivated+in+vitro.">Experiments on the development of chick and duck embryos, cultivated in vitro.</a> Phil. Trans. R. Soc. Lond. B. 221, 179-230 (1932).</li><li>Waddington, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+by+the+primitive+streak+and+its+derivatives+in+the+chick.">Induction by the primitive streak and its derivatives in the chick.</a> J. Exp. Biol. 10, 38-46 (1933).</li><li>Gallera, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excision+et+transplantation+des+diff&amp;eacute;rentes+r&amp;eacute;gions+de+la+ligne+primitive+chez+le+poulet.">Excision et transplantation des diff&amp;eacute;rentes r&amp;eacute;gions de la ligne primitive chez le poulet.</a> C. R. Ass. Anat. 49, 632-639 (1964).</li><li>Gallera, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+induction+in+birds.">Primary induction in birds.</a> Adv. Morph. 9. , 149-180 (1971).</li><li>Serbedzija, G. N., Fraser, S. E., Bronner-Fraser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathways+of+neural+crest+cell+migration+in+the+mouse+embryo+as+revealed+by+vital+dye+labeling.+Development+108.">Pathways of neural crest cell migration in the mouse embryo as revealed by vital dye labeling. Development 108.</a> , 605-612 (1990).</li><li>Psychoyos, D., Stern, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restoration+of+the+organizer+after+radical+ablation+of+Hensen's+node+and+the+anterior+primitive+streak+in+the+chick+embryo.+Development+122.">Restoration of the organizer after radical ablation of Hensen's node and the anterior primitive streak in the chick embryo. Development 122.</a> , 3263-3273 (1996).</li><li>Joubin, K., Stern, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+interactions+continuously+define+the+organizer+during+the+cell+movements+of+gastrulation.">Molecular interactions continuously define the organizer during the cell movements of gastrulation.</a> Cell. 98, 559-571 (1999).</li><li>Streit, A., Berliner, A. J., Papanayotou, C., Sirulnik, A., Stern, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiation+of+neural+induction+by+FGF+signaling+before+gastrulation.">Initiation of neural induction by FGF signaling before gastrulation.</a> Nature. , 406-474 (2000).</li><li>Psychoyos, D., Finnell, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+Culture+of+Early+Chick+Embryos+ex+vivo+(New+Culture.">Method for Culture of Early Chick Embryos ex vivo (New Culture.</a> JoVE. 20, 10-3791 (2008).</li></ol>

