Este trabajo fue financiado por la Deutsche Forschungsgemeinschaft SFB688 (a A.G.).

Cría de ratón fue aprobado por el Regierung Unterfranken, y todos los análisis se llevaron a cabo estrictamente de acuerdo con todos los alemanes y la Unión Europea las leyes y reglamentos sobre uso y cuidado de animales de laboratorio.
1. capa de microtitulación las placas con la integrina α4β1 Ex útero alantoides explante cultura
<ol><li>Reconstituir la integrina α4β1 murino liofilizado 200 μg/ml en tampón fosfato salina (PBS). Diluir hasta una concentración final de 10 μg/mL en precalentado (37 ° C) PBS. Pipetear 20 μL/pocillo de la solución de la integrina α4β1 diluido en el número de pocillos de una placa de microtitulación (alantoides 1/pocillo). Nota: Evite la formación de burbujas de aire al recubrimiento de los pocillos de la microplaca.</li>
	<li>Incubar las placas de microtitulación durante 60 min a 37 ° C por colocarlos en una incubadora humidificada cultivo de tejidos.</li>
	<li>Cuidadosamente aspirar el sobrenadante con una pipeta. Usar una succión suave e inclinar la placa para evitar tocar el fondo del pozo con la punta de la pipeta. Lavar los pocillos dos veces añadiendo 50 μl de medio de cultivo de precalentado (37 ° C) como de Dulbecco modificado Eagle Medium (DMEM) y quitarlo del medio de lavado por succión suave.</li>
	<li>Paso crítico: bloque gratis sitios de Unión mediante la adición de 50 μL/pocillo de 0.1% (p/v) de albúmina sérica bovina (BSA) / DMEM e incube durante ≥ 30 min a 37 ° C mediante la colocación de las placas de microtitulación en una incubadora humidificada cultivo de tejidos. Nota: Explantes alantoides pueden unir a varias superficies en presencia de suero. Por lo tanto es imperativo para bloquear sitios de Unión inespecífica, después de cubrir los pozos con la integrina y antes de agregar el alantoides disecada para explantación cultura contiene suero en medio (ver paso 4.4).</li>
	<li>Mantenga los pocillos recubiertos en la solución de bloqueo a 37 ° C hasta que se necesite en el paso 4.4.</li>
</ol>2. útero disección de un ratón embarazada en Dpc 8
Nota: Reducir las tasas de falsos positivos embarazo peso ganancia discriminación15.
<ol><li>El ratón de sacrificio mediante dislocación cervical. Desinfectar la capa en la zona abdominal mediante pulverización con etanol al 70% (v/v). Nota: Lleve guantes al manipular ratones. Eutanasia por dislocación cervical sin anestesia evita la contaminación química de la alantoides, que pueden interferir con el accesorio de la alantoides.</li>
	<li>Haciendo una pequeña incisión en la línea media con unas tijeras para abrir la piel abdominal. Use los dedos enguantados para sostener la piel por encima y por debajo de la incisión y separe la piel hacia el pecho y la cola del ratón.</li>
	<li>Agarre el peritoneo con pinzas. Con la otra mano, hacen una incisión en forma de V desde anterior hacia posterior con unas tijeras y mueva el tejido para exponer la cavidad abdominal. Use las pinzas para empujar los intestinos a un lado para acceder al útero.</li>
	<li>Localice los dos cuernos uterinos. Sostenga el cuello del útero (el segmento caudal) con pinzas y corte los ligamentos con tijeras finas. Luego separar ambos cuernos uterinos, cortar los oviductos con tijeras.</li>
	<li>Utilice pinzas para transferir al útero en un plato de 10 cm estéril que contiene estéril, PBS de temperatura (RT). Eliminar el tejido adiposo, vasos y nervios que rodean los cuernos uterinos con finas pinzas o tijeras (figura 1A).</li>
</ol>3. embrión disección
Nota: Realice los siguientes pasos bajo un estereomicroscopio equipado con una gama zoom 8:1.
<ol><li>Separar los embriones por el corte entre los sitios de implantación con las tijeras. Sujete la guarnición uterina muscular de los cogollos de útero con pinzas y tire de ella aparte para exponer la decidua (figura 1B).</li>
	<li>Con unas pinzas, transferencia de los brotes del útero en una nueva placa de 10 cm que contienen estéril, RT PBS.</li>
	<li>Fijar la decidua en el embrión con las puntas del fórceps.</li>
	<li>Hacer una incisión en la parte de anti-mesometrial de la decidua con pinzas finas (figura 1) y retire la decidua con fórceps para exponer el embrión (figura 1). Nota: El polo mesometrial de la decidua está altamente vascularizado y puede ser más ancho en su base que la parte anti-mesometrial, que contiene el embrión y aparece más pálido.</li>
	<li>Diseccionar el embrión con el fórceps (Figura 1E, F).</li>
	<li>Usando una cuchara de micro, transferir el embrión en una gota de PBS estéril contenida en un recipiente estéril de 3,5 cm.</li>
	<li>Transferir el embrión en una dulce gota de PBS en el mismo plato de 3,5 cm con una cuchara de micro y disecar el embrión del saco de yema de huevo con unas pinzas (figura 1, H).</li>
