We gratefully acknowledge Dr. Deborah L. Gumucio as our advisor and for her invaluable support in defining the culture and imaging methods. We also thank Dr. Jim Brodie, Dr. Hong-Xiang Lu, Dr. Charlotte Mistretta, and Dr. Ann Grosse for their contributions to the development of the whole intestine organ culture system. Helpful discussions on imaging provided excellent advice from Dr. Chip Montrose, Michael Czerwinski and Sasha Meshinchi. All imaging was performed in the Microscopy and Image Analysis Laboratory at the University of Michigan. Funding support was provided by NIH R01 DK065850.

NOTA: Todos los ratones fueron manejados con humanidad utilizando los protocolos aprobados por la Universidad de Michigan Medical School Unidad de Laboratorio de Medicina Animal y de acuerdo con las directrices del Comité de la Universidad sobre el uso y cuidado de los animales. 1. System Organ Culture Whole <ol><li> Preparación de medios de comunicación y placas de cultivo <ol><li> En una campana de cultivo de tejidos, eliminar 5 ml de medio BGJb de la botella stock y añadir 5 ml de penicilina / estreptomicina. Prepare una solución de trabajo de los medios de comunicación en un tubo cónico de 50 ml, mediante la adición de 1 ml de 5 mg / ml de ácido ascórbico a 49 ml de medio BGJb (con Pen / Strep). </li><li> Preparar una placa de cultivo de 6 pocillos mediante la adición de 1 ml por debajo de cada uno de los cultivos polarizados. NOTA: Si no se utilizan los 6 transwells, mover transwells adicionales en una placa de 6 pocillos estéril fresco para su uso posterior. Añadir 3 ml de los medios de trabajo BGJb a cada una de tres placas de Petri de 10 cm estériles. </li></ol></li><li> Preparación para la disección <ol><li> Remoje las herramientas de disección en el 70% ethanol y asegúrese de mantener las herramientas limpias durante el trabajo. </li><li> Configurar la zona de disección con un gran cubo de hielo y colocar la placa de cultivo de 6 pocillos y 10 cm placas de Petri con medios BGJb sobre hielo. Trabajar en el hielo tanto como sea posible mientras que la disección y preparación de los intestinos para la cultura. </li><li> Anestesiar a los ratones hembra adulta en una cámara con isofluorano (100 l) antes de la eutanasia por dislocación cervical para la recolección de los intestinos fetales. </li><li> Después de cosechar los fetos de su madre, etapa cada feto cuidadosamente según la Theiler puesta en escena gráfica 13. No confíe en los días post-coito para determinar la edad de cada feto como variaciones de hasta 24 horas en la etapa de desarrollo se observan habitualmente en una sola camada. </li></ol></li><li> La disección de los intestinos fetales <ol><li> La eutanasia a los fetos por decapitación antes de la extracción de los intestinos. </li><li> Diseccionar cada intestino en 1x de Dulbecco tamponada con fosfato salino (DPBS) y luego place en una placa de Petri de 10 cm con el trabajo de medios BGJb. </li><li> Retire cuidadosamente el bazo desde el estómago y el páncreas desde el estómago y el duodeno superior. </li><li> Separar suavemente el epiplón teniendo cuidado de dejar el epiplón adjunta tanto como sea posible y la separación de las conexiones sólo lo suficiente para permitir que el intestino para ser enderezada. NOTA: Para intestinos mayores de E14.5 o los que están siendo cultivados más de 24 horas, separar suavemente el intestino en 3 segmentos iguales para permitir el contenido luminal fluyan a través del intestino y ser expulsados ​​de los extremos cortados. Sin embargo, para que las culturas se inició antes de E13.5, espere hasta después de 24 horas de cultivo antes de la separación de los 3 segmentos del intestino para permitir la serosa crezca adecuadamente en toda