Este video muestra los diferentes pasos en la realización de un ensayo para la inducción neural; Este ensayo se utiliza esencialmente para la caracterización de las moléculas de supuesta inducción neural en el pollo, y por lo tanto puede ser utilizado para una amplia variedad de aplicaciones, que van desde micromanipulaciones embriológico 1-4; 6 para desentrañar nuevas cascadas de señalización 7,8, todos con el objetivo de la comprensión de la etapa inicial en la formación del cerebro y ...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Eggs</td>
<td>Animal</td>
<td>Charles River Laboratories</td>
<td>Premium Fertile</td>
<td>Fertilized, HH3+ (14 hr)</td>
</tr>
<tr>
<td>Stereomicroscope</td>
<td>Microscope</td>
<td>Leica Microsystems</td>
<td>MZ9.5 or similar</td>
<td>&nbsp;</td>
<tr>
<td>Hybridization Incubator</td>
<td>Equipment</td>
<td>Robbins Scientific</td>
<td>M1000</td>
<td>Use with inverted Pyrex dish and 500 ml ddH2O beaker</td>
</tr>
<tr>
<td>Marsh Automatic Incubator</td>
<td>Equipment</td>
<td>Lyon</td>
<td>RX</td>
<td>&nbsp;</td>
<tr>
<td>Pyrex dish </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Watchmaker's glass 50mm</td>
<td>Tool</td>
<td>VWR</td>
<td>66112-060</td>
<td>&nbsp;</td>
<tr>
<td>Glass rings</td>
<td>Tool</td>
<td>Physical Plant facility</td>
<td>&nbsp;</td>
<td>cut 4 mm thick sections of glass tubing (27 mm outer diam, 25 mm inner diam). Do not fine polish.</td>
</tr>
<tr>
<td>Curved Forceps (1)</td>
<td>Tool</td>
<td>Electron Microscopy Sciences</td>
<td>72991-4C</td>
<td>&nbsp;</td>
<tr>
<td>Forceps (2)</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>11002-13</td>
<td>blunt ended using sharpening Stone and 100ul mineral oil</td>
</tr>
<tr>
<td>Fine scissors</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>14161-10</td>
<td>&nbsp;</td>
<tr>
<td>Plastic dishes</td>
<td>Tool</td>
<td>Falcon</td>
<td>353001</td>
<td>&nbsp;</td>
<tr>
<td>Rubber Bulb</td>
<td>Tool</td>
<td>Electron Microscopy Sciences</td>
<td>70980</td>
<td>&nbsp;</td>
<tr>
<td>DiI</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>D-282</td>
<td>&nbsp;</td>
<tr>
<td>Aspirator tube assembly</td>
<td>Tool</td>
<td>Sigma</td>
<td>A5177-5EA</td>
<td>&nbsp;</td>
<tr>
<td>Microelectrode puller</td>
<td>Equipment</td>
<td>Sutter Instruments</td>
<td>Sutter Instruments	P-97 Flaming/Brown Micropipette</td>
<td>&nbsp;</td>
<tr>
<td>Pasteur Capillary Pipette</td>
<td>Tool</td>
<td>Electron Microscopy Sciences</td>
<td>70950-12</td>
<td>round edge under flame</td>
</tr>
<tr>
<td>Culture chamber</td>
<td>Tool</td>
<td>Pioneer Plastics</td>
<td>030C</td>
<td>&nbsp;</td>
<tr>
<td>Microcapillary tube</td>
<td>Tool</td>
<td>Sigma</td>
<td>P1049-1PAK</td>
<td>&nbsp;</td>
<tr>
<td>Microdissecting knife</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>10056-12</td>
<td>Use to puncture cavities prior to in situ hybridization</td>
</tr>
<tr>
<td>Minuten pins 0.2mm diam</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>26002-20</td>
<td>Mix 1 part Curing Agent, 9 parts Base; set O/N at 37C</td>
</tr>
<tr>
<td>Diethylpyrocarbonate (depc)</td>
<td>Reagent</td>
<td>Electron Microscopy Sciences</td>
<td>15710</td>
<td>&nbsp;</td>
<tr>
<td>Sylgard 184 Silicon Elastomer Curing Agent and Base</td>
<td>&nbsp;</td>
<td>Dow Corning</td>
<td>0001986475</td>
<td>Mix 1 part Curing Agent, 9 parts Base; set O/N at 37C</td>
</tr>
<tr>
<td>Diethylpyrocarbonate (depc)</td>
<td>&nbsp;</td>
<td>Acros Organics</td>
<td>10025025</td>
<td>Add 1ml depc to 1l PBS; shake; autoclave</td>
</tr>
<tr>
<td>16% PFA</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>15710</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

assay for neural induction in the chick embryo

El embrión de pollo es una herramienta valiosa en el estudio del desarrollo embrionario temprano. Su transparencia, accesibilidad y facilidad de manipulación, lo convierten en una herramienta ideal para el estudio de la formación inicial y los patrones del sistema nervioso. Este video muestra la forma de tejido organizador del injerto en un huésped, un método por el cual nódulo de Hensen s (el organizador en el embrión de pollo) se injerta a un ectodermo competente de acogida. El injerto organizador instruye cubre na he tejido para adoptar un destino neuronal a través de señales de inducción neural. Este mecanismo se conoce como inducción neural, y constituye el paso inicial en la formación del cerebro y la médula espinal en amniotas. Este método se utiliza esencialmente para la caracterización de las moléculas de supuesta inducción neural en el pollo. Este video muestra los diferentes pasos en el ensayo de inducción neural; En primer lugar, el embrión es donnor explantados y puestas en un plato. Luego, el embrión de acogida se prepara para la nueva cultura. El injerto se extirpa y se trasplanta a la zona pelúcida margen de acogida. El anfitrión se cultiva durante 18-22 horas. El conjunto se fija y se procesaron para otras aplicaciones (por ejemplo, hibridación in situ). Este método fue concebido originalmente por Waddington<sup&gt; 1,2</sup&gt; Y la Gallera<sup&gt; 3,4</sup&gt;.

