Agradezco a Jeannie T. Lee por la supervisión de este proyecto cuando estaba en el laboratorio Lee y a Tyler Broering por editar el manuscrito. Este trabajo fue apoyado por el Basil O'Connor Starter Scholar Award de march of dimes Foundation y la nih grant GM098605.

1.3D Preparación de diapositivas
<ol><li>Prepare los siguientes reactivos:<ol>
<li>Tampón CSK: 100 mM NaCl, 300 mM sacarosa, 10 mM PIPAS, 3 mM MgCl2. Ajuste el pH a 6,8 con NaOH de 1 M. Almacene el búfer CSK a 4 °C.</li>
		<li>Tampón CSK con Tampón Tritón X-100 al 0,5%: Añadir 200 μl de Tampón X-100 a 40 ml de Tampón CSK. Mezcle la solución con agitador magnético hasta que Triton X-100 se disuelva por completo. Almacene el tampón CSK con 0,5% tritón X-100 a 4 °C.</li>
		<li>Paraformadehído al 4% (PFA)–PBS, pH 7.4: Disolver 4 g de PFA en aproximadamente 70-80 ml de agua. Para acelerar el proceso de disolución, añadir aproximadamente 20 μl de NaOH de 4 N y mantener la solución en un baño de agua a 60 °C hasta que el PFA se haya disuelto por completo y la solución sea transparente. Este proceso toma aproximadamente 1 hora. Después de que la PFA se haya disuelto por completo, añadir 10 ml de PBS 10x. Ajustar el volumen final a 100 ml con agua y enfriar la solución a temperatura ambiente (25 °C). Utilice una solución recién preparada para cada nuevo experimento. Nota: Obtenga el permiso institucional para todo el trabajo animal y se adhiera a las pautas relevantes del cuidado animal.</li>
	</ol>
</li>
	<li>Eutanasiar ratones machos. Usando las autopsas y las tijeras, elimine el testículo del ratón. Coloque el testículo en PBS (o RPMI 1640). Después de la eliminación del testículo, lleve a cabo rápidamente los siguientes pasos 1.3-1.4 para prevenir la degradación del ARN.</li>
	<li>Romper el testículo en una pequeña placa de Petri y retirar la túnica albugínea. Desentrañar los túbulos seminíferos en PBS sobre hielo usando pórceps.</li>
	<li>Usando fórceps, transfiera varios túbulos (varios trozos de túbulos de aproximadamente 5-10 mm de longitud) en un pocillo de un plato de 4 pocillos que contenga 500 ml de tampón CSK + 0,5% Triton X-100 en hielo, e incube durante 6 min. La sobreexposición al buffer CSK puede dar lugar a la interrupción de células.</li>
	<li>Usando pórceps, transfiera todos los túbulos a un pocillo de un plato de 4 pocillos que contenga una solución de PFA-PBS al 4%. Este paso se puede preformar sin un estereomicroscopio, pero opcionalmente se puede utilizar. Incubar durante 10 min a temperatura ambiente. Simultáneamente comenzar a preparar 12 cámaras de citoespino durante los períodos de incubación.</li>
	<li>Usando pórceps, transfiera todos los túbulos a un pocillo de un plato de 4 pocillos que contenga PBS. Incubar durante 5 min a temperatura ambiente.</li>
	<li>En la parte posterior de un portaobjetos de vidrio, transfiera todos los túbulos a 30 μl de PBS (la parte superior del portaobjetos debe evitarse debido a la carga catónica). Usando las puntas de dos tórceps, rompa los túbulos en pedazos. Corte los túbulos en una dirección horizontal recortando los túbulos entre las puntas de dos pinzas y tirando de los pinzas horizontalmente. Continúe con este paso durante aproximadamente 10-20 segundos.</li>
	<li>Mezcle la suspensión mediante pipeteo con una pipeta P20.</li>
	<li>Transferir la suspensión a un tubo de microcentrífuga, diluir con PBS a aproximadamente 1,3 ml. Aplicar 100 μl de suspensión en cada una de las 12 cámaras de citoespino (1,2 ml en total). En este paso, no es necesario determinar la concentración de células.</li>
