Die Autoren danken den Beitrag der Hammond Bay Biologische Station, Great Lakes Science Center, US Geological Survey. Wir danken auch Dr. Melinda Rahmen des Center for Advanced Microscopy an der Michigan State University für ihre technische Unterstützung bei der konfokalen Laser-Scanning-Mikroskopie. Diese Studie wird durch Zuschüsse aus dem Great Lakes Kommission für die Fischerei zu YWCD und WML unterstützt.

1. Herstellung der Lösung <ol><li> Auffüllen auf 1 l 10x 0,1 M Phosphatpufferlösung (PBS, pH 7,4): Gewicht 26,2 g Natriumphosphat (einbasischen), 115 g Natriumphosphat (dibasisches) und 87,66 g NaCl. Löst in etwa 800 ml destilliertem H 2 O, pH-Einstellung und bringt das Volumen auf 1 l mit destilliertem H 2 O. <ol><li> Stellen Sie 1 L 0,1 M phosphatgepufferter Kochsalzlösung (pH 7,4): Nehmen Sie 100 ml 10x PBS, und fügen Sie 900 ml destilliertem H 2 O. </li><li> Stellen Sie 1 L 0,1 M phosphatgepufferter Kochsalzlösung (pH 7,4) mit 0,1% Triton X-100: Nehmen Sie 100 ml 10x PBS, 1 ml Triton X-100, und bringen das Volumen auf 1 L mit destilliertem H 2 O. </li></ol></li><li> Auffüllen auf 1 l 10x 0,1 M Phosphatpuffer (PB, pH 7,4): Gewicht 26,2 g Natriumphosphat (einbasischen) und 115 g Natriumphosphat (dibasisches). Löst in etwa 800 ml destilliertem H 2 O, pH-Einstellung und bringt das Volumen auf 1 l mit destilliertem H 2 O. <ol><li> Stellen Sie 1 L 0,1 M Phosphatpuffer (pH 7,4): Nehmen Sie 100 ml 10x PB,und fügen Sie 900 ml destilliertem H 2 O. </li><li> Stellen Sie 1 L 0,1 M Phosphatpuffer (pH 7,4) mit 0,1% Triton X-100: Nehmen Sie 100 ml 10x PB, 1 ml Triton X-100, und bringen das Volumen auf 1 L mit destilliertem H 2 O. </li></ol></li><li> Machen 400 ml Hydrogel-Monomer-Lösung (4% Acrylamid, 0,25% Bis-Acrylamid, 4% Paraformaldehyd, 0,0075% Ammoniumpersulfat, 0,0005% Saponin in 0,1 M PBS): Man wiegt 0,3 g Ammoniumpersulfat und 0,2 g Saponin. Add 210 ml destilliertem H 2 O, 40 ml 40% Acrylamid, 10 ml 2% Bis-Acrylamid, 40 ml 10x PBS (pH 7,4), 100 ml 16% igem Paraformaldehyd, und gut mischen. </li><li> Stellen 4 L Clearing-Lösung (4% SDS in 0,2 M Borsäure): Wiegen 49,464 g Borsäure und 160 g SDS. Löst in etwa 3,5 L destilliertem H 2 O, pH auf 8,5 mit NaOH, und bringen das Volumen bis zu 4 L. </li><li> Stellen Sie 1 L 80% Glycerin-Lösung: Messen 800 ml Glycerin, 200 ml destilliertem H 2 O, und gut mischen. </li></ol> 2. Gewebepräparation <ol><li> Hydrogel-Monomer-Lösung herzustellen, und 10 ml Aliquots zu je 15 ml Zentrifugenröhrchen. </li><li> Präparieren Sie die ganze Gewebe und zu beheben in dem Hydrogel für 2 Tage bei 4 ° C Stellen Sie sicher, dass das Hydrogel-Lösung mindestens 10-fache Volumen des Gewebes. </li><li> Bringen Sie die Hydrogel-fixierten Gewebe (in der 15-ml-Zentrifugenrohr) zu einem Abzug. </li><li> Polymerisieren das Hydrogel fixiertem Gewebe durch Zugabe von 5 ul TEMED, gut mischen, und lassen Sie sich im Abzug bei Raumtemperatur für 2-3 Stunden. </li><li> Entfernen Sie das überschüssige Gel gründlich. Hinweis: Extra-Gel wird den Clearing-Prozess und den Antikörper Penetration während der Färbung zu verhindern. </li><li> Inkubieren Sie die fixierten Gewebe in 10 ml Clearing-Lösung in einem Inkubator Schüttler bei 70 rpm und 50 ° C eingestellt, für einige Tage, bis das Gewebe wird transparent. Ändern Clearing-Lösung mindestens einmal pro Tag und entfernen Sie das überschüssige Gel aus dem Gewebe, wenn die Änderung der Clearing-Lösung. </li><li> Spülen des Gewebes in 10 ml 0,1 M PB mit 0,1% Triton X-100 bei 70 rUhr und 37 ° C über Nacht. Wiederholen Sie diesen Schritt 3 Mal und entfernen Sie das überschüssige Gel aus dem Gewebe, wenn die Änderung der Pufferlösung. </li><li> Inkubieren der geklärten Gewebe in der primären Antikörperlösung in PB mit 0,1% Triton X-100 bei 70 rpm und 37 ° C für 2 Tage. Hinweis: Stellen Sie genug primären Antikörperlösung, die ganze Gewebe einzutauchen. </li><li> Mit 0,1% Triton X-100 spült das Gewebe in 10 ml PB bei 70 rpm und 37 ° C über Nacht. Wiederholen Sie diesen Schritt 3 Mal und entfernen Sie das überschüssige Gel aus dem Gewebe, wenn die Änderung der Pufferlösung. </li><li> Führen Sie die folgenden Schritte in einem dunklen Raum oder unter schwachem Licht. Decken Sie die Proben mit Folie auf ein Foto, Bleich verhindern. </li><li> Inkubieren der geklärten Gewebe mit dem fluoreszierenden Zweitantikörper-Lösung in PB mit 0,1% Triton X-100 und der Blockierungsserum bei 70 rpm und 37 ° C für 1 Tag. </li><li> Mit 0,1% Triton X-100 spült das Gewebe in 10 ml PB bei 70 rpm und 37 ° C über Nacht. Wiederholen Sie diesen Schritt 3 Mal und entfernen Sie das überschüssige Gel from das Gewebe beim Wechsel der Pufferlösung. </li><li> Spülen Sie das Gewebe in 10 ml PBS bei 70 min und 37 ° C über Nacht. Wiederholen Sie diesen Schritt 3 Mal und entfernen Sie das überschüssige Gel aus dem Gewebe, wenn die Änderung der Pufferlösung. </li><li> Inkubieren geklärt Gewebe in 80% Glycerin bei 70 min und 37 ° C über Nacht. </li><li> Bewahren Sie die fluoreszierenden Buntgewebe bei 4 ° C vor der konfokalen Mikroskopie. </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. 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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>