	<li>Opcionalmente, si es necesario (por ejemplo, para la genotipificación por reacción en cadena de polimerasa), traslado del saco de yema de huevo en un tubo estéril aspirando con ~ 5 μl PBS en una punta de pipeta de orificio grande 200 μl. Almacenar a-20 ° C. Nota: Tejido materno (es decir, el saco vitelino parietal como membrana de Reichert y el cono de ectoplacental) que podría contaminar la preparación saco vitelino puede conducir a resultados erróneos de genotipificación. Eliminación de estos tejidos es más fácil antes de la disección del embrión desde el saco vitelino.</li>
</ol>4. alantoides disección y cultura Ex Utero en la integrina α4β1 inmovilizados
<ol><li>Corte el alantoides desde su sitio de inserción basal en el extremo posterior del embrión con unas pinzas finas (figura 1 H).</li>
	<li>Opcionalmente, si se requiere el desarrollo puesta en escena, contar los pares somite del embrión por inspección visual utilizando un microscopio equipado con óptica de contraste de fase y un 5 × 10 × objetivo. Nota: El embrión puede ser extendido con fórceps para facilitar el conteo del somite. Fusión y accesorio corioalantoideas ocurren entre E 8.0 y 9.0 E en embriones con pares ≥ 6 somite. Giro comienza en embriones con 6-8 pares de somite.</li>
	<li>Pipeta de 40 μL/pocillo de DMEM suplementado con suero bovino fetal 10% (v/v), L-glutamina y antibióticos en el número requerido de prelacado pocillos de la placa (véase sección 1).</li>
	<li>Recoger el alantoides flotante aspirando con ~ 5 μl PBS en un gran orificio de una pipeta de 200 μl. Transferencia 1 alantoides/bien.</li>
	<li>Incubar el allantoides colocando las placas de microtitulación en una incubadora humidificada cultivo de tejidos (37 ° C, 5% CO2) para p. ej., 6, 12 y 24 h. Para evitar interferencias con el accesorio de la alantoides, no se mueven las placas de microtitulación entre estos puntos de tiempo.</li>
	<li>En cada momento, anotar accesorio alantoides (p. ej., no alantoides accesorio/flotante alantoides parcial o totalmente Unido; totalmente propagación alantoides) utilizando un microscopio equipado con óptica de contraste de fase contraste o diferencial de interferencia y un x 5 o 10 x objetivo (figura 2). Nota: Ejemplos representativos de allantoides recién aislados y totalmente conectados se muestran en la figura 2. Ex utero, la punta distal del allantoides aislados de ratones C57BL/6J de tipo salvaje se fija dentro de las 4-6 h (esto se denomina fijación parcial de la alantoides) y accesorio completo de allantois todo se logra dentro de ~ 12 h de la cultura. Por 18-24 h, el explante ha aplanado y asumió una forma circular (que se refiere como totalmente extendido alantoides) con CD31-positivo del plexo vascular9,16.</li>
</ol>5. confirmación la coloración de la alantoides Explanted de la célula endotelial marcador CD31
<ol><li>Aspire el medio de cultivo de los explantes de extensión completamente y fijar las células añadiendo 40 μL/pocillo de paraformaldehído al 4% (w/v) / PBS. Nota: Paraformaldehido es tóxico. Use guantes y gafas de seguridad para evitar contacto con los ojos y la piel.</li>
	<li>Incubar por 10 min a temperatura ambiente y luego aspirar el sobrenadante. Lavar tres veces al agregar 40 μL/pocillo de PBS y quitarlo del medio de lavado por succión suave.</li>
	<li>Permeabilizar las células y bloquear sitios de Unión inespecíficos mediante la adición de 40 μL/pocillo de 0,1% (v/v) suero de cabra normal BSA/10% Triton X-100/0.1% (w/v) (v/v) en PBS durante 30 min a RT. Wash dos veces añadiendo 40 μL/pocillo de PBS y quitarlo del medio de lavado por succión suave. Nota: El protocolo puede hacer una pausa durante la noche en este punto.</li>
	<li>Mancha las células añadiendo 20 μL/pocillo de anticuerpos primarios anti-CD31 diluido 1:50 en 0.1% (p/v) BSA/PBS durante la noche a 4 ° C. Lavar tres veces al agregar 40 μL/pocillo de PBS y quitarlo del medio de lavado por succión suave.</li>
	<li>Detectar anticuerpos primarios Unidos añadiendo 20 μL/pocillo de marcada con fluorescencia secundaria cabra anticuerpos anti rata diluido 1: 200 en 0.1% (p/v) BSA/PBS para 1 h en placas de microtitulación RT. proteger de la luz por envolver en papel de aluminio o colocando en una cámara oscura. Lavar tres veces al agregar 40 μL/pocillo de PBS y quitarlo del medio de lavado por succión suave.</li>
	<li>Incrustar las células añadiendo 20 μL/pocillo de medio de montaje. Incubar durante ~ 1 h a temperatura ambiente hasta que haya solidificado el medio de montaje. Almacenar las placas de microtitulación a temperatura ambiente o a 4 ° C, protegido de la luz.</li>
	<li>Imagen el plexo vascular de la alantoides explantes bajo un microscopio de fluorescencia equipado un × 5 o 10 × objetivo y filtros de fluorescencia adecuado. Nota: Imágenes representativas del plexo vascular CD31-positivo de un alantoides completamente propagación se muestran en la figura 3. Otros marcadores para el plexo vascular, tales como anticuerpos dirigen contra Flk-1, capitán, Tie-1 Tie-2 puede utilizarse o bien16.</li>
</ol>