	<li>Cytospin las muestras a 2.000 rpm durante 10 min a temperatura ambiente. Después del citoespinning, seque los portaobjetos en el banco del laboratorio durante unos minutos a temperatura ambiente. Después de que se haya dejado que los portaobjetos se sequen por completo durante unos minutos, vaya al siguiente paso.</li>
	<li>Usando un frasco de Coplin que contenga PBS, lave los portaobjetos durante 5 minutos a temperatura ambiente.</li>
	<li>Transfiera los portaobjetos a un frasco de Coplin que contenga etanol al 70% y espere al menos 2 minutos antes del inicio de RNA FISH. Los portaobjetos son estables durante unas semanas en etanol al 70% a 4 °C. Para el almacenamiento a largo plazo, deshidrate los portaobjetos mediante tratamiento en serie con etanol al 80% y 100% en frascos coplin durante 2 min cada uno y se seque al aire completamente deshidratar los portaobjetos. Almacenar a -80 °C en una caja deslizante.</li>
</ol>2. Preparación de la sonda de ADN Cot-1 por cebado aleatorio
<ol><li>Prepare los siguientes reactivos:<ol>
<li>Tampón de hibridación: Mezcle los componentes de procedimiento para preparar 900 ml de tampón de hibridación; esta solución se utilizará para almacenar sondas. Esta acción tampón de hibridación permite la adición de un volumen del 10% de solución inhibidora de la ARNasa (Ribonucleósido Complejo Vanadyl) antes de la hibridación.<table><tbody>
<tr>
<td>componente</td>
					<td>Cantidad (μl)</td>
					<td>Concentración final</td>
				</tr>
<tr>
<td>formamida</td>
					<td>500</td>
					<td>50% (v/v)</td>
				</tr>
<tr>
<td>50% Dextrano</td>
					<td>200</td>
					<td>10% (p/v)</td>
				</tr>
<tr>
<td>20x SSC</td>
					<td>100</td>
					<td>2x</td>
				</tr>
<tr>
<td>1% BSA</td>
					<td>100</td>
					<td>0.1%</td>
				</tr>
</tbody></table>Antes de la hibridación, agregue 1/10 vol de 200 mM Ribonucleoside Vanadyl Complex o agua a la acción tampón de hibridación para que coincida con la concentración final. A -20 °C, esta mezcla es estable durante más de un año.</li>
		<li>20x SSC: Disolver 88,2 g de citrato de sodio y 175,3 g de NaCl en aproximadamente 800 ml de agua. Ajuste el pH a 7.0 usando unas gotas de 1 M HCl. Agregue agua para ajustar el volumen final a 1 L. Esterilice por autoclave. Después de la preparación, 20x SSC se puede almacenar a temperatura ambiente.</li>
		<li>50% (p/v) Dextrano: Remover agua y añadir lentamente 50 g de sulfato de dextrano. Siga revolviendo durante la noche a temperatura ambiente. Añadir agua para ajustar el volumen final a 100 ml. Conservar a temperatura ambiente.</li>
	</ol>
</li>
	<li>Agregue los siguientes componentes que se enumeran a continuación a un tubo de PCR. Usando una máquina de PCR, mezcle e incube la solución durante 5 min a 95 °C.<table><tbody>
<tr>
<td>componente</td>
				<td align="center">Cantidad por reacción (μl)</td>
				<td align="center">final</td>
			</tr>
<tr>
<td>ADN de Cot-1: 1 mg/ml</td>
				<td align="center">1</td>
				<td align="center">1 μg</td>
			</tr>
<tr>
<td>Imprimación aleatoria de 9mers (del kit)</td>
				<td align="center">10</td>
				<td align="center"></td>
			</tr>
<tr>
<td>Agua</td>
				<td align="center">27</td>
				<td align="center"></td>
			</tr>
</tbody></table>
</li>
	<li>Centrífuga brevemente y colocar sobre hielo.</li>
	<li>Agregue los siguientes componentes. Mezclar e incubar durante 30 min a 37 °C utilizando una máquina de PCR. (Para más detalles del kit utilizado ver la tabla de reactivos y equipos).<table><tbody>
<tr>
<td>componente</td>
				<td align="center">Cantidad por reacción (μl)</td>
				<td align="center">Concentración final</td>
			</tr>
<tr>
<td>Mezcla preparada a partir de la etapa 2.2</td>
				<td align="center">38</td>
				<td align="center"></td>
			</tr>
<tr>
<td>Tampón de nucleótidos 5x (del kit)</td>
				<td align="center">9.2</td>
				<td align="center"></td>
			</tr>
<tr>
<td>Cy3-dUTP (1 mM)</td>