Watch this Scientific Journal Video about Assay for Neural Induction in the Chick Embryo at JoVE.com

Assay for Neural Induction in the Chick Embryo

Inducción neural es el primer paso en la formación del cerebro. Se trata de un mecanismo por el cual el nodo de Hensen (organizador), instruye a los tejidos adyacentes a la adopción de un destino neuronal, es decir, para dar lugar al sistema nervioso. Este video muestra un ensayo de inducción neural en el embrión de pollo.

Ensayo de inducción neural en el embrión de pollo

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

assay-for-neural-induction-in-the-chick-embryo

Research

JoVE Journal

Biology

17.1K vistas.  Texas A&M University (TAMU). Inducción neural es el primer paso en la formación del cerebro. Se trata de un mecanismo por el cual el nodo de Hensen (organizador), instruye a los tejidos adyacentes a la adopción de un destino neuronal, es decir, para dar lugar al sistema nervioso. Este video muestra un ensayo de inducción neural en el embrión de pollo.

Video: Assay for Neural Induction in the Chick Embryo

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

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

Electrophysiological Recording in the Drosophila Embryo

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of &lt;8 hours (at 25&deg;C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. Whole-cell patch-clamp techniques can then be employed to record from individually-identifiable neurons and somatic muscles. These recording configurations have been used to track the appearance and maturation of ionic currents and action potential propagation in both neurons and muscles. Genetic mutants affecting these electrical properties have been characterized to reveal the molecular composition of ion channels and associated signaling complexes, and to begin exploration of the molecular mechanisms of functional differentiation. A particular focus has been the assembly of synaptic connections, both in the central nervous system and periphery. The glutamatergic neuromuscular junction (NMJ) is most accessible to a combination of optical imaging and electrophysiological recording. A glass suction electrode is used to stimulate the peripheral nerve, with excitatory junction current (EJC) recordings made in the voltage-clamped muscle. This recording configuration has been used to chart the functional differentiation of the synapse, and track the appearance and maturation of presynaptic glutamate release properties. In addition, postsynaptic properties can be assayed independently via iontophoretic or pressure application of glutamate directly to the muscle surface, to measure the appearance and maturation of the glutamate receptor fields. Thus, both pre- and postsynaptic elements can be monitored separately or in combination during embryonic synaptogenesis. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.

Electrophysiological Recording in the Drosophila Embryo

Electrophysiological recordings from Drosophila embryos allow analyses of developing muscle and neuron electrical properties, as well as characterization of functional synaptogenesis at the glutamatergic neuromuscular junction and central cholinergic and GABAergic synapses.

Registro electrofisiológico en el Drosophila Embrión

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite ...

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of the Drosophila melanogaster syncytial embryo for studying mitosis1. Drosophila has useful genetics with a sequenced genome, and it can be easily maintained and manipulated. Many mitotic mutants exist, and transgenic flies expressing functional fluorescently (e.g. GFP) - tagged mitotic proteins have been and are being generated. Targeted gene expression is possible using the GAL4/UAS system2. 

The Drosophila early embryo carries out multiple mitoses very rapidly (cell cycle duration, &asymp;10 min). It is well suited for imaging mitosis, because during cycles 10-13, nuclei divide rapidly and synchronously without intervening cytokinesis at the surface of the embryo in a single monolayer just underneath the cortex. These rapidly dividing nuclei probably use the same mitotic machinery as other cells, but they are optimized for speed; the checkpoint is generally believed to not be stringent, allowing the study of mitotic proteins whose absence would cause cell cycle arrest in cells with a strong checkpoint. Embryos expressing GFP labeled proteins or microinjected with fluorescently labeled proteins can be easily imaged to follow live dynamics (Fig. 1). In addition, embryos can be microinjected with function-blocking antibodies or inhibitors of specific proteins to study the effect of the loss or perturbation of their function3. These reagents can diffuse throughout the embryo, reaching many spindles to produce a gradient of concentration of inhibitor, which in turn results in a gradient of defects comparable to an allelic series of mutants. Ideally, if the target protein is fluorescently labeled, the gradient of inhibition can be directly visualized4. It is assumed that the strongest phenotype is comparable to the null phenotype, although it is hard to formally exclude the possibility that the antibodies may have dominant effects in rare instances, so rigorous controls and cautious interpretation must be applied. Further away from the injection site, protein function is only partially lost allowing other functions of the target protein to become evident.

