Los autores agradecen la contribución de la Estación Biológica de Hammond Bay, Great Lakes Science Center, Servicio Geológico de EE.UU.. También agradecemos a la Dra. Melinda Frame en el Centro de Microscopía Avanzada de la Universidad del Estado de Michigan por su apoyo técnico en la microscopía confocal de barrido láser. Este estudio es apoyado por becas de la Comisión de Pesca de Lagos a YWCD y WML.

1. Preparación de la Solución <ol><li> Hacer 1 L de 10x 0,1 M solución salina tamponada con fosfato (PBS, pH 7,4): Pesar 26,2 g de fosfato de sodio (monobásico), 115 g de fosfato de sodio (dibásico), y 87,66 g de NaCl. Disolver en aproximadamente 800 ml de H 2 O destilada, ajustar el pH, y llevar el volumen hasta 1 litro con H2O destilada <ol><li> Hacer 1 L de solución salina tampón fosfato 0,1 M (pH 7,4): Tomar 100 ml de 10x PBS y añadir 900 ml de H2O destilada </li><li> Hacer 1 L de 0,1 M de tampón fosfato salino (pH 7,4) con 0,1% de Triton X-100: Tomar 100 ml de 10x de PBS, añadir 1 ml de Triton X-100, y llevar el volumen hasta 1 L con H2O destilada </li></ol></li><li> Hacer 1 L de 0,1 M de 10x tampón de fosfato (PB, pH 7,4): Pesar 26,2 g de fosfato de sodio (monobásico) y 115 g de fosfato de sodio (dibásico). Disolver en aproximadamente 800 ml de H 2 O destilada, ajustar el pH, y llevar el volumen hasta 1 litro con H2O destilada <ol><li> Hacer 1 L 0,1 M tampón fosfato (pH 7,4): Tome 100 ml 10x PB,y añadir 900 ml de agua destilada H 2 O. </li><li> Hacer 1 L 0,1 M tampón fosfato (pH 7,4) con 0,1% Triton X-100: Tomar 100 ml 10x PB, añadir 1 ml de Triton X-100, y llevar el volumen hasta 1 litro con H2O destilada </li></ol></li><li> Hacer solución de 400 ml de monómero de hidrogel (4% de acrilamida, 0,25% de bis-acrilamida, 4% de paraformaldehído, persulfato de amonio 0,0075%, 0,0005% de saponina en PBS 0,1 M): Pesar 0,3 g de persulfato de amonio y 0,2 g de saponina. Añadir 210 ml de H2O destilada, 40 ml de 40% de acrilamida, 10 ml de 2% de bis-acrilamida, 40 ml de 10x de PBS (pH 7,4), 100 ml de 16% de paraformaldehído, y mezclar bien. </li><li> Haga solución clarificadora 4 L (4% SDS en ácido bórico 0,2 M): Pesar 49,464 g de ácido bórico y 160 g de SDS. Disolver en aproximadamente 3,5 l de H 2 O destilada, ajustar el pH a 8,5 con NaOH, y llevar el volumen hasta 4 L. </li><li> Hacer 1 L de solución de glicerol al 80%: Medir 800 ml de glicerol, añadir 200 ml de H 2 O destilada y mezclar bien. </li></ol> 2. Preparación de Tejido <ol><li> Preparar la solución de monómero de hidrogel y 10 ml de alícuota a