la longitud del intestino. </li><li> Usar una pipeta de boca para transferir las piezas intestinales en los cultivos polarizados de la placa de cultivo (1.1.2). </li><li> Reposicionar suavemente los intestinos a medida que sea necesario para que estén en línea recta la Transwell. NOTA: Una vez que los intestinos comienzan a moverse contenido luminal a través del tubo, cualquier torcedura en el intestino provocarán una copia de seguridad y daños en el intestino. </li></ol></li><li> Cultura y tratamiento <ol><li> Retire el papel de debajo de la transwell, añadir 700 l de medio de tratamiento (los medios de comunicación que trabajan con proteínas recombinantes añadidos o inhibidores farmacológicos) bajo la transwell. </li><li> Añadir 300 l de medio de tratamiento gota a gota en la parte superior de los intestinos y deje que penetre a través de la transwell. Vuelva a colocar el intestino en caso necesario. </li><li> Cambie medio de tratamiento por lo menos una vez al día o más a menudo, dependiendo de la vida media del reactivo de tratamiento. </li></ol></li><li> Preparación de proteínas empapado perlas de agarosa para una fuente localizada de reactivo de tratamiento <ol><li> Resuspender las perlas de agarosa en su recipiente de almacenamiento y la transferencia de 100 l de perlas de agarosa en un tubo de microcentrífuga de 1,5 ml. </li><li> Llenar el volumen restante del tubo con 1x estéril Phosphate-Buffered Saline (PBS) y mezclar las perlas para lavarlos. Colocar el tubo de microcentrífuga en un bastidor por unos pocos minutos para permitir que las perlas se depositan en el fondo y luego pipetee de la PBS en la parte superior de la de las perlas. Vuelva a llenar el tubo de microcentrífuga con 1x PBS estéril y repetir el proceso de lavado dos veces más. </li><li> Preparar la proteína de interés a dos veces la concentración deseada para el tratamiento. </li><li> Mezclar 5 l de perlas de agarosa lavadas con 5 l de la proteína de stock 2x y agite suavemente las perlas en la proteína. Se coloca el tubo en hielo durante 1 hora, mezclando suavemente cada 10-15 min. </li><li> Enjuague las perlas brevemente en PBS 1x estéril antes de colocar las perlas en los intestinos. </li></ol></li><li> La colocación de perlas de agarosa <ol><li> Con suavidad, coloque las bolas de agarosa en la parte superior de los intestinos usando una pipeta de boca con agujas capilares tirados. Coloca las bolitas con extremo cuidado ya que el manejo brusco puede dañar el intestino y prevenir el desarrollo de vellosidades en el área lesionada. </li><li> Coloque el doble de cuentas que sean necesarios para el análisis, ya que los intestinos se someterán a los movimientos peristálticos y aproximadamente la mitad de los granos colocados rodará fuera de los intestinos durante el tiempo de cultivo. </li></ol></li><li> La fijación de los intestinos cultivadas <ol><li> Retire con cuidado los medios de tratamiento de debajo de la transwell y permitir que los intestinos se sequen al aire durante 3 min. </li><li> Añadir 1 ml de fijador bajo el transwell durante 2 min, luego cubrir la parte superior del intestino con 2 ml de fijador para el resto del tiempo de fijación. Fijar con paraformaldehído al 4% O / N a 4 ° C o durante 2 horas a temperatura ambiente. </li></ol></li></ol> 2. Imaging de intestinos fijos <ol><li> Imágenes Wholemount <ol><li> Coloque el conjunto de los intestinos (con fluorescencia endógena) en un portaobjetos con 100 l de 1x DPBS. El uso de fórceps para arreglar suavemente los intestinos. </li><li> Utilice un pañuelo de papel de laboratorio para retirar cuidadosamente los DPBS y añadir inmediatamente 100 l de 70% de glicerol para hacer el mo tejidovolver transparente y aumentar la posible profundidad