Die Autoren haben nichts zu offenbaren.

Dieses Protokoll wird von einer neuen Methode namens &quot;Klarheit&quot; 12, die intakten Gewebes mit Polyacrylamid vernetzt, um ein nanoporöses Hydrogel zu bilden, und streift die Plasmamembran des Gewebes, um optische Transparenz und Durchlässigkeit zu erreichen makromolekularen dann modifiziert. &quot;CLARITY&quot; können intakte Gewebe Bildgebung von Langstrecken-Projektion und lokale Verdrahtung im Nervensystem. Diese neue Methode kann verwendet werden, um die gesamte Gallensystems in Meerneunauge Le...

Meerneunauge durch drei verschiedene Leben zu entwickeln Stufen 1,2. Larven Meerneunauge (L) verbringen die meiste Zeit in Höhlen benthische Filtrierer. Nachdem man durch sieben metamorphen Phasen der dramatischen Veränderungen in der Morphologie und äußere Umgestaltung in die inneren Organe 3, die daraus resultierenden Jugendliche (JV) geben eine parasitäre Phase, während der sie auf Blut-und Gewebeflüssigkeiten von Host-Fische zu füttern, die Erhöhung der Körpermasse mehr als 100 Mal . Nach 1,0 bis 1,5 years Fütterung auf die Wirtsfische im Meer oder großen Seen, Erwachsene aufhören Fütterung während des Frühjahrs und wandern zum Laichen in die Flüsse und dann sterben 1,2. Während der Metamorphose, verliert das Meerneunauge Leber die Gallenblase und der Gallenwege, eine evolutionäre mutierten Phänotyp, die den menschlichen Säugling Krankheit Gallengangsatresie nachahmt. Infant Gallengangsatresie ist eine seltene pädiatrische Lebererkrankung mit schweren medizinischen Komplikationen 4,5,6,7, 8.9.10, aber die Pathogenese und Ätiologie der Gallengangsatresie sind weitgehend unbekannt 4. Patienten mit Gallengangsatresie sterben innerhalb von zwei Jahren nach der Geburt, wenn ein chirurgischer Eingriff (Kasai-Verfahren) durchgeführt wird 5. Anschließend werden diese Patienten erfordern umfangreiche klinische Behandlung und oft eine Lebertransplantation sechs. Viele Theorien Gallenatresie Etiopathogenese vorgeschlagen worden, wie virale Infektionen, kongenitale Fehlbildungen, Autoimmunerkrankungen und toxischen Insult. Allerdings bleibt der Beitrag der jeweils für die Entwicklung der Gallengangsatresie nicht schlüssig 7,8,9,10. Im Gegensatz zu Kindern, die pathologische Gallengangsatresie leiden, unterziehen Meerneunauge entwicklungs programmiert Gallengangsatresie ohne umfangreiche necroinflammation, Fibrose oder Zirrhose 10. Die Tiere können vorübergehende Cholestase während dieses Prozesses 10 leiden, aber passen sich diesem Entwicklungszustanddurch de novo-Synthese und Sekretion von Gallensäuren im Darm nach der Entwicklungs biliäre Atresie, zusätzlich zu bekannten Mechanismen, wie die Verringerung der Gallensalz-Synthese in der Leber 11. Dieser Entwicklungsprozess in Meerneunauge bietet die einzige bekannte Möglichkeit, das Fortschreiten der Gallengangsatresie zu untersuchen. Eine neu entwickelte Methode namens &quot;Klarheit&quot; ermöglicht hochauflösende Bildgebung in komplexen Nervensystem von Säugetieren durch die Umwandlung intakten Gewebe in einem optisch transparenten nanoporöse Hydrogel-12. Mit Meerneunauge Leber und eine modifizierte CLARITY-Protokoll können intakte Gewebe-Bildgebung der Leber und Gallen Degeneration ganzen Metamorphose dokumentiert werden.