				<td align="center">0.8</td>
				<td align="center">μM 16</td>
			</tr>
<tr>
<td>Klenow (de kit)</td>
				<td align="center">2</td>
				<td align="center"></td>
			</tr>
</tbody></table>
</li>
	<li>Añadir 2 μl de tampón de parada (desde el kit) para interrumpir la reacción.</li>
	<li>Purificar la reacción detenida usando columnas de microspin.</li>
	<li>Añadir 50 μg (5 μl) de ADN de espermatozoides arenqueros (50 veces más de ADN Cot-1 plantilla) a la fracción purificada.</li>
	<li>Mida el volumen total con una pipeta. Añadir 0,7x volumen de etanol al 100% y 0,1x volumen de acetato de sodio 3 M (pH 5,2), mezclar bien y centrífuga durante 15 min a velocidad máxima (17.000 x g) utilizando una microcentrífuga a 4 °C.</li>
	<li>Retire el líquido, y agregue 200 μl de etanol al 70%, mezcle bien y centrífuga durante 5 min a la velocidad máxima a 4 °C.</li>
	<li>Retire el exceso de líquido. Seque el precipitado durante 10-30 min a temperatura ambiente.</li>
	<li>Disuelva el pellet en 20 μl de hibridación de tampón. Debido a que el pellet es a menudo difícil de disolver, pipeta repetidamente. Como opción alternativa, un mezclador de vórtice también se puede utilizar para disolver eficientemente el pellet. Las sondas se pueden mantener a -20 °C hasta su uso.</li>
</ol>3. Cuna-1 ARN PESCADO
<ol><li>Agregue los componentes que se enumeran a continuación a un tubo de PCR. Usando una máquina de PCR, incubar durante 10 min a 80 °C, seguido de un paso previo de aproximadamente 10-30 min a 42 °C. Mantener a 42 °C hasta la hibridación. Ribonucleoside Vanadyl Complex es opcional; se puede reemplazar con agua.<table><tbody>
<tr>
<td>componente</td>
				<td align="center">Cantidad por reacción (μl)</td>
				<td align="center">final</td>
			</tr>
<tr>
<td>Sonda de ADN Cot-1 (50 ng/μl)</td>
				<td align="center">2</td>
				<td align="center">100 ng</td>
			</tr>
<tr>
<td>Complejo Ribonucleósido Vanadyl (200 mM)</td>
				<td align="center">2</td>
				<td align="center">20 mM</td>
			</tr>
<tr>
<td>Búfer de hibridación</td>
				<td align="center">agregado a 20</td>
				<td align="center"></td>
			</tr>
</tbody></table>
</li>
	<li>Deshidrate los portaobjetos en un frasco de Coplin que contiene etanol del 80% por 2 minutos, seguido por un frasco de Coplin que contiene el etanol del 100% por 2 minutos. Secar completamente en el banco de laboratorio a temperatura ambiente.</li>
	<li>Precaliente una cámara de caja de punta (llena de 2-3 cm de agua) en una incubadora de 42 °C por adelantado. Coloque la diapositiva en la cámara de la caja de la punta. Centrifugar brevemente las sondas preannealed y pipeta directamente sobre las diapositivas deshidratadas. Evite hacer burbujas. Cubra suavemente la corredera con un resbalón de cubierta.</li>
	<li>Hibridar durante 6 horas a la noche (14-15 horas) a 42 °C.</li>
	<li>Prepare soluciones de lavado en frascos Coplin (dos frascos con 2x SSC y 50% formamida, y dos frascos con 2x SSC). Precalentar a 42 °C durante 30 min en un baño de agua.</li>
	<li>Utilice una cuchilla de afeitar para quitar el resbalón de la cubierta del borde de la corredera. Evite rascarse las muestras. Lave los portaobjetos en un frasco de Coplin que contenga 2x SSC y 50% de formamida durante 5 min a 45 °C. Repita este paso una vez.</li>
	<li>Lave los portaobjetos en un frasco de Coplin que contenga 2x SSC durante 5 minutos a 45 °C. Repita este paso una vez.</li>
	<li>Agregue 20 μl de soporte de montaje DAPI a las guías y cubra con un resbalón de cubierta. Presione suavemente el vidrio de la cubierta para quitar los medios de montaje adicionales. Blot medios de montaje adicionales con tejido de limpieza. Los portaobjetos están listos para la evaluación de microscopía.</li>
</ol>