de formación de imágenes. NOTA: Si se desea una mayor penetración del intestino, en lugar de montar el intestino en un 70% de glicerol, empape el intestino en unas gotas de Focus Claro solución para 10-30 min. Pipeta de fuera de la solución de enfoque claro y montar el intestino con el Monte Claro. </li><li> Sumerja cada una de las cuatro esquinas de un cubreobjetos en plastilina y recoger una pequeña cantidad de arcilla para usar como separadores entre el portaobjetos y cubreobjetos para mantener el cubreobjetos de aplanamiento del intestino. </li><li> Sellar el cubreobjetos sobre el portaobjetos con valap derretida aplicada con un bastoncillo de algodón. </li><li> Imagen de los intestinos a ambos un microscopio confocal en posición vertical o invertida. Por favor refiérase a la discusión para más detalles. </li></ol></li><li> Seccionamiento Vibratome de intestinos fijos (para vista en sección transversal de las estructuras intestinales) <ol><li> Intestinos Insertar en el 7% de agarosa en pequeños moldes de plástico (10 x 10 x 15 mm) y permitir que los bloques de agarosa aenfriar y solidificar. </li><li> Retire los bloques de agarosa de los moldes de plástico y pegamento bloques de agarosa a la etapa de recolección vibratome con pegamento instantáneo. </li><li> Llenar la etapa de recolección de frío hielo 1x DPBS. </li><li> Cortar secciones con un espesor de entre 100-150 micras. Por secciones más gruesas utilizan las soluciones de compensación (que se describen en la sección 2.1.2) para visualizar más en la sección transversal. </li><li> Vierta vibratome secciones de la etapa de recolección en un pocillo de una placa de 6 pocillos para el almacenamiento a 4 ° C. </li></ol></li><li> La tinción de inmunofluorescencia de tejidos, vibratome seccionadas fijos <ol><li> Coloque 5-12 secciones vibratome por pocillo en una placa de 24 pocillos con 1x DPBS. </li><li> Retire 1x DPBS y añadir 500 l de solución de permeabilización por pocillo. Agite suavemente las muestras durante 25 minutos a temperatura ambiente. </li><li> Después de permeabilización, lavar las muestras 3 veces con 1 × PBS. </li><li> Bloquee las muestras durante al menos 30 min en 500 l de disolución de bloqueo. </li><li> Después del bloqueo, eXchange la solución de bloqueo con 500 l de anticuerpos primarios diluidos en solución de bloqueo y mantener las muestras a 4 ° CO / N. NOTA: Las diluciones de anticuerpos primarios que funcionan bien en las secciones congeladas y parafina generalmente funcionan bien en vibratome secciones también, sin embargo, los anticuerpos que requiere la recuperación de antígeno nuclear generalmente no funcionan bien con este protocolo (por ejemplo, BrdU). </li><li> El día siguiente, se lavan las muestras 3 veces durante 15 min cada vez con solución de bloqueo. </li><li> Después del lavado, se aplican 500 l del anticuerpo secundario diluido en solución de bloqueo. Envolver la placa de 24 pocillos en papel de aluminio para proteger los fluoróforos de la luz y el rock las muestras durante 45 min a 2 horas a RT. </li><li> Lávese las muestras 3 veces con 1 × PBS durante 15 minutos cada uno y montar las secciones vibratome en las diapositivas como se describe en la Sección 2.4. </li></ol></li><li> Montaje vibratome secciones <ol><li> Preparar en otro portaobjetos para vibratome montaje cortando lonchas finas de doble stick y la colocación de la cinta a lo largo de los lados largos de las diapositivas. </li><li> Coloque cuidadosamente 5-10 secciones vibratome entre la cinta de doble cara en las diapositivas en una gota de 100 l de PBS 1x. </li><li> Desplegar todas las secciones y se extendió a cabo en una sola capa en toda la diapositiva. </li><li> Elimine cualquier exceso de agarosa o trozos