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of microinjection and high resolution imaging in the Drosophila melanogaster syncytial embryo to study mitosis.

Técnicas de microinyección para el estudio de la mitosis en el Drosophila melanogaster Embrión sincitial

This protocol describes the use of the Drosophila melanogaster syncytial embryo  for studying mitosis1.  Drosophila has useful genetics with a sequenced ...

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study angiogenesis but also to study tumorigenesis. After the chick chorioallantoic membrane (CAM) has developed, its blood vessel network can be easily accessed, manipulated and observed and therefore provides an optimal setting for angiogenesis assays. Since the lymphoid system is not fully developed until late stages of incubation, the chick embryo serves as a naturally immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition to nurturing developing allo- and xenografts, the CAM blood vessel network provides a uniquely supportive environment for tumor cell intravasation, dissemination, and vascular arrest and a repository where arrested cells extravasate to form micro metastatic foci.
For experimental purposes, in most of the recent studies the CAM was exposed by cutting a window through the egg shell and experiments were carried out in ovo, resulting in significant limitations in the accessibility of the CAM and possibilities for observation and photo documentation of effects. When shell-less cultures of the chick embryo were used1-4, no experimental details were provided and, if published at all, the survival rates of these cultures were low. We refined the method of ex ovo culture of chick embryos significantly by introducing a rationally controlled extrusion of the egg content. These ex ovo cultures enhance the accessibility of the CAM and chick embryo, enabling easy in vivo documentation of effects and facilitating experimental manipulation of the embryo. This allows the successful application to a large number of scientific questions: (1) As an improved angiogenesis assay5,6, (2) an experimental set up for facilitated injections in the vitreous of the chick embryo eye7-9, (3) as a test environment for dissemination and intravasation of dispersed tumor cells from established cell lines inoculated on the CAM10-12, (4) as an improved sustaining system for successful transplantation and culture of limb buds of chicken and mice13 as well as (5) for grafting, propagation, and re-grafting of solid primary tumor tissue obtained from biopsies on the surface of the CAM14.
In this video article we describe the establishment of a refined chick ex ovo culture and CAM assay with survival rates over 50%. Besides we provide a step by step demonstration of the successful application of the ex ovo culture for a large number of scientific applications. 
Daniel S. Dohle, Susanne D. Pasa, and Sebastian Gustmann contributed equally to this study.

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

The chick chorioallantoic membrane (CAM) is a unique, naturally immunodeficient supportive culture environment to study angiogenesis and tumorigenesis. This video article demonstrates the different steps in chick ex ovo culture, application of potentially angiogenic substances and successful inoculation of tumor cells and tissues on the surface of the CAM.

Pollo Ex ovo Cultura y Ex ovo CAM Ensayo: cómo funciona realmente

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study ...

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

Employment of enhancer elements to drive expression of reporter genes in neurons is a widely used paradigm for tracking axonal projection. For tracking axonal projection of spinal interneurons in vertebrates, germ line-targeted reporter genes yield bilaterally symmetric labeling. Therefore, it is hard to distinguish between the ipsi- and contra-laterally projecting axons. Unilateral electroporation into the chick neural tube provides a useful means to restrict expression of a reporter gene to one side of the central nervous system, and to follow axonal projection on both sides 1 ,2-5. This video demonstrates first how to handle the eggs prior to injection. At HH stage 18-20, DNA is injected into the sacral level of the neural tube, then tungsten electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator. The egg is sealed with tape and placed back into an incubator for further development. Three days later (E6) the spinal cord is removed as an open book preparation from embryo, fixed, and processed for whole mount antibody staining. The stained spinal cord is mounted on slide and visualized using confocal microscopy.

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

This video demonstrates how to visualize axonal pathways of genetically defined groups of neurons in the embryonic chick spinal cord utilizing in ovo electroporation of reporter genes under the control of specific enhancer elements.

Descifrar Caminos axonal de los grupos definidos genéticamente las neuronas en el tubo neural del polluelo Utilizando In ovo La electroporación

Employment of enhancer elements to drive expression of reporter genes in neurons is a widely used paradigm for tracking axonal projection. For tracking ...

