Les auteurs reconnaissent la contribution de la Station biologique de Hammond Bay, Great Lakes Science Center, US Geological Survey. Nous remercions également le Dr Melinda cadre au Centre de microscopie avancée à l&#39;Université d&#39;État du Michigan pour son soutien technique à balayage laser microscopie confocale. Cette étude est financée par des subventions de la Commission des pêcheries des Grands Lacs à YWCD et WML.

Une. Préparation Solution <ol><li> Faire une L 10x 0,1 M de solution saline tamponnée au phosphate (PBS, pH 7,4): Peser 26,2 g de phosphate de sodium (monobasique), 115 g de phosphate de sodium (dibasique), et 87,66 g de NaCl. Dissoudre dans environ 800 ml de H 2 O distillée, ajuster le pH, et porter le volume à 1 L avec de l&#39;eau distillée 2 O. <ol><li> Faire une solution saline L 0,1 M de tampon phosphate (pH 7,4): Prendre 100 ml 10x PBS et ajouter 900 ml de H 2 O. distillée </li><li> Assurez 1 L 0,1 M de tampon phosphate salin (pH 7,4) avec 0,1% de Triton X-100: Prendre 100 ml 10x PBS, ajouter 1 ml de Triton X-100, et porter le volume à 1 L avec de l&#39;eau distillée 2 O. </li></ol></li><li> Assurez 1 L 10x 0,1 M tampon phosphate (PB, pH 7,4): Peser 26,2 g de phosphate de sodium (monobasique) et 115 g de phosphate de sodium (dibasique). Dissoudre dans environ 800 ml de H 2 O distillée, ajuster le pH, et porter le volume à 1 L avec de l&#39;eau distillée 2 O. <ol><li> Assurez 1 L 0,1 M de tampon phosphate (pH 7,4): Prendre 100 ml 10x PB,et ajouter 900 ml d&#39;eau distillée H 2 O. </li><li> Assurez 1 L 0,1 M de tampon phosphate (pH 7,4) avec 0,1% de Triton X-100: Prendre 100 ml 10x PB, ajouter 1 ml de Triton X-100, et porter le volume à 1 L avec de l&#39;eau distillée 2 O. </li></ol></li><li> Ajoutez la solution de monomère d&#39;hydrogel de 400 ml (4% d&#39;acrylamide, 0,25% de bis-acrylamide, du paraformaldéhyde 4%, 0,0075% de persulfate d&#39;ammonium, 0,0005% de saponine dans du PBS 0,1 M): Peser 0,3 g de persulfate d&#39;ammonium et 0,2 g de saponine. Ajouter 210 ml de H 2 O distillée, 40 ml 40% d&#39;acrylamide, 10 ml 2% de bis-acrylamide, 40 ml 10x PBS (pH 7,4), 100 ml 16% de paraformaldéhyde, et bien mélanger. </li><li> Préparer une solution de compensation de 4 L (4% de SDS dans 0,2 M d&#39;acide borique): Peser 49,464 g d&#39;acide borique et 160 g de SDS. Dissoudre dans environ 3,5 L H 2 O distillée, ajuster le pH à 8,5 avec NaOH, et porter le volume à 4 L. </li><li> Assurez 1 L solution de glycérol à 80%: Mesurer 800 ml de glycérol, ajouter 200 ml de H 2 O distillée, et bien mélanger. </li></ol> 2. Préparation des tissus <ol><li> Préparer la solution de monomère d&#39;hydrogel et aliquote de 10 ml de chaque tube de centrifugation de 15 ml. </li><li> On dissèque le tissu et fixer l&#39;ensemble dans l&#39;hydrogel pendant 2 jours à 4 ° C. Assurez-vous que la solution d&#39;hydrogel est d&#39;au moins 10 fois le volume du tissu. </li><li> Amener le tissu fixé d&#39;hydrogel (dans le tube de centrifugation de 15 ml) à une hotte de laboratoire. </li><li> Polymériser l&#39;hydrogel tissu fixé en ajoutant 5 pi de TEMED, bien mélanger et laisser reposer dans la hotte à température ambiante pendant 2-3 heures. </li><li> Retirer soigneusement le gel excès. Remarque: gel supplémentaire sera entraver le processus de compensation et de la pénétration d&#39;anticorps pendant la coloration. </li><li> Incuber le tissu fixé dans 10 ml de solution de compensation dans un shaker incubateur réglé à 70 tours par minute et 50 ° C pendant plusieurs jours jusqu&#39;à ce que le tissu devient transparent. Changer solution de compensation au moins une fois par jour et enlever le gel en excès du tissu lors du changement de la solution de compensation. </li><li> Rincer le tissu dans 10 ml de 0,1 M de PB avec 0,1% de Triton X-100 à 70 rh et à 37 ° C pendant une nuit. Répéter cette étape trois fois, et enlever le gel en excès du tissu lors du changement de la solution tampon. </li><li> Faire incuber le tissu clarifié dans la solution d&#39;anticorps primaire dans PB avec 0,1% de Triton X-100 à 70 rpm et 37 ° C pendant 2 jours. Remarque: solution suffisamment d&#39;anticorps primaire pour submerger les tissus. </li><li> Rincer le tissu dans 10 ml de PB avec 0,1% de Triton X-100 à 70 rpm et 37 ° C pendant une nuit. Répéter cette étape trois fois, et enlever le gel en excès du tissu lors du changement de la solution tampon. </li><li> Effectuez les étapes suivantes dans une pièce sombre ou en faible lumière. Couvrir les échantillons de papier d&#39;aluminium pour éviter photoblanchiment. </li><li> Faire incuber le tissu avec la solution clarifiée fluorescent de l&#39;anticorps secondaire en PB avec 0,1% de Triton X-100 et le sérum de blocage 70 tours par minute et à 37 ° C pendant 1 jour. </li><li> Rincer le tissu dans 10 ml de PB avec 0,1% de Triton X-100 à 70 rpm et 37 ° C pendant une nuit. Répétez cette étape 3 fois et enlever le gel excès felon le tissu lors du changement de la solution tampon. </li><li> Rincer le tissu dans 10 ml de PBS à 70 rpm et 37 ° C pendant une nuit. Répéter cette étape trois fois, et enlever le gel en excès du tissu lors du changement de la solution tampon. </li><li> Faire incuber le tissu clarifié dans 80% de glycerol à 70 rpm et 37 ° C pendant une nuit. </li><li> Stocker le tissu teinté fluorescente à 4 ° C avant la microscopie confocale. </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>