irregulares que impida un cubreobjetos de sentarse plana. </li><li> Con cuidado, usar un pañuelo desechable de laboratorio para absorber el exceso de 1x PBS desde alrededor de las secciones vibratome. </li><li> Añadir una gota de medio de montaje acuoso en la parte superior de las secciones de vibratome y luego cubreobjetos. </li><li> Sellar el cubreobjetos sobre los portaobjetos con derretida valap aplica con un algodón. </li><li> Imagen secciones vibratome a ambos un microscopio confocal vertical o invertida. Por favor refiérase a la discusión para más detalles sobre la selección de un sistema de imágenes. </li></ol></li></ol> 3. imágenes en vivo de los intestinos enteros Intestinos cosechado de fetuses después de E12.5, con el epiplón conectado deja intacto, desarrollar con éxito las vellosidades en la cultura con el sistema anterior. Por lo tanto, este sistema ex vivo permite la captura de imágenes de sección z vivo de los intestinos en desarrollo que pueden ser reconstruidas para una vista tridimensional de la morfogénesis en el tiempo. <ol><li> La creación de imágenes en directo <ol><li> Utilice pegamento instantáneo para adherir una membrana de policarbonato individuo a una pantalla de malla fina. Esto proporciona un andamio de apoyo para mantener el intestino en su lugar debajo de la lente de inmersión de microscopio confocal vertical. Por favor refiérase a la sección de discusión para más detalles sobre la selección de los microscopios de imágenes en directo. </li><li> Coloque la pantalla de malla en el centro de los pocillos de una placa de cultivo. </li><li> Coloque el intestino diseccionado sobre la membrana de policarbonato y añadir una gota de pegamento instantáneo en cada extremo. Utilice sólo el pegamento suficiente para colocar el intestino, pero no abrigo! </li><li> Añadir cultura libre medios de rojo fenol por debajo del policarbonatomembrana en el centro pocillo de la placa de cultivo. </li><li> Añadir agua hasta el borde exterior de la placa de cultivo para proporcionar humedad. </li><li> Desde el intestino fetal se somete a ondas peristálticas-como de contracción en cultivo, añadir 100 ml de una solución 1: 5 de xilazina en 1x PBS a 3 ml de medio para controlar el movimiento del intestino mientras que las imágenes. Esta dosis será controlar los movimientos intestinales por alrededor de 8-10 horas sin comprometer el desarrollo de las vellosidades. </li><li> Para una vista transversal del intestino, incrustar los intestinos vivos en una solución de agarosa al 3-4% y cortar secciones gruesas (150-500 M) en un vibratome. Coincidir con la rigidez de la agarosa con la rigidez del intestino de ser cortado y determinado empíricamente la etapa de desarrollo. Por lo general, una solución de agarosa al 3% funciona bien para los intestinos menores de E14.5 y una solución al 4% funciona bien para los intestinos E15-E16. </li><li> Pega las secciones vibratome a una membrana de policarbonato colocada en un tamiz de malla (como en la Sección 3.1.1) yla cultura como se describe en la Sección 3.1.2-3.1.6. </li><li> Llevar a cabo la formación de imágenes en vivo usando un microscopio confocal de fluorescencia de dos fotones (láser de excitación entre 690-1,040 nm) en un soporte vertical con una tabla ajustable. </li><li> Láser de dos fotones sintonizado a 900 nm proporciona una penetración profunda con la mejor resolución para las señales de las buenas prácticas agrarias. Reduce el daño a los tejidos, mientras que la formación de imágenes. Además, el establecimiento de la potencia del láser para la emisión más bajo posible reduce el daño del tejido. Típicamente para obtener una buena excitación de GFP, utilizar la energía 12% en el láser de dos fotones. </li></ol></li></ol>