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Los autores no tienen nada que revelar.

En presencia de suero, que es una rica fuente de moléculas de matriz extracelular adhesiva Pro como la fibronectina, alantoides explantes concederá fácilmente a varios plástico, vidrio o filtro superficies9,16. Por lo tanto, si del análisis realizado se pretende investigar específicamente el efecto de una modificación genética o cualquier otro tratamiento accesorio de la alantoides a inmovilizado α4β1, es fundamental para bloquear sitios de Unión inesp...

La placenta es indispensable para el crecimiento embrionario y desarrollo en el ambiente uterino. Constituye la interfaz para oxígeno, metabolitos y nutrientes intercambio entre la circulación fetal y materna y también funciona como un órgano endocrino e inmunológico. Cualquier insulto endógeno o exógeno que deteriora el desarrollo placentario puede conducir a retraso de crecimiento intrauterino, pérdida fetal o complicaciones del embarazo, tanto en ratones como en seres humanos1. Mientras que el número de mutaciones de ratón específicos causantes de la fisiología placentaria anormal continúa aumentando2, con 219 genotipos actualmente listados en el sitio web de informática de genoma de ratón (MGI) (http://www.informatics.jax.org/vocab/ mp_ontology / MP:0010038), muchos de estos fenotipos no están bien entendidas desde un punto de vista mecanicista. Además de alteraciones genéticas, fármacos de molécula pequeña, anticuerpos monoclonales, toxinas, patógenos, exceso metabolitos o diversos agentes ambientales pueden afectar desarrollo de placenta y función3,4,5 , 6 , 7. sin embargo, simple en vitro ensayos que pasos críticos del modelo en el desarrollo de la placenta son escasos.
Desarrollo placentario murino se inicia en el midgestation temprano, cuando el alantoides (precursor del desarrollo del cordón umbilical) emerge desde el extremo posterior del embrión como un brote que crece hacia el corion8. Chorioadhesive las células en la capa externa de la yema alantoidea median el accesorio y separarse de la alantoides en la superficie del corión (corioalantoideas "fusión"). El corion posteriormente dobla en las vellosidades en las que crece la vasculatura de la alantoides para formar el laberinto, la capa interna de placenta donde se intercambian nutrientes y oxígeno entre la sangre materna estrechamente yuxtapuestas sinusoides y vasos embrionarios9 ,10.
La fijación de la alantoides al corión es el paso inicial y fundamental en la formación del laberinto, y defectos en este proceso se encuentran entre las causas más comunes de mortalidad embrionaria en el midgestation1. Aunque un número de mutantes de ratón se ha descrito donde corioalantoideas accesorio no ocurren10, y la base de datos MGI enumera actualmente 108 mutantes de ratón que se caracterizan por la fusión anormal corioalantoideas (http:// www.informatics.Jax.org/Vocab/mp_ontology/MP:0002824), la interacción intercelular entre el vascular cell adhesión molecule-1 (VCAM-1, expresado en el mesodermo alantoideo) y la integrina α4β1 (expresado en el mesotelio coriónico, que es derivado de mesoderm extraembrionarias) parece ser indispensable para la fijación corioalantoideas y fusión11,12,13. La coloración de Immunohistochemical de montaje en todo tipo de salvaje embriones ha mostrado que VCAM-1 se expresa dentro de los dos tercios distales del tallo alantoideo por día aproximadamente embrionario (E) 7.513 (1-4 etapa de somite), y que su expresión sigue siendo alta durante corioalantoideas apego y fusión11,12. Normalmente se detecta fallo de accesorio alantoideo en el corion por análisis histológicos del útero brotes. Sin embargo, los tamaños alantoides son fisiológicamente muy variables entre y dentro de camadas de la misma etapa de desarrollo14y allantois solamente puede conectar al corion cuando ha alcanzado un tamaño suficiente para hacer contacto físico. Reflejando esta variabilidad natural, corioalantoideas accesorio ocurre en algún momento entre ~ E 8.0 y 9.0 E en el úteroy una evaluación estadísticamente fiable de este proceso por histología, por tanto, es dependiente en el análisis de un gran número de conceptuses obtener suficientes muestras en la etapa de desarrollo apropiada.
Aquí, describimos un método para evaluar apego alantoideo ex utero que es menos dependiente en tamaño del alantoides. Demostramos la disección de embriones de ratón y sus allantoides de los úteros de ratones embarazadas ~ 8 días post coitum (dpc; dpc 0.5 señala la detección de un tapón vaginal), y la posterior cultura del alantoides explantes de α4β1 inmovilizados integrinas. Este método permite una evaluación rápida y funcional de la fijación y propagar el comportamiento de los explantes de allantois múltiples en paralelo y en diferentes momentos sucesivos. El protocolo puede ser utilizado para detectar los efectos de las mutaciones específicas, drogas o diversos factores ambientales sobre alantoides accesorio ex vivo.