Les auteurs n&#39;ont rien à révéler.

Ce protocole est modifié à partir d&#39;une nouvelle méthode appelée «clarté» 12, qui réticule tissu intact avec polyacrylamide pour former un hydrogel nanoporeux, puis supprime le membrane plasmique des tissus pour assurer la transparence optique et la perméabilité macromoléculaire. «Clarté» permet l&#39;imagerie des tissus intacts de projection à long terme et le câblage du circuit local dans le système nerveux. Cette nouvelle méthode peut être utilisée pour visualiser l&#39;ensemble du ...

La lamproie marine se développer à travers trois étapes distinctes vie 1,2. Larvaire lamproie (L) passent plus de temps dans des terriers comme filtreurs benthiques. Après avoir traversé sept étapes métamorphiques de changements dramatiques dans la morphologie externe et la réorganisation des organes internes 3, les mineurs résultant (JV) entrent dans un stade parasitaire au cours de laquelle ils se nourrissent de sang et les liquides tissulaires de poisson hôte, l&#39;augmentation de la masse corporelle de plus de 100 fois . Après 1.0 à 1,5 ans d&#39;alimentation sur le poisson hôte dans l&#39;océan ou les grands lacs, les adultes cessent de s&#39;alimenter au début du printemps et migrent dans les rivières pour frayer et mourir 1,2. Au cours de métamorphose, le foie de la lamproie marine perd la vésicule biliaire et l&#39;ensemble de l&#39;arbre biliaire, un phénotype mutant évolutif qui imite la maladie infantile humaine atrésie des voies biliaires. Atrésie des voies biliaires infantile est une maladie du foie pédiatrique rare avec de graves complications médicales 4,5,6,7, 8,9,10, mais la pathogénie et l&#39;étiologie de l&#39;atrésie des voies biliaires sont en grande partie inconnus 4. Les patients présentant une atrésie des voies biliaires meurent dans les deux ans après la naissance, sauf si une intervention chirurgicale (procédure Kasai) est effectuée 5. Par la suite, ces patients ont besoin d&#39;une vaste prise en charge clinique et souvent une transplantation du foie 6. De nombreuses théories de l&#39;atrésie des voies biliaires étiopathogenèse ont été proposées, telles que l&#39;infection virale, une malformation congénitale, maladie auto-immune, et insulte toxique. Toutefois, la contribution de chacun à l&#39;élaboration d&#39;atrésie des voies biliaires restent peu concluantes 7,8,9,10. Contrairement nourrissons qui souffrent atrésie des voies biliaires pathologique, la lamproie marine subir de développement programmé atrésie des voies biliaires sans necroinflammation vaste, fibrose ou de cirrhose 10. Les animaux peuvent souffrir cholestase transitoire pendant ce processus 10, mais s&#39;adapter à cet état ​​de développementpar l&#39;intermédiaire de la synthèse de novo et de la sécrétion de sels biliaires dans l&#39;intestin après l&#39;atrésie biliaire développementale, en plus des mécanismes connus tels que la réduction de la synthèse des sels biliaires dans le foie 11. Ce processus de développement dans la grande lamproie marine fournit la seule occasion connue pour examiner la progression de l&#39;atrésie des voies biliaires. Une méthode nouvellement développé appelé «clarté» permet imagerie à haute résolution dans les systèmes nerveux des mammifères complexes en transformant tissu intact dans un hydrogel nanoporeux optiquement transparent 12. Utilisation de la lamproie marine foie et un protocole de CLARITY modifié, l&#39;imagerie des tissus intacts de la dégénérescence biliaire peut être documentée tout au long de la métamorphose du foie.