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The authors have no financial conflicts to disclose.

La naturaleza dinámica e interacciones de tejidos complejos del intestino en desarrollo requiere la visualización en 3D para tener una apreciación más completa de estos eventos morfogenéticos. Con la evolución de la tecnología de imagen, la capacidad de examinar las vellosidades morfogénesis en detalle está desarrollando / mejora y con ello, la comprensión de la comunicación espacial y la interacción durante la organogénesis es mucho mayor. Los métodos alternativos para el cult...

Each intestinal villus is composed of two main tissue compartments: an epithelial surface layer and a mesenchymal core. The mouse small intestine is formed at embryonic day 10 when a sheet of endoderm closes and seals to form a tube of epithelium surrounded by mesenchymal cells3. This flat tube of epithelium undergoes rapid proliferation, growing both in length and girth and undergoes dramatic rearrangements involving dynamic cell shape changes1. At the same time, the surrounding mesenchyme also undergoes many developmental processes including the formation of the vascular plexus, differentiation of smooth muscle and recruitment of enteric neurons4. In the proximal small intestine at embryonic day 14.5, condensations (clusters) of Hedgehog- and PDGF-responsive cells begin to form adjacent to the epithelium2,5. Formation of mesenchymal clusters continues to spread along the length of the intestine so that they cover the entirety of the small intestine by embryonic day 16.52. As mesenchymal clusters form, the epithelial cells closest to the clusters begin to withdraw from the cell cycle, while the other epithelial cells continue to proliferate. Those cells directly above the mesenchymal cluster that have withdrawn from the cell cycle begin to change shape as the emerging villus buckles into the lumen. Further growth of the villus is driven in part by the continued proliferation of the epithelium between the emerging villi. The mesenchymal clusters remain tightly adhered to the epithelium of the growing villus and continue to express a variety of signaling molecules. The wave of villus emergence propagates along the length of the small intestine following the formation of mesenchymal clusters. As the intestine continues to grow and the intervillus region extends between emerging villi, new mesenchymal clusters form adjacent to the intervillus epithelium and further rounds of villus emergence and growth ensue6.
Synchronized development of the epithelium and mesenchyme is essential for villus morphogenesis. Signaling molecules are secreted from one layer to the other where receptors receive and transduce the signal message in order to coordinate development between the epithelium and mesenchyme. Mesenchymal clusters act as signaling centers and express a variety of developmental morphogens7-10. Disruption of cluster formation or pattern results in loss of villus emergence and pattern. Inhibition of PDGF signaling results in fewer clusters and fewer villi and those villi that do form are misshapen following the abnormal clusters11. Loss of Hedgehog signaling results in complete loss of cluster formation and failure of villus emergence2,12. Together, these data demonstrate that clusters coordinate development of the villus epithelium with its mesenchymal core.
Using this whole organ culture system, we are able to alter signaling involved in epithelial-mesenchymal cluster cross-talk to determine the role of those signals in villus morphogenesis. Two-photon confocal optical sectioning and reconstruction afford the ability to visualize cluster formation and villus emergence in three-dimensions and better understand the spatial relationships between the mesenchymal clusters and their overlying epithelium. Extending the culture system to four dimensions, we can capture z-stacks of developing clusters and villi over time and observe these interactions. Ultimately, the ability to follow villus development in this manner and observe changes that occur with altered signaling will revolutionize the understanding of epithelial mesenchymal interactions in villus morphogenesis.