cada tubo de centrífuga de 15 ml. </li><li> Diseccionar todo el tejido y fijar en el hidrogel durante 2 días a 4 ° C. Asegúrese de que la solución de hidrogel es al menos 10 veces el volumen del tejido. </li><li> Llevar el tejido fijado de hidrogel (en el tubo de centrífuga de 15 ml) a una campana de humos. </li><li> Polimerizar el tejido fijado hidrogel añadiendo 5 l de TEMED, mezclar bien y dejar reposar en la campana de extracción a temperatura ambiente durante 2-3 horas. </li><li> Retire el exceso de gel a fondo. Nota: gel extra obstaculizará el proceso de compensación y la penetración de anticuerpos durante la tinción. </li><li> Incubar el tejido fijado en 10 ml de compensación solución en un agitador incubador a 70 rpm y 50 ° C durante varios días hasta que el tejido se vuelve transparente. Cambie solución de limpieza de al menos una vez por día y eliminar el exceso de gel a partir del tejido cuando se cambia la solución de aclarado. </li><li> Enjuague el tejido en 10 ml de PB 0,1 M con 0,1% de Triton X-100 al 70 rpm y de 37 ° C durante la noche. Repita este paso 3 veces y eliminar el exceso de gel a partir del tejido cuando se cambia la solución tampón. </li><li> Incubar el tejido aclarado en la solución de anticuerpo primario en PB con 0,1% de Triton X-100 a 70 rpm y 37 ° C durante 2 días. Nota: Asegúrese de suficiente solución de anticuerpo primario para sumergir todo el tejido. </li><li> Enjuague el tejido en 10 ml de PB con 0,1% de Triton X-100 a 70 rpm y 37 ° C durante la noche. Repita este paso 3 veces y eliminar el exceso de gel a partir del tejido cuando se cambia la solución tampón. </li><li> Realice los pasos siguientes en un cuarto oscuro o con poca luz. Cubra las muestras con papel de aluminio para evitar la decoloración foto. </li><li> Incubar el tejido aclarado con la solución de anticuerpo secundario fluorescente en PB con 0,1% de Triton X-100 y el suero de bloqueo a 70 rpm y 37 ° C durante 1 día. </li><li> Enjuague el tejido en 10 ml de PB con 0,1% de Triton X-100 a 70 rpm y 37 ° C durante la noche. Repita este paso 3 veces y retire el exceso de gel fesde el tejido cuando el cambio de la solución tampón. </li><li> Enjuague el tejido en 10 ml de PBS a 70 rpm y 37 ° C durante la noche. Repita este paso 3 veces y eliminar el exceso de gel a partir del tejido cuando se cambia la solución tampón. </li><li> Incubar el tejido se aclara en 80% de glicerol a 70 rpm y 37 ° C durante la noche. </li><li> Guarde el tejido teñido fluorescente a 4 ° C antes de la microscopía confocal. </li></ol>