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			<td height="23" style="height:23px;width:319px;">C57BL/6J wildtype mice</td>
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			<td style="width:256px;">Fine Science Tools</td>
			<td>15012-12</td>
			<td>Noyes spring scissors with 14 mm blades for the dissection of uterus buds.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Microspoon</td>
			<td style="width:256px;">Carl Roth</td>
			<td>AT18.1</td>
			<td>5 mm Spoon diameter.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Microtiter plates</td>
			<td style="width:256px;">ibidi</td>
			<td style="width:119px;">81501</td>
			<td>We use uncoated, sterile angiogenesis slides (internal volume 50 &#181;L) with a hydrophobic surface that is not tissue-culture treated to reduce non-a4b1-mediated binding to plastic.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">10 cm dish</td>
			<td style="width:256px;">Nunc</td>
			<td style="width:119px;">150350</td>
			<td>Sterile tissue culture dishes.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">3.5 cm dish</td>
			<td style="width:256px;">Nunc</td>
			<td style="width:119px;">150288</td>
			<td>Sterile tissue culture dishes.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Large orifice pipette tips&#160;</td>
			<td style="width:256px;">Biozym</td>
			<td style="width:119px;">VT0140X</td>
			<td>Low binding pipette tips, 200 &#181;L.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">a4b1 integrin</td>
			<td style="width:256px;">R&#38;D Systems</td>
			<td style="width:119px;">6054-A4</td>
			<td>Murine a4b1 integrin recombinantly expressed and purified from a Chinese hamster ovary cell line.</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:319px;">Dulbecco&#39;s Phosphate Buffered Saline (PBS)</td>
			<td style="width:256px;">Sigma Aldrich</td>
			<td style="width:119px;">D8537</td>
			<td>Without Ca2+ and Mg2+.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:319px;">Dulbecco&#8217;s Modified Eagle Medium (DMEM)</td>
			<td style="width:256px;">PAN Biotech</td>
			<td style="width:119px;">P04-03600</td>
			<td style="width:915px;">The formulation contains 4.5 g/L glucose, 110 mg/L sodium pyruvate and 3.7 g/L NaHCO3 but no L-glutamine, which is added separately.</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:319px;">Penicillin/Streptomycin</td>
			<td style="width:256px;">Gibco (ThermoFisher Scientific)</td>
			<td style="width:119px;">15140-122</td>
			<td>100 x (10,000 U/mL Penicillin and 10,000 &#181;g/mL streptomycin) stock solution.&#160;Prepare and store aliquots at -20 &#176;C to avoid freeze/thaw.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">L-Glutamine</td>
			<td style="width:256px;">PAN Biotech</td>
			<td style="width:119px;">P04-80100</td>
			<td>100 x (200 mM) Stock solution. Prepare and store aliquots at -20 &#176;C to avoid freeze/thaw.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Fetal bovine serum&#160;</td>
			<td style="width:256px;">Biochrom</td>
			<td style="width:119px;">S 0115</td>
			<td>We use heat-inactivated FBS (heated to 56 &#176;C for 30 min with mixing to inactivate complement).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Bovine serum albumin (BSA)</td>
			<td style="width:256px;">Sigma Aldrich</td>
			<td style="width:119px;">A7030</td>
			<td>We use protease- and fatty acid-free albumin. Prepare a 0.1 % (w/v) solution in DMEM. Sterile filter the solution through a disposable cell culture filter with a 0.22 &#181;m pore size and low protein-binding membrane, and store aliquots at -20 &#176;C.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">para-Formaldehyde</td>