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			<td height="21" style="height:21px;width:508px;">Fine dissecting forceps&#160;</td>
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			<td style="width:444px;">2 pairs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70% Ethanol</td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1x sterile Dulbecco&#39;s Phosphate-Buffered Saline (DPBS)</td>
			<td>Gibco&#160;</td>
			<td>14040-133</td>
			<td style="width:444px;">500 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6 well plates</td>
			<td>Costar</td>
			<td>3516</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24 well plates</td>
			<td>Costar&#160;</td>
			<td>3524</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">60 x 15 mm petri dishes</td>
			<td>Falcon</td>
			<td>&#160;451007</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Transwell plates, 24 mm inserts, 8.0 mm polycarbonate membranes</td>
			<td>Corning Costar&#160;</td>
			<td>3428</td>
			<td style="width:444px;">6 inserts per plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BGJb media</td>
			<td>Invitrogen&#160;</td>
			<td>12591-038</td>
			<td style="width:444px;">500 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PenStrep (10,000U/ml Penicillin; 10,000 mg/ml Streptomycin)</td>
			<td>Gibco</td>
			<td>&#160;15140</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ascorbic Acid</td>
			<td>Sigma&#160;</td>
			<td>A0278</td>
			<td style="width:444px;">make 5 mg/ml stock, filter, aliquot and store at -20 &#176;C</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Mouth pipet (Drummond 1-15 inch aspirator tube assembly)</td>
			<td>Fisher</td>
			<td style="width:133px;">&#160;21-180-10</td>
			<td style="width:444px;">remove the aspirator assembly and replace it with a 1000 &#181;l pipet tip which acts as an adaptor to plug in a 6 inch glass Pasteur pipet.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6 inch glass pasteur pipets</td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose beads</td>
			<td>BioRad&#160;</td>
			<td>153-7301</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Capillary Tubes</td>
			<td>World Precision Instruments&#160;</td>
			<td>TW100F-4</td>
			<td style="width:444px;">pull to needles</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">4% Paraformaldehyde</td>
			<td></td>
			<td></td>
			<td style="width:444px;">made in 1 x PBS, pH to 7.3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;"></td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Live Imaging Materials</td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name of Material/Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td style="width:444px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Culture plates</td>
			<td>Falcon&#160;</td>
			<td>353037</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fine mesh stainless steel screen</td>
			<td>purchase at hardware store</td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Polycarbonate membranes</td>
			<td>Thomas scientific&#160;</td>
			<td>4663H25</td>
			<td style="width:444px;">alternatively, cut Corning Costar 3428 membranes off of transwell supports</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Instant glue</td>
			<td>purchase at hardware store</td>
			<td style="width:133px;">gel based preferrably</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">35 x 10 mm plates</td>
			<td>Falcon&#160;</td>
			<td style="width:133px;">351008</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">7% agarose</td>
			<td>Sigma</td>
			<td>&#160;A9414</td>
			<td style="width:444px;">prepare w/v in 1x DPBS, heating to dissolve in a waterbath</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">minutien pins</td>
			<td>Fine Science Tools&#160;</td>
			<td style="width:133px;">26002-20</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phenol red free media (DMEM)</td>
			<td>Gibco&#160;</td>
			<td style="width:133px;">21063-029</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Xylazine (100 mg/ml)</td>
			<td>AnaSed&#160;</td>
			<td style="width:133px;">139-236</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Matrigel</td>
			<td>BD</td>
			<td style="width:133px;">356231</td>
			<td style="width:444px;">basement membrane matrix, growth factor reduced, phenol red-free</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">3-4% agarose</td>
			<td>Sigma&#160;</td>
			<td>A9414</td>
			<td style="width:444px;">prepare w/v in 1x DPBS, heating to dissolve in a waterbath</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;"></td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Imaging of fixed intestines</td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name of Material/Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td style="width:444px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">vaseline</td>
			<td style="width:223px;">purchase at pharmacy</td>
			<td></td>
			<td>used to make VALAP: equal parts vaseline, lanolin, paraffin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">lanolin</td>
			<td style="width:223px;">Sigma&#160;</td>
			<td>L7387</td>
			<td>used to make VALAP: equal parts vaseline, lanolin, paraffin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">paraffin</td>
			<td style="width:223px;">Surgipath</td>
			<td>39601006</td>
			<td>used to make VALAP: equal parts vaseline, lanolin, paraffin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70% glycerol in 1 x PBS</td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Focus clear and Mount Clear</td>
			<td>CelExplorer Labs Co.</td>
			<td>&#160;F101-KIT</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Modeling clay</td>
			<td>purchase at art supply store</td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">double stick tape</td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">cotton applicator swabs</td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">plastic molds, 10mm x 10mm x 5 mm)</td>
			<td>Tissue Tek&#160;</td>
			<td>4565</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">slides</td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">coverslips</td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">lab wipe</td>
			<td>Kimberly Clark</td>
			<td>34155</td>
			<td style="width:444px;">lint free delicate task wipe</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Theiler staging chart&#160;</td>
			<td></td>
			<td></td>
			<td style="width:444px;"><a href="http://www.emouseatlas.org/emap/ema/theiler_stages/%20downloads/theiler2.pdf">http://www.emouseatlas.org/emap/ema/theiler_stages/ downloads/theiler2.pdf</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leica SP5X confocal microscope&#160;</td>
			<td>Leica</td>
			<td></td>
			<td style="width:444px;">Used to conduct the live imaging&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leica DMI 6000 stand&#160;</td>
			<td>Leica</td>
			<td></td>
			<td style="width:444px;">Used to conduct the live imaging&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Aqueous mounting medium (Prolong Gold)</td>
			<td>Molecular Probes&#160;</td>
			<td>P36930</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;"></td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Materials for Immunofluorescence staining of fixed, vibratome sectioned intestines</td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name of Material/Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td style="width:444px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24 well plate</td>
			<td>Costar</td>
			<td>&#160;3524</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Triton X-100</td>
			<td>Sigma&#160;</td>
			<td>T-8787</td>
			<td style="width:444px;">used to make Permeabilization solution: 0.5% Triton X-100 in 1 x PBS</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Goat serum</td>
			<td></td>
			<td></td>
			<td style="width:444px;">used to make Blocking Solution: 4% Goat serum, 0.1% Tween20 in 1x PBS</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Tween20&#160;</td>
			<td>Sigma</td>
			<td>&#160;P9416</td>
			<td style="width:444px;"></td>
		</tr>
	</tbody>
</table>

mouse fetal whole intestine culture system for ex vivo manipulation of signaling pathways and three-dimensional live imaging of villus development