<ol>
	<li>Applegate, V. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+history+of+the+sea+lamprey+(Petromyzon+marinus)+in+Michigan.">Natural history of the sea lamprey (Petromyzon marinus) in Michigan.</a> US Fish and Wildlife Service Special Science Report on Fishery Service. (55), (1950).</li><li>Hardisty, M. W., Potter, I. C., Hardisty, M. W., Potter, I. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+general+biology+of+adult+lampreys.">The general biology of adult lampreys.</a> The biology of lampreys. 1, 127-206 (1971).</li><li>Youson, J. H., Potter, I. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+description+of+the+stages+in+the+metamorphosis+of+the+anadromous+sea+lamprey,+Petromyzon+marinus+L.">A description of the stages in the metamorphosis of the anadromous sea lamprey, Petromyzon marinus L.</a> Can. J. Zool. 57, 1808-1817 (1979).</li><li>Boomer, L. A., 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=Cholangiocyte+apoptosis+is+an+early+event+during+induced+metamorphosis+in+the+sea+lamprey,+Petromyzon+marinus+L.">Cholangiocyte apoptosis is an early event during induced metamorphosis in the sea lamprey, Petromyzon marinus L.</a> J. Pediatr. Surg. 45, 114-120 (2010).</li><li>Kasai, M., Suzuki, H., Ohashi, E., Ohi, R., Chiba, T., Okamoto, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technique+and+results+of+operative+management+of+biliary+atresia.">Technique and results of operative management of biliary atresia.</a> World J. Surg. 2, 571-580 (1978).</li><li>Suzuki, T., Hashimoto, T., Kondo, S., Sato, Y., Hussein, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+patients&#8217;+outcome+post-Kasai+operation:+a+19-year+experience+with+modification+of+the+hepatic+portoenterostomy+and+applying+a+novel+steroid+therapy+regimen.">Evaluating patients&#8217; outcome post-Kasai operation: a 19-year experience with modification of the hepatic portoenterostomy and applying a novel steroid therapy regimen.</a> Pediatr. Surg. Int. 26, 825-830 (2010).</li><li>Hartley, J. L., Davenport, M., Kelly, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biliary+atresia.">Biliary atresia.</a> Lancet. 374, 1704-1713 (2009).</li><li>Morecki, R., Glaser, J. H., Cho, S., Balistreri, W. F., Horwitz, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biliary+atresia+and+reovirus+type+3+infection.">Biliary atresia and reovirus type 3 infection.</a> New Engl. J. Med. 307, 481-484 (1982).</li><li>Shimadera, S., Iwai, N., Deguchi, E., Kimura, O., Fumino, S., Yokoyama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+inv+mouse+as+an+experimental+model+of+biliary+atresia.">The inv mouse as an experimental model of biliary atresia.</a> J. Pediatr. Surg. 42, 1555-1560 (2007).</li><li>Sidon, E. W., Youson, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+changes+in+the+liver+of+the+sea+lamprey,+Petromyzon+marinus+L.,+during+metamorphosis:+I.+Atresia+of+the+bile+ducts.">Morphological changes in the liver of the sea lamprey, Petromyzon marinus L., during metamorphosis: I. Atresia of the bile ducts.</a> J. Morphol. 177, 109-124 (1983).</li><li>Yeh, C. -. Y., Chung-Davidson, Y. -. W., Wang, H., Li, K., Li, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+synthesis+and+secretion+of+bile+salts+as+an+adaptation+to+developmental+biliary+atresia+in+the+sea+lamprey.">Intestinal synthesis and secretion of bile salts as an adaptation to developmental biliary atresia in the sea lamprey.</a> Proc. Nat. Acad. Sci. U.S.A. 109, 11419-11424 (2012).</li><li>Chung, K., 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=Structural+and+molecular+interrogation+of+intact+biological+systems.">Structural and molecular interrogation of intact biological systems.</a> Nature. 497, 332-337 (2013).</li><li>Alarc&#243;n, V. B., Filosa, M. F., Youson, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokeratins+in+the+liver+of+the+sea+lamprey+(Petromyzon+marinus)+before+and+after+metamorphosis.">Cytokeratins in the liver of the sea lamprey (Petromyzon marinus) before and after metamorphosis.</a> Cell Tissue Res. 287, 365-374 (1997).</li><li>Youson, J. H., Ogilvie, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructural+features+of+degeneration+of+the+gallbladder+during+lamprey+biliary+atresia.">Ultrastructural features of degeneration of the gallbladder during lamprey biliary atresia.</a> Tissue and Cell. 22, 477-492 (1990).</li><li>Morii, M., 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=Onset+of+apoptosis+in+the+cystic+duct+during+metamorphosis+of+a+Japanese+lamprey,+Lethenteron+reissneri.">Onset of apoptosis in the cystic duct during metamorphosis of a Japanese lamprey, Lethenteron reissneri.</a> Anat. Rec. 293, 1155-1166 (2010).</li></ol>