<ol>
	<li>Peters, A. H., Plug, A. W., van Vugt, M. J., de Boer, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+drying-down+technique+for+the+spreading+of+mammalian+meiocytes+from+the+male+and+female+germline.">A drying-down technique for the spreading of mammalian meiocytes from the male and female germline.</a> Chromosome Res. 5, 66-68 (1997).</li><li>Namekawa, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postmeiotic+sex+chromatin+in+the+male+germline+of+mice.">Postmeiotic sex chromatin in the male germline of mice.</a> Curr Biol. 16, 660-667 (2006).</li><li>Namekawa, S. H., Lee, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+nascent+RNA,+single-copy+DNA+and+protein+localization+by+immunoFISH+in+mouse+germ+cells+and+preimplantation+embryos.">Detection of nascent RNA, single-copy DNA and protein localization by immunoFISH in mouse germ cells and preimplantation embryos.</a> Nat Protoc. 6, 270-284 (2011).</li><li>Mahadevaiah, S. K., Costa, Y., Turner, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+RNA+FISH+to+study+gene+expression+during+mammalian+meiosis.">Using RNA FISH to study gene expression during mammalian meiosis.</a> Methods in molecular biology. 558, 433-444 (2009).</li><li>Hall, L. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ectopic+human+XIST+gene+can+induce+chromosome+inactivation+in+postdifferentiation+human+HT-1080+cells.">An ectopic human XIST gene can induce chromosome inactivation in postdifferentiation human HT-1080 cells.</a> Proc Natl Acad Sci U S A. 99, 8677-8682 (2002).</li><li>Huynh, K. D., Lee, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inheritance+of+a+pre-inactivated+paternal+X+chromosome+in+early+mouse+embryos.">Inheritance of a pre-inactivated paternal X chromosome in early mouse embryos.</a> Nature. 426, 857-862 (2003).</li><li>Namekawa, S. H., VandeBerg, J. L., McCarrey, J. R., Lee, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex+chromosome+silencing+in+the+marsupial+male+germ+line.">Sex chromosome silencing in the marsupial male germ line.</a> Proceedings of the National Academy of Sciences of the United States of America. 104, 9730-9735 (2007).</li><li>Namekawa, S. H., Payer, B., Huynh, K. D., Jaenisch, R., Lee, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-step+imprinted+X+inactivation:+repeat+versus+genic+silencing+in+the+mouse.">Two-step imprinted X inactivation: repeat versus genic silencing in the mouse.</a> Mol Cell Biol. 30, 3187-3205 (2010).</li><li>Ichijima, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MDC1+directs+chromosome-wide+silencing+of+the+sex+chromosomes+in+male+germ+cells.">MDC1 directs chromosome-wide silencing of the sex chromosomes in male germ cells.</a> Genes Dev. 25, 959-971 (2011).</li><li>Sin, H. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNF8+regulates+active+epigenetic+modifications+and+escape+gene+activation+from+inactive+sex+chromosomes+in+post-meiotic+spermatids.">RNF8 regulates active epigenetic modifications and escape gene activation from inactive sex chromosomes in post-meiotic spermatids.</a> Genes Dev. 26, 2737-2748 (2012).</li></ol>

