Os autores reconhecem a contribuição da Estação Biológica Hammond Bay, Great Lakes Science Center, EUA Geological Survey. Agradecemos também a Dr. Melinda quadro do Centro de Microscopia Avançada na Universidade Estadual de Michigan pelo apoio técnico em microscopia confocal de varredura a laser. Este estudo é suportado por concessões do Great Lakes Comissão das Pescas para YWCD e WML.

1. Solução Preparação <ol><li> Adicione 1 L de 10x de 0,1 M de solução salina de tampão fosfato (PBS, pH 7,4): Pesar 26,2 g de fosfato de sódio (monobásico), fosfato de sódio a 115 g (dibásico), e 87,66 g de NaCl. Dissolver em cerca de 800 ml de H2O destilada, ajustar o pH, e trazer o volume até 1 litro com H2O destilada <ol><li> Adicione uma solução salina L 0,1 M de tampão fosfato (pH 7,4): Tome 100 ml 10x PBS, e adicionar 900 mL de H2O destilada </li><li> Adicione 1 L de 0,1 M de tampão fosfato salino (pH 7,4) com 0,1% de Triton X-100: Tome 100 ml 10x PBS, adicionar 1 ml de Triton X-100, e trazer o volume até 1 litro com H2O destilada </li></ol></li><li> Adicione 1 L de tampão de fosfato 0,1 M de 10x (PB, pH 7,4): Pesar 26,2 g de fosfato de sódio (monobásico) e fosfato de sódio a 115 g (dibásico). Dissolver em cerca de 800 ml de H2O destilada, ajustar o pH, e trazer o volume até 1 litro com H2O destilada <ol><li> Faça 1 L de 0,1 M de tampão fosfato (pH 7,4): Tomar 100 ml 10x PB,e adicionar 900 mL de água destilada H 2 O. </li><li> Adicione 1 L de 0,1 M de tampão fosfato (pH 7,4) com 0,1% de Triton X-100: Tome 10x 100 ml de PB, adicionar 1 ml de Triton X-100, e trazer o volume até 1 litro com H2O destilada </li></ol></li><li> Adicione 400 ml de solução de monómeros de hidrogel (4% de acrilamida, 0,25% de bis-acrilamida, paraformaldeído a 4%, 0,0075% de persulfato de amónio, 0,0005% de saponina em PBS a 0,1 M): Pesar 0,3 g de persulfato de amónio e 0,2 g de saponina. Adicionar 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 paraformaldeído, e misturar bem. </li><li> Faça solução compensação 4 L (4% SDS em ácido bórico 0,2 M): Pesar 49,464 g de ácido bórico e 160 g SDS. Dissolve-se em cerca de 3,5 L de H2O destilada, ajustar o pH para 8,5 com NaOH, e perfazer o volume até 4 L. </li><li> Faça 1 L solução de glicerol 80%: Medir 800 ml de glicerol, adicionar 200 ml de H 2 O destilada, e misture bem. </li></ol> 2. Preparação de Tecidos <ol><li> Preparar a solução de monómeros de hidrogel e uma alíquota de 10 ml a cada tubo de centrífuga de 15 ml. </li><li> Dissecar todo o tecido e fixar no hidrogel durante 2 dias a 4 ° C. Certifique-se de que a solução de hidrogel é, pelo menos, 10 vezes o volume do tecido. </li><li> Traga o tecido fixado hidrogel (no tubo de centrífuga de 15 ml) para um extractor de fumo. </li><li> Polimerizar o tecido fixado hidrogel por adição de 5 ul de TEMED, misturar bem, e deixar assentar, na hotte, à temperatura ambiente durante 2-3 horas. </li><li> Remover o excesso de gel completamente. Nota: gel extra irá dificultar o processo de compensação ea penetração de anticorpos durante a coloração. </li><li> Incubar o tecido fixado em 10 ml de solução de compensação num agitador de incubador ajustado a 70 rpm e 50 ° C durante vários dias até que o tecido torna-se transparente. Alterar solução limpar pelo menos uma vez por dia e remover o excesso de gel a partir do tecido quando se muda a solução de compensação. </li><li> Lavar o tecido em 10 ml de 0,1 M PB, com 0,1% de Triton X-100 a 70 rpm e 37 ° C durante a noite. Repetir esta etapa 3 vezes e remover o excesso de gel a partir do tecido quando se muda a solução tampão. </li><li> Incubar o tecido clarificado na solução de anticorpo primário em PB com 0,1% de Triton X-100 a 70 rpm e 37 ° C durante 2 dias. Nota: Certifique-suficiente solução de anticorpo primário para submergir todo o tecido. </li><li> Lavar o tecido em 10 ml de PB com 0,1% de Triton X-100 a 70 rpm e 37 ° C durante a noite. Repetir esta etapa 3 vezes e remover o excesso de gel a partir do tecido quando se muda a solução tampão. </li><li> Execute as seguintes etapas em um quarto escuro ou sob luz fraca. Cubra as amostras com papel alumínio para evitar o branqueamento foto. </li><li> Incubar o tecido clarificado com a solução de anticorpo secundário fluorescente nas PB com 0,1% de Triton X-100 e soro de bloqueio, a 70 rpm e 37 ° C durante 1 dia. </li><li> Lavar o tecido em 10 ml de PB com 0,1% de Triton X-100 a 70 rpm e 37 ° C durante a noite. Repita este passo 3 vezes e remover o excesso de gel from o tecido quando se muda a solução tampão. </li><li> Lavar o tecido em 10 ml de PBS, a 70 rpm e 37 ° C durante a noite. Repetir esta etapa 3 vezes e remover o excesso de gel a partir do tecido quando se muda a solução tampão. </li><li> Incubar o tecido clarificada em 80% de glicerol, a 70 rpm e 37 ° C durante a noite. </li><li> Armazenar o tecido manchado fluorescente, a 4 ° C antes de microscopia 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>