Los autores no tienen nada que revelar.

Este protocolo se modificó a partir de un nuevo método llamado &quot;claridad&quot; 12, que se reticula tejido intacto con poliacrilamida para formar un hidrogel nanoporoso, y luego despoja de la membrana plasmática del tejido para conseguir la transparencia óptica y la permeabilidad macromolecular. &quot;CLARIDAD&quot; permite obtener imágenes de los tejidos intactos de proyección de largo alcance y el cableado del circuito local en el sistema nervioso. Este nuevo método se puede utilizar para visuali...

Lamprea de mar desarrollarse a través de tres etapas de la vida distinta 1,2. Lamprea marina larvas (L) pasan la mayor parte de tiempo en madrigueras como filtradores bentónicos. Después de pasar por siete etapas metamórficas de cambios drásticos en la morfología externa y la reorganización de los órganos internos 3, a los juveniles (JV) entran en una etapa parasitaria durante el cual se alimentan de sangre y fluidos de los tejidos del pez hospedador, el aumento de la masa corporal de más de 100 veces . 1.0 del a 1,5 años que se alimentan de los peces de acogida en el mar o grandes lagos, los adultos dejan de alimentación durante el comienzo de la primavera y migran hacia los ríos para desovar y luego mueren 1,2. Durante la metamorfosis, el hígado lamprea de mar pierde la vesícula biliar y el árbol biliar, un fenotipo mutante evolutivo que imita la enfermedad infante humano atresia biliar. Atresia biliar infantil es una enfermedad hepática pediátrica rara con complicaciones médicas graves 4,5,6,7, 8.9.10, sin embargo, la patogenia y etiología de la atresia biliar son en gran parte desconocidos 4. Los pacientes con atresia biliar mueren dentro de los dos años después del nacimiento a menos que la intervención quirúrgica (procedimiento de Kasai) se lleva a cabo 5. Posteriormente, estos pacientes requieren un amplio manejo clínico y, a menudo el trasplante hepático 6. Se han propuesto muchas teorías sobre la etiopatogenia atresia biliar, como la infección viral, malformación congénita, enfermedad autoinmune, y el insulto tóxico. Sin embargo, la contribución de cada uno para el desarrollo de la atresia biliar no son concluyentes 7,8,9,10. A diferencia de los niños que sufren atresia biliar patológica, la lamprea de mar se someten desarrollo programado atresia biliar sin una amplia necroinflamación, fibrosis o cirrosis 10. Los animales pueden sufrir colestasis transitoria durante este proceso 10, pero adaptarse a este trastorno del desarrolloa través de la síntesis de novo y de la secreción de sales biliares en el intestino después de la atresia biliar desarrollo, además de los mecanismos conocidos, tales como la reducción de la síntesis de sales biliares en el hígado 11. Este proceso de desarrollo de la lamprea de mar proporciona la única conocida oportunidad de examinar el progreso de la atresia biliar. Un nuevo método desarrollado la llamada &quot;CLARIDAD&quot; permite obtener imágenes de alta resolución en los sistemas nervioso de mamíferos complejos mediante la transformación de tejido intacto en un hidrogel nanoporoso ópticamente transparente 12. El uso de la lamprea de mar hígado y un protocolo CLARIDAD modificada, las imágenes intactas de tejido de la degeneración biliar puede ser documentado a lo largo de la metamorfosis de hígado.

<table border="0" cellpadding="0" cellspacing="0" width="702">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">40% acrylamide&#160;</td>
			<td style="width:200px;">Bio-Rad</td>
			<td style="width:119px;">161-0140</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2% bis-acrylamide&#160;</td>
			<td>Bio-Rad</td>
			<td>161-0142</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TEMED</td>
			<td>Bio-Rad</td>
			<td>161-0800</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ammonium persulfate&#160;</td>
			<td>Sigma</td>
			<td>A3678-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">boric acid</td>
			<td>Sigma</td>
			<td>B7901-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">saponin&#160;</td>
			<td>Sigma</td>
			<td>47036</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sodium dodecyl sulfate&#160;</td>
			<td>Sigma</td>
			<td style="width:119px;">L337-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sodium phosphate (monobasic)</td>
			<td>Sigma</td>
			<td>04269-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sodium phosphate (dibasic)</td>
			<td>Sigma</td>
			<td>S5136-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td>Sigma</td>
			<td>X100-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">glycerol&#160;</td>
			<td>Sigma</td>
			<td>G9012-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">16% paraformaldehyde&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710-S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaOH pellets&#160;</td>
			<td>EMD</td>
			<td>SX0590-3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15 ml centrifuge tubes</td>
			<td style="width:200px;">Any brand</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">dissecting tools&#160;</td>
			<td style="width:200px;">Any brand</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

a new clarification method to visualize biliary degeneration during liver metamorphosis in sea lamprey (petromyzon marinus)