La mayoría de los procesos morfogenéticos en el intestino fetal se han deducido a partir de secciones finas de tejidos fijados, proporcionando instantáneas de los cambios más etapas de desarrollo. Información tridimensional a partir de secciones delgadas de serie puede ser difícil de interpretar debido a la dificultad de la reconstrucción de las secciones de serie perfectamente y mantener la orientación adecuada de los tejidos en las secciones de serie. Las investigaciones recientes de Grosse et al., 2011 ponen de manifiesto la importancia de la información dimensional tres en la comprensión de la morfogénesis de las vellosidades del intestino en desarrollo 1. Reconstrucción tridimensional de las células intestinales individualmente etiquetados demostró que la mayoría de las células epiteliales intestinales en contacto con ambas superficies apicales y basales. Además, la reconstrucción tridimensional del citoesqueleto de actina en la superficie apical del epitelio demostró que el lumen intestinal es continua y que los lúmenes secundarios son un artefacto de sectioning. Esos dos puntos, junto con la demostración de la migración nuclear interkinetic en el epitelio intestinal, definen el epitelio intestinal como el desarrollo de un epitelio pseudoestratificado y no estratificados como se pensaba anteriormente 1. La capacidad de observar el epitelio tridimensionalmente era seminal a demostrar este punto y la redefinición de la morfogénesis epitelial en el intestino fetal. Con la evolución de la tecnología de imágenes multi-fotón y software de reconstrucción en tres dimensiones, la capacidad de visualizar intacta, el desarrollo de órganos está mejorando rápidamente. Excitación de dos fotones permite la penetración más profunda menos perjudiciales en los tejidos con alta resolución. Dos fotones de imágenes y reconstrucción 3D de todo el intestino del feto de ratón en Walton et al., 2012 ayudaron a definir el patrón de vellosidades consecuencia 2. Aquí se describe un sistema de cultivo de órgano entero que permite ex vivo el desarrollo de las vellosidades y las extensiones de ese sistema de cultivo para permitirlos intestinos sean tridimensionalmente fotografiado durante su desarrollo.

Cultura de explantes enteros de intestinos fetales permite el análisis de la ubicación, la distribución, y la duración de las moléculas de señalización que coordinan el desarrollo intestinal, ya que este sistema permite la manipulación de la señalización con reactivos farmacológicos o proteínas recombinantes. El sistema de cultivo transwell (Figura 1A, reproducida de Walton et al., 2012) 2 proporciona una interfaz aire-líquido, lo que hace posible colocar de drogas o de ...

Watch this Scientific Journal Video about Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development at JoVE.com

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Improved imaging technology is allowing three-dimensional imaging of organs during development. Here we describe a whole organ culture system that allows live imaging of the developing villi in the fetal mouse intestine.

Sistema de Cultura ratón fetal Whole Intestino para<em&gt; Ex Vivo</em&gt; Manipulación de vías de señalización y tridimensional de imágenes en vivo del desarrollo de vellosidades

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over ...

mouse-fetal-whole-intestine-culture-system-for-ex-vivo-manipulation

Research

JoVE Journal

Biology

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

14.8K vistas.  University of Michigan. Improved imaging technology is allowing three-dimensional imaging of organs during development. Here we describe a whole organ culture system that allows live imaging of the developing villi in the fetal mouse intestine. 

Video: Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

Método para la Cultura de primeros embriones del polluelo ex vivo (Nueva Cultura)

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

Investigación

Biología

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

Un sistema para la<em&gt; Ex vivo</em&gt; Cultivo de páncreas embrionario

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations1. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in2). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1.
The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones3,4, forming foci within 5-15 minutes5. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair6. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins7-9. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs6. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis10.
In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1's behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs11. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis12. Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.