Os autores não têm nada a revelar.

Este protocolo modificado a partir de um novo método denominado &quot;clareza&quot; 12, que reticula tecido intacto com poliacrilamida para formar um hidrogel nanoporoso, e depois tira da membrana plasmática de tecidos para alcançar a transparência óptica e a permeabilidade de macromoléculas. &quot;Clareza&quot; permite imagens de tecidos intactos de projeção de longo alcance e fiação do circuito local no sistema nervoso. Este novo método pode ser usado para visualizar todo o sistema biliar no fíg...

Lampreia Mar desenvolver-se através de três fases distintas de vida 1,2. Lampreia mar larval (L) passam a maior parte do tempo em tocas como filtradores bentónicos. Depois de passar por sete estágios metamórficos de mudanças dramáticas na morfologia externa e reorganização em órgãos internos 3, os juvenis resultantes (JV) entrar numa fase parasitária durante o qual eles se alimentam de sangue e fluidos de tecido de peixe hospedeiro, aumentando a massa corporal mais de 100 vezes . Depois de 1,0 a 1,5 anos se alimentando no peixe hospedeiro no mar ou grandes lagos, os adultos deixam alimentação durante o início da primavera e migram para os rios para desovar e morrer 1,2. Durante a metamorfose, o fígado lampreia mar perde a vesícula biliar e toda a árvore biliar, um fenótipo mutante evolutivo que imita a doença bebê humano atresia biliar. Atresia biliar infantil é uma doença hepática pediátrica rara, com graves complicações médicas 4,5,6,7, 8,9,10, no entanto, a patogênese e etiologia da atresia de vias biliares são em grande parte desconhecida 4. Os pacientes com atresia biliar morrer dentro de dois anos após o nascimento, a menos que a intervenção cirúrgica (cirurgia de Kasai) é realizada 5. Posteriormente, estes pacientes necessitam de extensa gestão clínica e muitas vezes o transplante de fígado 6. Muitas teorias de etiopatogenia atresia biliar têm sido propostas, como infecção viral, malformação congênita, doença auto-imune, e insulto tóxico. No entanto, a contribuição de cada um para o desenvolvimento de atresia biliar permanece inconclusiva 7,8,9,10. Ao contrário de crianças que sofrem atresia biliar patológico, lampreia de mar sofrem programada pelo desenvolvimento atresia biliar sem necroinflammation extensa, fibrose ou cirrose 10. Os animais podem sofrer colestase transitória durante este processo de 10, mas se adaptar a esta condição de desenvolvimentoVia de síntese de novo e secreção de sais biliares no intestino, após a atresia biliar de desenvolvimento, para além de mecanismos conhecidos, tais como a redução da síntese de sais biliares no fígado 11. Este processo de maturação na lampreia mar oferece a oportunidade única conhecida para analisar a progressão da atresia biliar. Um método recém-desenvolvido chamado &quot;clareza&quot; permite imagens de alta resolução no sistema nervoso de mamíferos complexos, transformando tecido intacto em um hidrogel nanoporous opticamente transparente 12. Usando lampreia fígado mar e um protocolo CLAREZA modificado, de imagem de tecido intacto de degeneração biliar pode ser documentada em toda metamorfose fígado.