La atresia biliar es una rara enfermedad de la infancia, con un estimado de 1 en 15.000 de frecuencia en el sureste de los Estados Unidos, pero más común en los países del este de Asia, con una frecuencia reportada de 1 en 5000 en Taiwán. Aunque se sabe mucho sobre la gestión de la atresia biliar, su patogenia sigue siendo difícil de alcanzar. La lamprea marina (Petromyzon marinus) ofrece una oportunidad única para examinar el mecanismo y la progresión de la degeneración biliar. Lamprea de mar se desarrollan a través de tres etapas de la vida distintas: larva, parasitarias y adultos. Durante la transición de larvas a juveniles parasitaria, la lamprea de mar sufren metamorfosis con dramática reorganización y remodelación en la morfología externa y los órganos internos. En el hígado, todo el sistema se pierde biliar, incluyendo la vesícula biliar y el árbol biliar. Un nuevo método desarrollado la llamada &quot;CLARIDAD&quot; se modificó para aclarar todo el hígado y el cruce con el intestino en metamórfica lamprea de mar. Laproceso de degeneración biliar fue visualizado y discernido durante la lamprea de mar metamorfosis mediante el uso de microscopía confocal de barrido láser. Este método proporciona una poderosa herramienta para estudiar la atresia biliar en un modelo animal único.

Varios acontecimientos importantes del desarrollo se producen en el sistema hepatobiliar durante la lamprea de mar metamorfosis. El conducto biliar y la vesícula biliar sufren apoptosis y degenerado (Figura 1). Combinando el método de clarificación modificado y tinción con hígado marcador de células citoqueratina 19 (CK19, presente en ambos cholangiocytes y hepatocitos antes y después de la metamorfosis 13) y Bcl2 marcador anti-apoptótica usando microscopía confocal, todo el sistema ...

Watch this Scientific Journal Video about A New Clarification Method to Visualize Biliary Degeneration During Liver Metamorphosis in Sea Lamprey Petromyzon marinus at JoVE.com

A New Clarification Method to Visualize Biliary Degeneration During Liver Metamorphosis in Sea Lamprey Petromyzon marinus

Lamprea de mar pierde la vesícula biliar y los conductos biliares durante la metamorfosis, un proceso similar a la atresia biliar humano. Un nuevo método de fijación y aclaración (CLARITY) se modificó para visualizar todo el árbol biliar mediante microscopía confocal de barrido láser. Este método proporciona una poderosa herramienta para estudiar la degeneración biliar.

Un método Aclaración Nueva visualizar biliar Degeneración Durante Hígado metamorfosis en la lamprea de mar ( Petromyzon marinus)

Biliary atresia is a rare disease of infancy, with an estimated 1 in 15,000 frequency in the southeast United States, but more common in East Asian ...

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Biology

A New Clarification Method to Visualize Biliary Degeneration During Liver Metamorphosis in Sea Lamprey (Petromyzon marinus)

13.7K vistas.  Michigan State University.  Lamprea de mar pierde la vesícula biliar y los conductos biliares durante la metamorfosis, un proceso similar a la atresia biliar humano. Un nuevo método de fijación y aclaración (CLARITY) se modificó para visualizar todo el árbol biliar mediante microscopía confocal de barrido láser. Este método proporciona una poderosa herramienta para estudiar la degeneración biliar. 