<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)

Atrésie des voies biliaires est une maladie rare de l&#39;enfance, avec une fréquence d&#39;environ 1 à 15 000 dans le sud-est des États-Unis, mais plus fréquente dans les pays en Asie de l&#39;Est, avec une fréquence déclarée de 1 à 5000 à Taiwan. Bien que beaucoup est connu au sujet de la gestion de l&#39;atrésie des voies biliaires, sa pathogénie est encore insaisissable. La lamproie marine (Petromyzon marinus) offre une occasion unique d&#39;examiner le mécanisme et la progression de la dégénérescence des voies biliaires. La lamproie marine développer à travers trois étapes de la vie distincts: larves, parasites, et adultes. Lors du passage de larves à des mineurs parasite, la lamproie marine subir la métamorphose de la réorganisation dramatique et le remodelage de la morphologie externe et les organes internes. Dans le foie, l&#39;ensemble du système biliaire est perdu, y compris la vésicule biliaire et des voies biliaires. Une méthode nouvellement développé appelé «clarté» a été modifié pour clarifier l&#39;ensemble du foie et la jonction avec l&#39;intestin dans métamorphique lamproie marine. Laprocessus de dégénérescence biliaire a été visualisée et discerné lors de la lamproie marine métamorphose en utilisant la numérisation laser microscopie confocale. Cette méthode fournit un outil puissant pour étudier atrésie des voies biliaires dans un modèle animal unique.

Plusieurs événements importants se produisent dans le développement du système hépatobiliaire au cours de la lamproie marine métamorphose. La voie biliaire et de la vésicule biliaire et subissent une apoptose dégénérée (Figure 1). La combinaison de la méthode de clarification modifié et coloration au foie marqueur de cellules cytokératine 19 (CK19, présents dans les deux cholangiocytes et les hépatocytes avant et après la métamorphose 13) et Bcl2 marqueur anti-apoptotiques e...

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

La lamproie marine perdre la vésicule biliaire et des voies biliaires au cours de la métamorphose, un processus similaire à l&#39;atrésie biliaire humaine. Une nouvelle méthode de fixation et de clarification (CLARITY) a été modifié pour visualiser l&#39;ensemble de l&#39;arbre biliaire en utilisant la numérisation laser microscopie confocale. Cette méthode fournit un outil puissant pour étudier la dégénérescence biliaire.

Une nouvelle méthode de clarification de visualiser biliaire dégénérescence Pendant foie métamorphose de la lamproie marine ( 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 vues.  Michigan State University.  La lamproie marine perdre la vésicule biliaire et des voies biliaires au cours de la métamorphose, un processus similaire à l'atrésie biliaire humaine. Une nouvelle méthode de fixation et de clarification (CLARITY) a été modifié pour visualiser l'ensemble de l'arbre biliaire en utilisant la numérisation laser microscopie confocale. Cette méthode fournit un outil puissant pour étudier la dégénérescence biliaire. 

Vidéo: 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.

Pyroséquençage: une méthode simple pour le génotypage précis

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

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Biologie

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.

Méthode pour la culture d&#39;embryons de poulet Early ex vivo (Nouvelle 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 ...

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.

L&#39;utilisation de la microscopie à Time Lapse Visualisez anoxie induite Suspended Animation en<em&gt; C. elegans</em&gt; Embryons

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

Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization

Fluorescent in situ hybridization using DNA probes on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA FISH) represents the most direct way to visualize the location of gene loci, chromosomal sub-regions or entire territories in individual cells. This type of analysis provides insight into the global architecture of the nucleus as well as the behavior of specific genomic loci and regions within the nuclear space. Immunofluorescence, on the other hand, permits the detection of nuclear proteins (modified histones, histone variants and modifiers, transcription machinery and factors, nuclear sub-compartments, etc). The major challenge in combining immunofluorescence and 3D DNA FISH is, on the one hand to preserve the epitope detected by the antibody as well as the 3D architecture of the nucleus, and on the other hand, to allow the penetration of the DNA probe to detect gene loci or chromosome territories 1-5. Here we provide a protocol that combines visualization of chromatin modifications with genomic loci in 3D preserved nuclei.

Here we describe a protocol for simultaneous detection of histone modifications by immunofluorescence and DNA sequences by DNA FISH followed by 3D microscopy and analyses (3D immuno-DNA FISH).

Immunofluorescence combinés et FISH ADN sur 3D préservé noyaux en interphase pour étudier les changements dans l&#39;organisation nucléaire 3D

Fluorescent in situ hybridization using DNA probes  on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA  FISH) represents the ...

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.

À haut débit microinjections oursins 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 ...

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 procédé d&#39;extraction Pigments de Squid<em&gt; Doryteuthis pealeii</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.

Utilisation d&#39;une méthode immunohistochimique intégrale pour étudier l&#39;inervation du tractus biliaire dans<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.

Bouillon Microdilution In Vitro Screening : une méthode simple et rapide pour détecter les nouveaux composés antifongiques

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.

Protéine fluorescente verte bibloc pour visualiser les effecteurs livré de bactéries lors de l’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 ...

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.

Induction et détection d'apoptosis dans une culture primaire des cellules intestinales de concombre de mer

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