			<td style="width:256px;">Carl Roth</td>
			<td>0335.2</td>
			<td style="width:915px;">To make a formaldehyde solution in PBS, weigh para-formaldehyde powder in a ventilated hood, and add it to PBS preheated to ca. 60 &#176;C in a beaker on a stir plate. Add 1 N NaOH dropwise until solution clears. Let solution cool to room temperature, and filter through 0.22 mm filter syringe. Adjust pH with diluted HCl to ca. 6.9, and adjust to final volume with PBS. We aliquot and freeze the solution, but it can also be stored at 4 &#176;C for approximately four weeks.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Triton X-100</td>
			<td style="width:256px;">Sigma Aldrich</td>
			<td style="width:119px;">X100</td>
			<td>Use wide-orifice pipette tips to handle undiluted Triton X-100.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Normal goat serum</td>
			<td style="width:256px;">Sigma Aldrich</td>
			<td>G9023</td>
			<td>To block non-specific binding of antibodies in immunofluorescence analyses.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">a-CD31 Antibodies</td>
			<td style="width:256px;">BD Biosciences</td>
			<td>550274</td>
			<td>Purified rat anti-mouse monoclonal antibodies, clone MEC 13.3.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Secondary goat anti-rat antibodies</td>
			<td style="width:256px;">Thermo Fisher</td>
			<td>A-11006</td>
			<td>Goat anti-rat IgG (heavy and light chains), cross-adsorbed, fluorescently labeled with Alexa Fluor 488. Other fluorescent labels are also possible.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Mowiol 4-88</td>
			<td style="width:256px;">Sigma Aldrich</td>
			<td>81381</td>
			<td>Mounting medium for cytochemistry. MW ~31,000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Labovert&#160;</td>
			<td style="width:256px;">Leitz</td>
			<td style="width:119px;">Labovert&#160;</td>
			<td>Inverted phase contrast microscope equipped with a 4 x and 10 x objective for somite pair counting. Other phase contrast microscopes (such as those that are routinely used in tissue culture) are also suitable.&#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">M80</td>
			<td style="width:256px;">Leica Microsystems</td>
			<td style="width:119px;">M80</td>
			<td>Stereomicroscope with 8 : 1 zoom range (yielding a magnification between 7.5 x and 60 x) for embryo and allantois dissection.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">DM4000B</td>
			<td style="width:256px;">Leica Microsystems</td>
			<td style="width:119px;">DM4000B</td>
			<td>Microscope system for histology with LED illumination. We use HCX PL FLUOTAR 5 x/0.15 and HC PL FLUOTAR 10 x/0.30 objectives. Other microscopes equipped phase contrast and fluorescence optics can be used to score allantois attachment.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Digital camera</td>
			<td style="width:256px;">JVC</td>
			<td style="width:119px;">KY-F75U</td>
			<td>3-CCD digital capture camera attached to the DM4000B LED microscope. We use Diskus software (Hilgers) to operate both the microscope and the camera.</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;width:319px;">Confocal microscope</td>
			<td style="width:256px;">Leica Microsystems</td>
			<td style="width:119px;">SP5</td>
			<td style="width:915px;">Confocal microscope for higher-resolution imaging of endothelia in the vascular plexus of allantois explants. Immunofluorescence images were taken with a 40 x objective.</td>
		</tr>
	</tbody>
</table>