Video: A New Clarification Method to Visualize Biliary Degeneration During Liver Metamorphosis in Sea Lamprey Petromyzon marinus

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

Pyrosequencing: Un método simple para determinar el genotipo exacto

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

Investigación

Biología

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 ...

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

Caenorhabdits elegans has been used extensively in the study of stress resistance, which is facilitated by the transparency of the adult and embryo stages as well as by the availability of genetic mutants and transgenic strains expressing a myriad of fusion proteins1-4. In addition, dynamic processes such as cell division can be viewed using fluorescently labeled reporter proteins. The study of mitosis can be facilitated through the use of time-lapse experiments in various systems including intact organisms; thus the early C. elegans embryo is well suited for this study. Presented here is a technique by which in vivo imaging of sub-cellular structures in response to anoxic (99.999% N2; &lt;2 ppm O2) stress is possible using a simple gas flow through setup on a high-powered microscope. A microincubation chamber is used in conjunction with nitrogen gas flow through and a spinning disc confocal microscope to create a controlled environment in which animals can be imaged in vivo. Using GFP-tagged gamma tubulin and histone, the dynamics and arrest of cell division can be monitored before, during and after exposure to an oxygen-deprived environment. The results of this technique are high resolution, detailed videos and images of cellular structures within blastomeres of embryos exposed to oxygen deprivation.

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

Described here is an in vivo technique to image sub-cellular structures in animals exposed to anoxia using a gas flow through microincubation chamber in conjunction with a spinning disc confocal microscope. This method is straightforward and flexible enough to suit a variety of experimental parameters and model systems.

El uso de microscopía lapso de tiempo para Visualizar anoxia inducida animación suspendida en<em&gt; C. elegans</em&gt; Los embriones

Caenorhabdits elegans has been used extensively in the study of stress resistance, which is facilitated by the transparency of the adult and embryo stages ...

High Throughput Microinjections of Sea Urchin Zygotes

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of treatment solution is loaded into a microinjection needle that is used to physically inject individual immobilized cells or embryos. Despite the need for initial training to perform this procedure for high-throughput delivery, microinjection offers maximum efficiency and reproducible delivery of a wide variety of treatment solutions (including complex mixtures of samples) into cells, eggs or embryos. Applications to microinjections include delivery of DNA constructs, mRNAs, recombinant proteins, gain of function, and loss of function reagents. Fluorescent or colorimetric dye is added to the injected solution to enable instant visualization of efficient delivery as well as a tool for reliable normalization of the amount of the delivered solution. The described method enables microinjection of 100-400 sea urchin zygotes within 10-15 min.

Microinjection is a common technique used to deliver DNA constructs, mRNAs, morpholino antisense oligonucleotides or other treatments into eggs, embryos, and cells of various species.

High Throughput microinyecciones de Sea Urchin cigotos

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of ...

A Method for Extracting Pigments from Squid Doryteuthis pealeii

Cephalopods can undergo rapid and adaptive changes in dermal coloration for sensing, communication, defense, and reproduction purposes. These capabilities are supported in part by the areal expansion and retraction of pigmented organs known as chromatophores. While it is known that the chromatophores contain a tethered network of pigmented granules, their structure-function properties have not been fully detailed. We describe a method for isolating the nanostructured granules in squid Doryteuthis pealeii chromatophores and demonstrate how their associated pigments can be extracted in acidic solvents. To accomplish this, the chromatophore containing dermal layer is first manually isolated using a superficial dissection, and the pigment granules are removed using sonication, centrifugation, and washing cycles. Pigments confined within the purified granules are then extracted via acidic methanol solutions, leaving nanostructures with smaller diameters that are void of visible color. This extraction procedure produces a 58% yield of soluble pigments isolated from granules. Using this method, the composition of the chromatophore pigments can be determined and used to provide insight into the mechanism of adaptive coloration in cephalopods.

A Method for Extracting Pigments from Squid Doryteuthis pealeii

A protocol for the extraction of pigments from the nanostructured granules in squid Doryteuthis pealeii chromatophores is presented.