El autor no tiene nada que revelar.

En la preparación de diapositivas 3D, el paso de permeabilización precede al paso de fijación. Estos pasos se realizan directamente sobre túbulos seminíferos, preservando así la estructura tridimensional de la cromatina. Una opción alternativa para preservar la estructura de la cromatina para el ARN/ADN FISH es realizar la etapa de permeabilización y la etapa de fijación simultáneamente. Esta técnica alternativa se ha realizado en células germinales marsupiales y en embriones preimplantacional de ratón7...

En los testículos, las células germinales se diferencian de la espermatogonia diploide a través de la meiosis en espermatozoides maduros y haploides. Durante este proceso, la estructura de la cromatina nuclear de las células germinales se remodela de forma continua y dinámica. Las separaciones superficiales son de uso general para la examinación citológica de células espermatogénicas individuales. Un método predominante de diseminaciones superficiales emplea el tratamiento hipotónico por el cual las células espermatogénicas individuales se propagan y aplanan1. Estas condiciones son óptimas para el análisis detallado de cromosomas meióticos. Las características cromosómicas tales como focos sinápticos de la situación y de la recombinación se observan fácilmente usando este método. Sin embargo, el tratamiento hipotónico interrumpe la arquitectura de la cromatina subnuclear, por lo que esta técnica no es adecuada para el análisis estructural de núcleos. Por lo tanto, un método mejorado se ha diseñado para preservar la estructura tridimensional de la cromatina de células de germen testiculares. Este método se ha denominado como el método de diapositiva tridimensional (3D) (Figura 1). El método de deslizamiento 3D ha permitido la detección de localización de ARN naciente en núcleos, ya que inicialmente se optimizó para examinar la expresión génica y los estados de cromatina durante la diferenciación de células germinales testiculares por hibridación in situ de fluorescencia de ARN (FISH)2,3. Este método 3D también es aplicable a la combinación de inmunofluorescencia, ADN y ARN FISH. Además, este método ha ayudado en el descubrimiento de la cromatina sexual postmeiótica (EMPS), un compartimento silencioso de los cromosomas sexuales que se encuentran en las espermicidas postmeióticas2.
El método de deslizamiento 3D se optimizó originalmente a través de la combinación de dos pasos esenciales de preparación de diapositivas comúnmente utilizados para la tinción nuclear: el paso de fijación diseñado para fijar materiales nucleares y el paso de permeabilización destinado a eliminar materiales citoplasmáticos para mejorar la accesibilidad de los reactivos de tinción, como anticuerpos y sondas FISH. Como se describió anteriormente en otra publicación3,se ha determinado que la etapa de permeabilización debe preceder a la etapa de fijación con el fin de obtener resultados óptimos con bajo fondo citoplasmático. En este método de deslizamiento 3D, el paso de permeabilización y el paso de fijación posterior se realizan directamente en túbulos seminíferos y son seguidos por la disociación mecánica de células germinales con fórceps antes de citoespinning en diapositivas. Un método alternativo para los PECES DE ARN de las células espermatogénicas se desarrolló en otro laboratorio4. En este método, consistente con el método 3D, el paso de permeabilización debe preceder al paso de fijación para obtener resultados óptimos de RNA FISH.
El siguiente protocolo describe el método de deslizamiento 3D y proporciona un ejemplo de una posible aplicación, Cot-1 RNA FISH para la detección de ARN nucleares nacientes. El ADN de cot-1 consiste en elementos repetitivos en el genoma. Aquí, una sonda de ADN Cot-1 se hibrida con un intrón y UTR de las transcripciones nacientes, detectando así las regiones transcripcionalmente activas en el núcleo5,6. Las diapositivas 3D son versátiles y se pueden aplicar a una combinación de técnicas de inmunofluorescencia, ADN y ARN FISH para obtener un examen detallado de las relaciones espaciales entre la arquitectura de la cromatina.

<table border="0" cellpadding="0" cellspacing="0" width="562">
	<colgroup>
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Cot-1 DNA&#160;</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:119px;">18440-016</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stratagene Prime-It Fluor Fluorescence Labeling Kit</td>
			<td style="width:147px;">Agilent</td>
			<td align="right" style="width:119px;">
			300380
			</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Herring sperm DNA Solution</td>
			<td style="width:147px;">Invitrogen</td>
			<td>15634-017</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol, 200 proof (absolute)</td>
			<td>Pharmco-AAPER</td>
			<td align="right">111000200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium acetate</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">S2889</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Formamide deionized</td>
			<td>American Bioanalytical</td>
			<td>AB00600-00500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium citrate tribasic dihydrate</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">C8532</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Bovine serum albumin</td>
			<td style="width:147px;">Lifeblood Medical</td>
			<td style="width:119px;">77110-100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dextran sulfate sodium salt from Leuconostoc spp.</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">D6001</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:119px;">A10491-01</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">S0389</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">PIPES</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">P6757</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium chloride hexahydrate</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">M0250</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">T8787</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td align="right" style="width:119px;">76240</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Sodium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td align="right" style="width:119px;">221465</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Phosphate buffered saline</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">P4417</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">VECTASHIELD Mounting Medium with DAPI</td>
			<td style="width:147px;">Vector Laboratories</td>
			<td style="width:119px;">H1200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ribonucleoside-vanadyl complex</td>
			<td>New England Biolabs</td>
			<td style="width:119px;">S1402S</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Illustra MicroSpin G-50 Columns</td>
			<td>GE Healthcare</td>
			<td style="width:119px;">27-5330-01</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Microcentrifuge</td>
			<td style="width:147px;">Eppendorf</td>
			<td align="right" style="width:119px;">5424</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Forceps</td>
			<td style="width:147px;">VWR</td>
			<td style="width:119px;">300-050</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Microcentrifuge tubes</td>
			<td style="width:147px;">Bioexpress</td>
			<td style="width:119px;">C-3262-1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Cytospin 3</td>
			<td style="width:147px;">Shandon</td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Coplin jar</td>
			<td style="width:147px;">Fisher</td>
			<td style="width:119px;">08-815</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Superfrost /Plus Microscope Slides</td>
			<td>Fisher</td>
			<td>12-550-15</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-well dish</td>
			<td>Fisher</td>
			<td style="width:119px;">12-566-301</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cover glass&#160;</td>
			<td>Fisher</td>
			<td style="width:119px;">12-544-G</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:297px;">Air incubator</td>
			<td style="width:147px;">Revolutionary Science</td>
			<td style="width:119px;">RS-IF-203</td>
		</tr>
	</tbody>
</table>