dissection and explant culture of murine allantois for the in vitro analysis of allantoic attachment

La placenta es fundamental para el crecimiento y desarrollo de los embriones mamíferos. Por esta razón, numerosas alteraciones genéticas e insultos también ambientales probables que perturbe el desarrollo de la placenta o función pueden causar la pérdida temprana del embarazo en ratones y seres humanos. Sin embargo, se carecen de ensayos simples en vitro para detectar posibles efectos en la formación de la placenta. Aquí, nos centramos en el primer y fundamental paso en la formación de la placenta, que consiste en la fijación de la alantoides en el corion de modelado. Se describe un método para evaluar rápidamente el accesorio de explantes alantoideo de la integrina α4β1 inmovilizados, que sirve como un substrato chorio-mimético. Este enfoque en vitro permite una evaluación cualitativa de la fijación y propagar el comportamiento de los explantes de allantois múltiples en diferentes momentos consecutivos. El protocolo puede utilizarse para investigar el efecto de las mutaciones de ratón específicos, drogas o diversos factores ambientales que se han relacionado con complicaciones del embarazo o pérdida fetal en el alantoides accesorio ex vivo.

Este protocolo describe un método para aislar y explante allantoides murino ex vivoy detalles de la evaluación cualitativa de la alantoides accesorio a la integrina α4β1, un proceso crítico en vivo corioalantoideas fusión. Los resultados representativos que se muestra en la figura 1A–H demuestran los pasos sucesivos de aislamiento alantoides a partir de útero durante el embarazo. Se retiran los teji...

Watch this Scientific Journal Video about Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment at JoVE.com

Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment

Se describe un ensayo en vitro al accesorio corioalantoideas de modelo, el primer paso en la formación de la placenta. El protocolo muestra la cultura disección y explante de murinos allantoides de la integrina α4β1 inmovilizados. Accesorio de la alantoides se evalúa microscópicamente en momentos predeterminados.

Disección y la cultura de explante de Allantois murino para el análisis In Vitro del accesorio alantoideo

The placenta is essential for the growth and development of mammalian embryos. For this reason, numerous genetic alterations and likely also environmental ...

dissection-explant-culture-murine-allantois-for-vitro-analysis

Research

JoVE Journal

Medicine

Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment

7.8K vistas.  University of Würzburg. Se describe un ensayo en vitro al accesorio corioalantoideas de modelo, el primer paso en la formación de la placenta. El protocolo muestra la cultura disección y explante de murinos allantoides de la integrina α4β1 inmovilizados. Accesorio de la alantoides se evalúa microscópicamente en momentos predeterminados.

Video: Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment

Dissection of Human Vitreous Body Elements for Proteomic Analysis

The vitreous is an optically clear, collagenous extracellular matrix that fills the inside of the eye and overlies the retina. 1,2 Abnormal interactions between vitreous substructures and the retina underlie several vitreoretinal diseases, including retinal tear and detachment, macular pucker, macular hole, age-related macular degeneration, vitreomacular traction, proliferative vitreoretinopathy, proliferative diabetic retinopathy, and inherited vitreoretinopathies. 1,2 The molecular composition of the vitreous substructures is not known. Since the vitreous body is transparent with limited surgical access, it has been difficult to study its substructures at the molecular level. We developed a method to separate and preserve these tissues for proteomic and biochemical analysis. The dissection technique in this experimental video shows how to isolate vitreous base, anterior hyaloid, vitreous core, and vitreous cortex from postmortem human eyes. One-dimensional SDS-PAGE analyses of each vitreous component showed that our dissection technique resulted in four unique protein profiles corresponding to each substructure of the human vitreous body. Identification of differentially compartmentalized proteins will reveal candidate molecules underlying various vitreoretinal diseases.

This video shows an effective technique for differentiating and dissecting the various semi-transparent structures of the human vitreous body in post mortem eyes.

La disección de los elementos humanos Cuerpo Vítreo para el análisis proteómico

The vitreous is an optically clear, collagenous extracellular matrix that fills the inside of the eye and overlies the retina. 1,2 Abnormal interactions ...

Investigación

Medicina

Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts in this protocol have been successfully used in the derivation of induced pluripotent stem cells to model disease1. Early passages of these fibroblasts can also be used for cell-based functional assays to study specific disease pathways, mechanisms2 and subsequent drug screening approaches. The advantage of the presented protocol over enzymatic procedures are 1) the reproducibility of the technique from small amounts of tissue derived from older patients, e.g. patients affected with Parkinson's disease, 2) the technically simple approach over more challenging methodologies using enzymatic treatments, and 3) the time consideration: this protocol takes 15-20 min and can be performed immediately after biopsy arrival. Enzymatic treatments can take up to 4 hr and have the problems of overdigestion, reduction of cell viability and subsequent attachment of cells when not handled properly. This protocol describes the dissection and preparation of a 4-mm human skin biopsy for derivation of a fibroblast culture and has a very high success rate which is important when dealing with patient-derived tissue samples. In this culture, keratinocytes migrate out of the biopsy tissue within the first week after preparation. Fibroblasts appear 7-10 days after the first outgrowth of keratinocytes. DMEM high glucose media supplemented with 20% FBS favors the growth of fibroblasts over keratinocytes and fibroblasts will overgrow the keratinocytes. After 2 passages keratinocytes have been diluted out resulting in relatively homogenous fibroblast cultures which expresses the fibroblast marker SERPINH1 (HSP-47). Using this approach, 15-20 million fibroblasts can be derived in 4-8 weeks for cell banking. The skin dissection takes about 15-20 min, cells are then monitored once a day under the microscope, and media is changed every 2-3 days after attachment and outgrowth of cells.

The fibroblast explant culture protocol from human skin punch biopsies is a technically robust and simple way to derive skin cells within 4-8 weeks for banking of about 15-20 million cells at a low passage number.