Un método para extraer pigmentos de calamar<em&gt; Pealeii Doryteuthis</em

Cephalopods can undergo rapid and adaptive changes in dermal coloration for sensing, communication, defense, and reproduction purposes. These capabilities ...

Using a Whole-mount Immunohistochemical Method to Study the Innervation of the Biliary Tract in Suncus murinus

This work describes the whole-mount immunohistochemistry staining method in detail, using neurofilament protein antibody to label the innervation of the biliary tract in Suncus murinus (S. murinus&#160;). First, the specimen was dissected from S. murinus and fixed in 4% paraformaldehyde (PFA). Second, an enzymatic treatment and potential endogenous peroxidase inactivation were performed. The specimen was then exposed to the primary antibody, anti-neurofilament protein antibody, for 3-6 days. It was then incubated with the secondary antibody conjugated with horseradish peroxidase. The color reaction was revealed by reacting the specimen with a 3,3&#39;-diaminobenzidine (DAB) substrate. This method can be applied to analyze the innervation of all visceral organs. Furthermore, this protocol can also be adapted to test other neuronal antibodies, but optimization of the antibodies should be done first. This method was originally introduced by Kuratani and Tanaka1,2,3.

Using a Whole-mount Immunohistochemical Method to Study the Innervation of the Biliary Tract in Suncus murinus

A whole-mount immunohistochemical approach, to visualize neurofilament protein expression in the extrahepatic biliary tract in Suncus murinus. is presented here. This protocol can be used to analyze the innervation of all visceral organs in S. murinus or other species.

Uso de un método inmunohistoquímico de montaje completo para estudiar la inervación del tracto biliar en<em&gt; Suncus murinus</em

This work describes the whole-mount immunohistochemistry staining method in detail, using neurofilament protein antibody to label the innervation of the ...

Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds

Fungal infections have become an important medical condition in the last decades, but the number of available antifungal drugs is limited. In this scenario, the search for new antifungal drugs is necessary. The protocol reported here details a method to screen peptides for their antifungal properties. It is based on the broth microdilution susceptibility test from the Clinical and Laboratory Standards Institute (CLSI) M27-A3 guidelines with modifications to suit the research of antimicrobial peptides as potential new antifungals. This protocol describes a functional assay to evaluate the activity of antifungal compounds and may be easily modified to suit any particular class of molecules under investigation. Since the assays are performed in 96-well plates using small volumes, a large-scale screening can be completed in a short amount of time, especially if carried out in an automation setting. This procedure illustrates how a standardized and adjustable clinical protocol can help the bench-work pursuit of new molecules to improve the therapy of fungal diseases.

Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds

An easy and adaptable broth microdilution method for screening antifungal compounds and extracts.

Microdilución en Vitro proyección de caldo: un método fácil y rápido para detectar nuevos compuestos antimicóticos

Fungal infections have become an important medical condition in the last decades, but the number of available antifungal drugs is limited. In this ...

Split Green Fluorescent Protein System to Visualize Effectors Delivered from Bacteria During Infection

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the plant immune system. During infection cytoplasmic effectors are delivered to the host cytosol via a type III secretion system (T3SS). After delivery into the plant cell, the effector(s) targets the specific compartment(s) to modulate host cell processes for survival and replication of the pathogen. Although there has been some research on the subcellular localization of effector proteins in the host cells to understand their function in pathogenicity by using fluorescent proteins, investigation of the dynamics of effectors directly injected from bacteria has been challenging due to the incompatibility between the T3SS and fluorescent proteins.
Here, we describe our recent method of an optimized split superfolder green fluorescent protein system (sfGFPOPT) to visualize the localization of effectors delivered via the bacterial T3SS in the host cell. The sfGFP11 (11th &#946;-strand of sfGFP)-tagged effector secreted through the T3SS can be assembled with a specific organelle targeted sfGFP1-10OPT (1-10th &#946;-strand of sfGFP) leading to fluorescence emission at the site. This protocol provides a procedure to visualize the reconstituted sfGFP fluorescence signal with an effector protein from Pseudomonas syringae in a particular organelle in the Arabidopsis and Nicotiana benthamiana plants.