slide preparation method to preserve three-dimensional chromatin architecture of testicular germ cells

Durante la diferenciación testicular de la célula de germen, la estructura de la cromatina nuclear cambia dinámicamente. A continuación se describe un método diseñado para preservar la disposición tridimensional de la cromatina de las células germinales testiculares que se encuentran en ratones; este método se ha denominado como el método de diapositiva tridimensional (3D). En este método, los túbulos testiculares se tratan directamente con un paso de permeabilización que elimina el material citoplasmático, seguido de un paso de fijación que fija los materiales nucleares. Los túbulos se disocian, la suspensión celular es citospun, y las células se adhieren a los portaobjetos. Este método mejora la sensibilidad hacia la detección de estructuras subnucleares y es aplicable para la inmunofluorescencia, el ADN y la hibridación in situ de fluorescencia de ARN (FISH) y la combinación de estos métodos de detección. Como ejemplo de una posible aplicación del método de deslizamiento 3D, se muestra que un COT-1 RNA FISH detecta ARN nacientes. El método de deslizamiento 3D facilitará el examen detallado de las relaciones espaciales entre la estructura de la cromatina, el ADN y el ARN durante la diferenciación testicular de las células germinativas.

Un resultado representativo de una diapositiva 3D se muestra en la Figura 2. En este experimento, cot-1 ARN FISH se realizó para detectar la transcripción naciente junto con inmunostaining utilizando anticuerpos anti CBX1 y γH2AX. Para combinar el ARN FISH y el immunostaining, el immunostaining fue realizado primero, seguido por el PESCADO del ARN según lo descrito en3. CBX1 es una proteína heterocromatina que se localiza en heterocromatina pericentromérica y cromosomas sexuales silencio...

Watch this Scientific Journal Video about Slide Preparation Method to Preserve Three-dimensional Chromatin Architecture of Testicular Germ Cells at JoVE.com

Slide Preparation Method to Preserve Three-dimensional Chromatin Architecture of Testicular Germ Cells

El material aquí describe un método desarrollado para preservar la estructura tridimensional de la cromatina de las células germinales testiculares. Esto se ha denominado el método de diapositiva tridimensional (3D). Este método mejora la sensibilidad para la detección de estructuras subnucleares y es aplicable para la inmunofluorescencia, el ADN y la hibridación in situ de fluorescencia de ARN (FISH).

Método de preparación de diapositivas para preservar la arquitectura tridimensional de la cromatina de las células germinales testiculares

During testicular germ cell differentiation, the structure of nuclear chromatin dynamically changes. The following describes a method designed to preserve ...

slide-preparation-method-to-preserve-three-dimensional-chromatin

Research

JoVE Journal

Biology

7.3K vistas.  Cincinnati Children's Hospital Medical Center. El material aquí describe un método desarrollado para preservar la estructura tridimensional de la cromatina de las células germinales testiculares. Esto se ha denominado el método de diapositiva tridimensional (3D). Este método mejora la sensibilidad para la detección de estructuras subnucleares y es aplicable para la inmunofluorescencia, el ADN y la hibridación in situ de fluorescencia de ARN (FISH).