<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)

A atresia biliar é uma doença rara da infância, com uma estimativa de 1 em 15.000 freqüência no sudeste dos Estados Unidos, mas é mais comum em países do leste asiático, com uma freqüência relatada de 1 em 5000 em Taiwan. Embora muito se sabe sobre a gestão da atresia biliar, sua patogênese ainda é indescritível. A lampreia mar (Petromyzon marinus) oferece uma oportunidade única para examinar o mecanismo e progressão da degeneração biliar. Mar lampreia desenvolver através de três fases da vida distintas: larval, parasitárias e adultos. Durante a transição de larva para juvenil parasitária, lampreia de mar sofrem metamorfose com reorganização dramática e remodelação na morfologia externa e órgãos internos. No fígado, a totalidade do sistema biliar é perdida, incluindo a vesícula biliar e das vias biliares. Um método recém-desenvolvido chamado &quot;clareza&quot; foi modificado para esclarecer todo o fígado e na junção com o intestino em lampreia mar metamórfica. Oprocesso de degeneração das vias biliares foi visualizado e discernir durante lampreia metamorfose mar por meio de microscopia confocal de varredura a laser. Este método fornece uma ferramenta poderosa para o estudo de atresia biliar em um modelo animal único.

Vários eventos importantes do desenvolvimento ocorrem no sistema hepatobiliar durante lampreia metamorfose mar. O ducto biliar e da vesícula biliar em apoptose e degenerado (Figura 1). Combinando o método de clarificação modificado e coloração com marcador de células de fígado de citoqueratina 19 (CK19, presente em ambas as cholangiocytes e hepatócitos, antes e após a metamorfose 13) e Bcl2 marcador anti-apoptótica utilizando microscopia confocal, todo o sistema biliar foi captura...

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

Mar lampreia perder a vesícula e canais biliares durante a metamorfose, um processo semelhante à atresia biliar humano. Um novo método de fixação e esclarecimento (clareza) foi modificado para visualizar toda a árvore biliar através de microscopia confocal de varredura a laser. Este método fornece uma ferramenta poderosa para o estudo degeneração biliar.

Um Método de Esclarecimento Novo para Visualize biliar Degeneração Durante fígado metamorfose em lampreia 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 visualizações.  Michigan State University.  Mar lampreia perder a vesícula e canais biliares durante a metamorfose, um processo semelhante à atresia biliar humano. Um novo método de fixação e esclarecimento (clareza) foi modificado para visualizar toda a árvore biliar através de microscopia confocal de varredura a laser. Este método fornece uma ferramenta poderosa para o estudo degeneração biliar. 

Vídeo: 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: Um método simples para genotipagem Accurate

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

Biologia

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 a Cultura de Embriões início pintainho ex vivo (Nova 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.

Uso de Microscopia Time Lapse para Visualize Anoxia induzida Suspended Animation em<em&gt; C. elegans</em&gt; Embriões

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 microinjeções de ouriço do mar zigotos

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.

Um método para extrair Pigmentos de Squid<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.

Usando um método imunohistoquímico de montagem total para estudar a preservação do trato biliar em<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.

Como caldo em Vitro rastreio: um método fácil e rápido para detectar novos compostos antifúngicos

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 de proteína verde fluorescente Split Visualizar efetores entregue de bactérias durante a infecção

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.

Imunolabellagem de degeneração da miofibra em biópsias 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.

Apoptose indução e detecção em uma cultura primária de células intestinais de pepino do mar

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