Xenotransplantation of Human Stem Cells into the Chicken Embryo

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the behavior of cells in a standardized in vivo environment. The main advantages of the chicken embryo include low cost, high accessibility, ease of surgical manipulation and lack of a fully developed immune system. Xenotransplantation into chicken embryos can provide valuable information about cell proliferation, differentiation and behavior, the responses of cells to signals in defined embryonic tissue niches, and tumorigenic potential. Transplanting cells into chicken embryos can also be a step towards transplantation experiments in other animal models. Recently the chicken embryo has been used to evaluate the neurogenic potential of human stem and progenitor cells following implantation into neural anlage1-6. In this video we document the entire procedure for transplanting human stem cells into the developing central nervous system of the chicken embryo. The procedure starts with incubation of fertilized eggs until embryos of the desired age have developed. The eggshell is then opened, and the embryo contrasted by injecting dye between the embryo and the yolk. Small lesions are made in the neural tube using microsurgery, creating a regenerative site for cell deposition that promotes subsequent integration into the host tissue. We demonstrate injections of human stem cells into such lesions made in the part of the neural tube that forms the hindbrain and the spinal cord, and into the lumen of the part of the neural tube that forms the brain. Systemic injections into extraembryonic veins and arteries are also demonstrated as an alternative way to deliver cells to vascularized tissues including the central nervous system. Finally we show how to remove the embryo from the egg after several days of further development and how to dissect the spinal cord free for subsequent physiological, histological or biochemical analyses.

In this paper we present a method for transplanting human stem cells into various regions of the central nervous system of the chicken embryo. This provides an in vivo model for assessing the proliferation and differentiation of various types of human stem cells in embryonic tissue environments.

Xenotrasplante de células madre humanas en el embrión de pollo

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the ...

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

Ensayo clonigénicas: las células adherentes

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

Modified Mouse Embryonic Stem Cell based Assay for Quantifying  Cardiogenic Induction Efficiency

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways. One of the hurdles to its understanding has been the varied methods in correlating changes of key signaling events to differentiation efficiency. We describe here the use of a mouse embryonic stem (ES) cell based assay to identify critical time windows for Wnt/&beta;-catenin and BMP signal activation during cardiogenic induction. By scoring for contracting embryonic bodies (EBs) in a 96-well plate format, we can quickly quantify cardiogenic efficiency and identify crucial time windows for Wnt/&beta;-catenin and BMP signal activation in a time course following specific modulator treatments. The principal outlined here is not limited to cardiac induction alone, and can be applied towards the study of many other cell lineages. In addition, the 96-well format has the potential to be further developed as a high throughput, automated assay to allow for the testing of more sophisticated experimental hypotheses.

We describe the use of a mouse ES cell based assay to identify critical time windows for Wnt/&#946;-catenin and BMP signal activation during cardiogenic induction. The method provides a standardized platform that reliably quantifies cardiogenic efficiency, and it is applicable to the study of other cell lineages.

Modificado madre embrionarias de ratón basado en células para la cuantificación de la eficiencia de inducción cardiogénico

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways.  One of the hurdles ...

Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. Tracking fiducial markers on the surfaces of these cellular sheets is a well-established method for estimating morphogenetic quantities such as growth, contraction, and shear. However, not all surface labeling techniques are readily adaptable to conventional imaging modalities and possess different advantages and limitations. Here, we describe two labeling methods and illustrate the utility of each technique. In the first method, hundreds of fluorescent labels are applied simultaneously to the embryo using magnetic iron particles. These labels are then used to quantity 2-D tissue deformations during morphogenesis. In the second method, polystyrene microspheres are used as contrast agents in non-invasive optical coherence tomography (OCT) imaging to track 3-D tissue deformations. These techniques have been successfully implemented in our lab to studythe physical mechanisms of early head fold, heart, and brain development, and should be adaptable to a wide range morphogenetic processes.

This article describes surface labeling and ex ovo tissue culture in the early chick embryo. Techniques amenable to time-lapse bright field, fluorescence, and optical coherence tomography imaging are presented. Tracking surface labels with high spatiotemporal resolution enables kinematic quantities such as morphogenetic strains (deformations) to be calculated in both two and three dimensions.

El seguimiento de deformaciones morfogenética del tejido en el embrión de pollo temprano

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. ...