This protocol describes a method for visualizing a DNA double-strand break signaling protein activated in response to DNA damage as well as its localization during mitosis.

De dos y tres dimensiones imágenes de células vivas de las proteínas de respuesta al daño del ADN

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

A Novel<em&gt; Ex vivo</em&gt; Método de la cultura para el corazón de embriones de ratón

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 &#176;&#8203;C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.

Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr&#160;exhibit morphological development comparable to embryos developing in utero.

Embriones de ratón con el desarrollo en todo un sistema de cultivo de embriones sin suero

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts migrate through the epidermis of the embryo where they subsequently colonize the developing hair follicles1,2. Neural crest cell migration is extensively studied in vitro but in vivo methods are still not well developed, especially in mammalian systems. One alternative is to use ex vivo organotypic culture3-6. Culture of mouse embryonic skin requires the maintenance of an air-liquid interface (ALI) across the surface of the tissue3,6. High resolution live-imaging of mouse embryonic skin has been hampered by the lack of a good method that not only maintains this ALI but also allows the culture to be inverted and therefore compatible with short working distance objective lenses and most confocal microscopes. This article describes recent improvements to a method that uses a gas permeable membrane to overcome these problems and allow high-resolution confocal imaging of embryonic skin in ex vivo culture6. By using a melanoblast specific Cre-recombinase expressing mouse line combined with the R26YFPR reporter line we are able to fluorescently label the melanoblast population within these skin cultures. The technique allows live-imaging of melanoblasts and observation of their behavior and interactions with the tissue in which they develop. Representative results are included to demonstrate the capability to live-image 6&#160;cultures in parallel.

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

We describe the dissection and ex vivo culture of mouse embryonic skin. The culture system maintains an air-liquid interface across the tissue surface and allows imaging on an inverted microscope. Melanoblasts, a component of the developing skin, are fluorescently labeled allowing their behavior to be observed using confocal microscopy.

Ex vivo Cultura de embriones de ratón con la piel y en vivo de imágenes de melanoblasto Migración

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts ...

An Ex vivo Culture System to Study Thyroid Development

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are produced by differentiated thyrocytes, organized in closed spheres called follicles, while calcitonin is synthesized by C-cells, interspersed in between the follicles and a dense network of blood capillaries. Although adult thyroid architecture and functions have been extensively described and studied, the formation of the &#8220;angio-follicular&#8221; units, the distribution of C-cells in the parenchyma and the paracrine communications between epithelial and endothelial cells is far from being understood.
This method describes the sequential steps of mouse embryonic thyroid anlagen dissection and its culture on semiporous filters or on microscopy plastic slides. Within a period of four days, this culture system faithfully recapitulates in vivo thyroid development. Indeed, (i) bilobation of the organ occurs (for e12.5 explants), (ii) thyrocytes precursors organize into follicles and polarize, (iii) thyrocytes and C-cells differentiate, and (iv) endothelial cells present in the microdissected tissue proliferate, migrate into the thyroid lobes, and closely associate with the epithelial cells, as they do in vivo.
Thyroid tissues can be obtained from wild type, knockout or fluorescent transgenic embryos. Moreover, explants culture can be manipulated by addition of inhibitors, blocking antibodies, growth factors, or even cells or conditioned medium. Ex vivo development can be analyzed in real-time, or at any time of the culture by immunostaining and RT-qPCR.
In conclusion, thyroid explant culture combined with downstream whole-mount or on sections imaging and gene expression profiling provides a powerful system for manipulating and studying morphogenetic and differentiation events of thyroid organogenesis.

An Ex vivo Culture System to Study Thyroid Development

This protocol describes dissection of mouse embryonic thyroid anlagen and the culture of explants on semiporous filters or on microscopy plastic slides. This system is ideal to study morphogenetic or differentiation events occurring during thyroid development of wild type or knockout embryos, and is amenable to gain- and loss-of-function experiments.

Un<em&gt; Ex vivo</em&gt; Sistema de Cultura para estudiar el desarrollo de la tiroides

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are ...

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 ...

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 ...