Fluorescent protein-based approaches to monitor effectors secreted by bacteria into host cells are challenging. This is due to the incompatibility between fluorescent proteins and the type-III secretion system. Here, an optimized split superfolder GFP system is used for visualization of effectors secreted by bacteria into the host plant cell.

Sistema proteína verde fluorescente para visualizar efectores entregado de las bacterias durante una infección

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the ...

Immunolabelling Myofiber Degeneration in Muscle Biopsies

The necrosis of muscle fibres (myonecrosis) plays a central role in the pathogenesis of several muscle conditions, including muscular dystrophies. Therapeutic options addressing the causes of muscular dystrophy pathogenesis are expected to alleviate muscle degeneration. Therefore, a method to assay and quantify the extent of cell death in muscle biopsies is needed. Conventional methods to observe myofiber degeneration in situ are either poorly quantitative or rely on the injection of vital dyes. In this article, an immunofluorescence protocol is described that stains necrotic myofibers by targeting immunoglobulin G (IgG) uptake by myofibers. The IgG uptake method is based on cell features characterizing the necrotic demise, including 1) the loss of plasma membrane integrity with the release of damage-associated molecular patterns and 2) the uptake of plasmatic proteins. In murine cross-sections, the co-immunolabelling of myofibers, extracellular matrix proteins, and mouse IgG allows clean and straightforward identification of myofibers with necrotic fate. This simple method is suitable for quantitative analysis and applicable to all species, including human samples, and does not require the injection of vital dye. The staining of necrotic myofibers by IgG uptake can also be paired with other co-immunolabelling.

Described here is a protocol for direct immunolabelling of necrotic myofibers in muscle cryosections. Necrotic cells are permeable to serum proteins, including immunoglobulin G (IgG). Revealing the uptake of IgG by myofibers allows the identification and quantification of myofibers that undergo necrosis regardless of muscle condition.

Inmunoetiquetado Desgeneración de miofibra en biopsias musculares

The necrosis of muscle fibres (myonecrosis) plays a central role in the pathogenesis of several muscle conditions, including muscular dystrophies. ...

Apoptosis Induction and Detection in a Primary Culture of Sea Cucumber Intestinal Cells

Primary cultured cells are used in a variety of scientific disciplines as exceptionally important tools for the functional evaluation of biological substances or characterization of specific biological activities. However, due to the lack of universally applicable cell culture media and protocols, well described cell culture methods for marine organisms are still limited. Meanwhile, the commonly occurring microbial contamination and polytropic properties of marine invertebrate cells further impede the establishment of an effective cell culture strategy for marine invertebrates. Here, we describe an easy-to-handle method for culturing intestinal cells from sea cucumber Apostichopus japonicus; additionally, we provide an example of in vitro apoptosis induction and detection in primary cultured intestinal cells. Moreover, this experiment provides details about the appropriate culture medium and cell collection method. The described protocol is compatible with a variety of widely available tissue samples from marine organisms including Echinodermata, Mollusca, and Crustacea, and it can provide sufficient cells for multiple in vitro experimental applications. This technique would enable researchers to efficiently manipulate primary cell cultures from marine invertebrates and to facilitate the functional evaluation of targeted biological materials on cells.

This protocol provides an easy-to-handle method to culture the intestinal cells from sea cucumber Apostichopus japonicus and is compatible with a variety of widely available tissue samples from marine organisms including Echinodermata, Mollusca, and Crustacea.

Inducción y detección de apoptosis en un cultivo primario de células intestinales de pepino marino

Primary cultured cells are used in a variety of scientific disciplines as exceptionally important tools for the functional evaluation of biological ...