Video: Slide Preparation Method to Preserve Three-dimensional Chromatin Architecture of Testicular Germ Cells

Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell cultivation. These technologies offer a wide spectrum of advantages not only for manufacturing but also for different applications. The size and shape of formed cell clusters can be influenced by the exact and reproducible architecture of the microfabricated scaffold and, therefore, the diffusion path length of nutrients and gases can be controlled.1 This is unquestionably a useful tool to prevent apoptosis and necrosis of cells due to an insufficient nutrient and gas supply or removal of cellular metabolites.
Our polymer chip, called CellChip, has the outer dimensions of 2 x 2 cm with a central microstructured area. This area is subdivided into an array of up to 1156 microcontainers with a typical dimension of 300 m edge length for the cubic design (cp- or cf-chip) or of 300 m diameter and depth for the round design (r-chip).2
So far, hot embossing or micro injection moulding (in combination with subsequent laborious machining of the parts) was used for the fabrication of the microstructured chips. Basically, micro injection moulding is one of the only polymer based replication techniques that, up to now, is capable for mass production of polymer microstructures.3 However, both techniques have certain unwanted limitations due to the processing of a viscous polymer melt with the generation of very thin walls or integrated through holes. In case of the CellChip, thin bottom layers are necessary to perforate the polymer and provide small pores of defined size to supply cells with culture medium e.g. by microfluidic perfusion of the containers.
In order to overcome these limitations and to reduce the manufacturing costs we have developed a new microtechnical approach on the basis of a down-scaled thermoforming process. For the manufacturing of highly porous and thin walled polymer chips, we use a combination of heavy ion irradiation, microthermoforming and track etching. In this so called "SMART" process (Substrate Modification And Replication by Thermoforming) thin polymer films are irradiated with energetic heavy projectiles of several hundred MeV introducing so-called "latent tracks" Subsequently, the film in a rubber elastic state is formed into three dimensional parts without modifying or annealing the tracks. After the forming process, selective chemical etching finally converts the tracks into cylindrical pores of adjustable diameter.

We present two processes for the microfabrication of porous polymer chips for three-dimensional cell cultivation. The first one is hot embossing combined with a solvent vapour welding process. The second one uses a recently developed microthermoforming process combined with ion track technology leading to a significant simplification of manufacture.

Microfabricación de chip del tamaño de andamios para el cultivo de células en tres dimensiones

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell ...

Investigación

Biología

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

Hi-C: Un método para estudiar la arquitectura tridimensional de los genomas.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

Aislamiento de células epiteliales mamarias de tridimensional mixto de células Esferoide Co-cultura

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

Simple and Efficient Technique for the Preparation of Testicular Cell Suspensions

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline cells. This heterogeneity is an important drawback concerning the study of the bases of mammalian spermatogenesis, as pure or enriched cell populations in certain stages of sperm development are needed for most molecular analyses1.
 Various strategies such as Staput2,3, centrifugal elutriation1, and flow cytometry (FC)4,5 have been employed to obtain enriched or purified testicular cell populations in order to enable differential gene expression studies. 
It is required that cells are in suspension for most enrichment/ purification approaches. Ideally, the cell suspension will be representative of the original tissue, have a high proportion of viable cells and few multinucleates - which tend to form because of the syncytial nature of the seminiferous epithelium6,7 - and lack cell clumps1 . Previous reports had evidenced that testicular cell suspensions prepared by an exclusively mechanical method clumped more easily than trypsinized ones1 . On the other hand, enzymatic treatments with RNAses and/or disaggregating enzymes like trypsin and collagenase lead to specific macromolecules degradation, which is undesirable for certain downstream applications. The ideal process should be as short as possible and involve minimal manipulation, so as to achieve a good preservation of macromolecules of interest such as mRNAs. Current protocols for the preparation of cell suspensions from solid tissues are usually time-consuming, highly operator-dependent, and may selectively damage certain cell types1,8 . 
	The protocol presented here combines the advantages of a highly reproducible and extremely brief mechanical disaggregation with the absence of enzymatic treatment, leading to good quality cell suspensions that can be used for flow cytometric analysis and sorting4, and ulterior gene expression studies9 .

A novel protocol for the mechanical preparation of testicular cell suspensions from rodent material, avoiding enzymes and detergents, is described. The method is very simple, fast, reproducible, and renders good quality cell suspensions, which are suitable for flow sorting and RNA extraction.

Técnica sencilla y eficaz para la preparación de suspensiones de células testiculares

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline ...

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues.
Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

We provide here an efficient and reliable protocol for immunostaining, Fluorescence in&#160;situ Hybridization, DNA staining followed by quantitative, high-resolution imaging in whole-mount Arabidopsis thaliana ovules. This method was successfully used to analyze chromatin modifications and nuclear architecture.