Piel biopsia en sacabocados explante cultura para Derivación de fibroblastos humanos primarios

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts ...

Quantitative Analysis and Characterization of Atherosclerotic Lesions in the Murine Aortic Sinus

Atherosclerosis is a disease of the large arteries and a major underlying cause of myocardial infarction and stroke. Several different mouse models have been developed to facilitate the study of the molecular and cellular pathophysiology of this disease. In this manuscript we describe specific techniques for the quantification and characterization of atherosclerotic lesions in the murine aortic sinus and ascending aorta. The advantage of this procedure is that it provides an accurate measurement of the cross-sectional area and total volume of the lesion, which can be used to compare atherosclerotic progression across different treatment groups. This is possible through the use of the valve leaflets as an anatomical landmark, together with careful adjustment of the sectioning angle. We also describe basic staining methods that can be used to begin to characterize atherosclerotic progression. These can be further modified to investigate antigens of specific interest to the researcher. The described techniques are generally applicable to a wide variety of existing and newly created dietary and genetically-induced models of atherogenesis.

We describe procedures to quantify and characterize atherosclerotic lesions in mouse models using precision sectioning of the aortic sinus and ascending aorta combined with histochemical and immunohistochemical analysis.

Atherosclerosis is a disease of the large arteries and a major underlying cause of myocardial infarction and stroke. Several different mouse models have ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

Infusiones de la arteria carótida para farmacocinéticas y farmacodinámicas Análisis de taxanos en ratones

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Whole Vitreous Humor Dissection for Vitreodynamic Analysis

The authors propose an effective technique to isolate whole, intact vitreous core and cortex from post mortem enucleated porcine eyes. While previous studies have shown the results of such dissections, the detailed steps have not been described, precluding researchers outside the field from replicating their methods. Other studies harvest vitreous either through aspiration, which does not maintain the vitreous structure anatomy, or through partial dissection, which only isolates the vitreous core. The proposed method isolates the whole vitreous body, with the vitreous core and cortex intact, while maintaining vitreous anatomy and structural integrity. In this method, a full thickness scleral flap in an enucleated porcine eye is first created and through this, the choroid tissue can be separated from the sclera. The scleral flap is then expanded and the choroid is completely separated from the sclera. Finally the choroid-retina tissue is peeled off the vitreous to leave an isolated intact vitreous body. The proposed vitreous dissection technique can be used to study physical properties of the vitreous humor. In particular, this method has significance for experimental studies involving drug delivery, vitreo-retinal oxygen transport, and intraocular convection.

The goal of this protocol is to show an effective technique to isolate whole, intact vitreous core and cortex from post mortem enucleated porcine eyes.

Todo el humor vítreo Disección de Análisis Vitreodynamic

The authors propose an effective technique to isolate whole, intact vitreous core and cortex from post mortem enucleated porcine eyes. While previous ...

Murine Prostate Micro-dissection and Surgical Castration

Mouse models are used extensively to study prostate cancer and other diseases. The mouse is an excellent model with which to study the prostate and has been used as a surrogate for discoveries in human prostate development and disease. Prostate micro-dissection allows consistent study of lobe-specific prostate anatomy, histology, and cellular characteristics in the absence of contamination of other tissues. Testosterone affects prostate development and disease. Androgen deprivation therapy is a common treatment for prostate cancer patients, but many prostate tumors become castration-resistant. Surgical castration of mouse models allows for the study of castration resistance and other facets of hormonal biology on the prostate. This procedure can be coupled with testosterone reintroduction, or hormonal regeneration of the prostate, a powerful method to study stem cell lineages in the prostate. Together, prostate micro-dissection and surgical castration opens up a multitude of opportunities for robust and consistent research of prostate development and disease. This manuscript describes the protocols for prostate micro-dissection and surgical castration in the laboratory mouse.

This manuscript describes the protocols for prostate micro-dissection and surgical castration in the laboratory mouse. We also depict representative results produced by these protocols. Finally, we discuss the advantages and utilization of these protocols.

Murino de próstata micro-disección y la castración quirúrgica

Mouse models are used extensively to study prostate cancer and other diseases. The mouse is an excellent model with which to study the prostate and has ...