Un método eficaz para Cuantitativo, Análisis de celda única de la cromatina modificación y Arquitectura Nuclear en Todo el montaje en Óvulos<em&gt; Arabidopsis</em

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the ...

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

FRET Imaging en hidrogeles tridimensionales

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...

Three-dimensional Reconstruction of the Vascular Architecture of the Passive CLARITY-cleared Mouse Ovary

The ovary is the main organ of the female reproductive system and is essential for the production of female gametes and for controlling the endocrine system, but the complex structural relationships and three-dimensional (3D) vasculature architectures of the ovary are not well described. In order to visualize the 3D connections and architecture of blood vessels in the intact ovary, the first important step is to make the ovary optically clear. In order to avoid tissue shrinkage, we used the hydrogel fixation-based passive CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/ Immunostaining/In situ-hybridization-compatible Tissue Hydrogel) protocol method to clear an intact ovary. Immunostaining, advanced multiphoton confocal microscopy, and 3D image-reconstructions were then used for the visualization of ovarian vessels and follicular capillaries. Using this approach, we showed a significant positive correlation (P &#60;0.01) between the length of the follicular capillaries and volume of the follicular wall.

Here we present an adaptation of the passive CLARITY and 3D reconstruction method for visualization of the ovarian vasculature and follicular capillaries in intact mouse ovaries.

Reconstrucción tridimensional de la arquitectura Vascular del ovario de ratón pasivo claridad-despejado

The ovary is the main organ of the female reproductive system and is essential for the production of female gametes and for controlling the endocrine ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipo-claro: Un tejido claro método de proyección de imagen tridimensional del tejido adiposo

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

Single-Cell Resolution Three-Dimensional Imaging of Intact Organoids

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development and function as well as disease. Fluorescence microscopy has played a major role in characterizing their cellular composition in detail and demonstrating their similarity to the tissue they originate from. In this article, we present a comprehensive protocol for high-resolution 3D imaging of whole organoids upon immunofluorescent labeling. This method is widely applicable for imaging of organoids differing in origin, size and shape. Thus far we have applied the method to airway, colon, kidney, and liver organoids derived from healthy human tissue, as well as human breast tumor organoids and mouse mammary gland organoids. We use an optical clearing agent, FUnGI, which enables the acquisition of whole 3D organoids with the opportunity for single-cell quantification of markers. This three-day protocol from organoid harvesting to image analysis is optimized for 3D imaging using confocal microscopy.

The entire 3D structure and cellular content of organoids, as well as their phenotypic resemblance to the original tissue can be captured using the single-cell resolution 3D imaging protocol described here. This protocol can be applied to a wide range of organoids varying in origin, size and shape.

Imagen tridimensional de resolución de una sola célula de organoides intactos

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development ...

Evaluating the Angiogenetic Properties of Ovarian Cancer Stem-Like Cells using the Three-Dimensional Co-Culture System, NICO-1

Cancer stem cells (CSCs) reside in a supportive niche, constituting a microenvironment comprised of adjacent stromal cells, vessels, and extracellular matrix. The ability of CSCs to participate in the development of endothelium constitutes an important characteristic that directly contributes to the general understanding of the mechanisms of tumorigenesis and tumor metastasis. The purpose of this work is to establish a reproducible methodology to investigate the tumor-initiation capability of ovarian cancer stem cells (OCSCs). Herein, we examined the neovascularization mechanism between endothelial cells and OCSCs along with the morphological changes of endothelial cells using the in vitro co-culture model NICO-1. This protocol allows visualization of the neovascularization step surrounding the OCSCs in a time course manner. The technique can provide insight regarding the angiogenetic properties of OCSCs in tumor metastasis.

Ovarian cancer stem cells (OCSC) are responsible for cancer initiation, recurrence, therapeutic resistance, and metastasis. The OCSC vascular niche is considered to promote self-renewal of OCSCs, leading to chemoresistance. This protocol provides the basis for establishing a reproducible OCSC vascular niche model in vitro.

Evaluación de las propiedades angiogenéticas de las células madre similares al cáncer de ovario utilizando el sistema de cocultivo tridimensional, NICO-1

Cancer stem cells (CSCs) reside in a supportive niche, constituting a microenvironment comprised of adjacent stromal cells, vessels, and extracellular ...