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic study in the liver. Here, numerous assays are described to comprehensively measure the phenotype and parameters of hepatic steatosis in mouse and hepatocyte models. 
Combining the physiological, histological, and biochemical methods, this system can be used to assess the progress of hepatic steatosis. In vivo, the measurements of body weight and nuclear magnetic resonance (NMR) provide a general understanding of mice in a non-invasive manner. Hematoxylin and Eosin (H&amp;E) and Oil Red O staining determine the histological morphology and lipid deposition of liver tissue under nutrient overload conditions, such as high-fat diet feeding. Next, the total lipid contents are isolated by chloroform/methanol extraction, which are followed by a biochemical analysis for triglyceride and cholesterol. Moreover, mouse primary hepatocytes are treated with high glucose plus insulin to stimulate lipid accumulation, an efficient in vitro model to mimic diet-induced hyperglycemia and hyperinsulinemia in vivo. Then, the lipid deposition is measured by Oil Red O staining and chloroform/methanol extraction. Oil Red O staining determines intuitive hepatic steatotic phenotypes, while the lipid extraction analysis determines the parameters that can be analyzed statistically. The present protocols are of interest to scientists in the fields of fatty liver diseases, insulin resistance, and type 2 diabetes.

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Here, optimized methods to generate in vivo and in vitro models of hepatic steatosis and to analyze the steatotic phenotypes and related physiological parameters are described.

Análisis optimizada de<em&gt; En Vivo</em&gt; y<em&gt; In Vitro</em&gt; La esteatosis hepática

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic ...

Dissection of the Mouse Pancreas for Histological Analysis and Metabolic Profiling

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at the 12 codon (Ptf1acre/+;LSL-KrasG12D/+) mouse strain as a model of human pancreatic cancer. The goal of our current studies is to identify novel metabolic biomarkers of pancreatic cancer progression. We have performed metabolic profiling of urine, feces, blood, and pancreas tissue extracts, as well as histological analyses of the pancreas to stage the cancer progression. The mouse pancreas is not a well-defined solid organ like in humans, but rather is a diffusely distributed soft tissue that is not easily identified by individuals unfamiliar with mouse internal anatomy or by individuals that have little or no experience performing mouse organ dissections. The purpose of this article is to provide a detailed step-wise visual demonstration to guide novices in the removal of the mouse pancreas by dissection. This article should be especially valuable to students and investigators new to research that requires harvesting of the mouse pancreas by dissection for metabolic profiling or histological analyses.

This video article provides a detailed demonstration of the procedures required to successfully remove the pancreas from a mouse by dissection for histological analysis and metabolic profiling.

Disección del páncreas de ratón para análisis histológico y perfiles metabólicos

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at ...

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a condition that typically involves damage to or loss of the delicate mechanosensory structures of the inner ear. Sophisticated in vitro and ex vivo assays have emerged in recent years, enabling the screening of an increasing number of potentially therapeutic compounds while minimizing resources and accelerating efforts to develop cures for SNHL. Though homogenous cultures of certain cell types continue to play an important role in current research, many scientists now rely on more complex organotypic cultures of murine inner ears, also known as cochlear explants. The preservation of organized cellular structures within the inner ear facilitates the in situ evaluation of various components of the cochlear infrastructure, including inner and outer hair cells, spiral ganglion neurons, neurites, and supporting cells. Here we present the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The careful preparation of these explants facilitates the identification of mechanisms that contribute to SNHL and constitutes a valuable tool for the hearing research community.

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

The goal of this protocol is to demonstrate the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The technique can be utilized as an in vitro screening tool in hearing research.

Técnica de Explante Coclear Murino Neonatal como<em&gt; In Vitro</em&gt; Herramienta de detección en la investigación de la audición

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a ...

Quantitative Micro-CT Analysis of Aortopathy in a Mouse Model of &#946;-aminopropionitrile-induced Aortic Aneurysm and Dissection

Aortic aneurysm and dissection is associated with significant morbidity and mortality in the population and can be highly lethal. While animal models of aortic disease exist, in vivo imaging of the vasculature has been limited. In recent years, micro-computerized tomography (micro-CT) has emerged as a preferred modality for imaging both large and small vessels both in vivo and ex vivo. In conjunction with a method of vascular casting, we have successfully used micro-CT to characterize the frequency and distribution of aortic pathology in &#946;-aminopropionitrile-treated C57/Bl6 mice. Technical limitations of this method include variations in the quality of the perfusion introduced by poor animal preparation, the application of proper methodologies for vessel size quantification, and the non-survivability of this procedure. This article details a methodology for the intravascular perfusion of a lead-based radiopaque silicone rubber for the quantitative characterization of aortopathy in a mouse model of aneurysm and dissection. In addition to visualizing aortic pathology, this method may be used for examining other vascular beds in vivo or vascular beds removed post-mortem.

This article describes a detailed methodology of using a radiopaque lead-based silicone rubber to perfuse the murine vasculature for aortic diameter quantification in a mouse model of aortic aneurysm and dissection.

Análisis de Micro-CT cuantitativa de Aortopathy en un modelo murino de disección y aneurisma de la aorta inducida por β-oxidasa

Aortic aneurysm and dissection is associated with significant morbidity and mortality in the population and can be highly lethal. While animal models of ...