<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)

Gallengangsatresie ist eine seltene Erkrankung des Kindesalters, mit einem geschätzten 1 in 15000 Frequenz im Südosten der Vereinigten Staaten, sondern in ostasiatischen Ländern häufiger mit einer Frequenz von 1 berichtet in 5000 in Taiwan. Obwohl viel über die Verwaltung der Gallengangsatresie bekannt ist, ist der Pathogenese noch ungeklärt. Das Meerneunauge (Petromyzon marinus) bietet eine einzigartige Gelegenheit, um den Mechanismus und den Verlauf von Gallendegeneration zu untersuchen. Meerneunauge entwickeln sich durch drei verschiedene Lebensphasen: Larven, Parasiten und Erwachsenen. Während des Übergangs von Larven von parasitären Jugend, Meerneunauge Metamorphose zu unterziehen mit dramatischen Sanierung und Umbau in äußere Morphologie und inneren Organen. In der Leber wird die gesamte Gallensystems verloren, einschließlich der Gallenblase und der Gallenwege. Eine neu entwickelte Methode namens &quot;Klarheit&quot; wurde modifiziert, um die gesamte Leber und die Kreuzung mit dem Darm in metamorphen Meerneunauge zu klären. DieProzess der Gallen Degeneration wurde durch Verwendung konfokaler Laser-Scanning-Mikroskopie visualisiert und Meerneunauge während der Metamorphose zu erkennen. Diese Methode bietet ein leistungsstarkes Werkzeug, um Gallengangsatresie in einer einzigartigen Tiermodell zu untersuchen.

Mehrere wichtige Entwicklungsschritte in der Leber-Gallen-System auftreten, während das Meerneunauge Metamorphose. Der Gallengang und die Gallenblase Apoptose und entarteten (Abbildung 1). Kombinieren des modifizierten Klärungsverfahren und Färbung mit Leberzellmarker Cytokeratin 19 (CK19, sowohl Cholangiozyten-und Hepatozyten vor und nach der Metamorphose 13 vorhanden) und anti-apoptotischen Bcl-2-Marker mittels konfokaler Mikroskopie wurde die gesamte Gallensystems entlang der Z-Achse (f...

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

Meerneunauge verlieren die Gallenblase und der Gallenwege während der Metamorphose, ein Prozess ähnlich dem menschlichen Gallengangsatresie. Eine neue Methode der Fixierung und Klärung (CLARITY) wurde modifiziert, um die Gallenwege mit konfokaler Laser-Scanning-Mikroskopie visualisieren. Diese Methode bietet ein leistungsstarkes Werkzeug, um Gallen Degeneration zu studieren.

Eine neue Methode zur Klärung biliäre Leberdegeneration Visualisieren Während Metamorphosis in Seeneunauge ( 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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Research

JoVE Journal

Biology

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

13.7K Aufrufe.  Michigan State University.  Meerneunauge verlieren die Gallenblase und der Gallenwege während der Metamorphose, ein Prozess ähnlich dem menschlichen Gallengangsatresie. Eine neue Methode der Fixierung und Klärung (CLARITY) wurde modifiziert, um die Gallenwege mit konfokaler Laser-Scanning-Mikroskopie visualisieren. Diese Methode bietet ein leistungsstarkes Werkzeug, um Gallen Degeneration zu studieren. 

Serie: 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: Eine einfache Methode für genaue Genotypisierung

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

Forschung

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.

Method for Culture of Early Hühnerembryonen 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 ...

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.

Verwendung von Zeitraffer-Mikroskopie zur Anoxie-induzierten Suspended Animation in Visualisieren<em&gt; C. elegans</em&gt; Embryonen

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.

Hochdurchsatz-Mikroinjektionen von 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 ...

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.

Ein Verfahren zum Extrahieren Pigmente aus 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.

Mit einer vollständigen immunhistochemischen Methode, um die Innervation der Gallenwege zu studieren<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.

Brühe Microdilution In Vitro Screening: eine einfache und schnelle Methode, um neue Antimykotika Verbindungen erkennen

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.

Split-grün fluoreszierendes Protein System Effektoren geliefert von Bakterien während der Infektion zu visualisieren

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.

Immunolabelling Myofiber Degeneration in Muskelbiopsien

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-Induktion und Detektion in einer primärer Kultur von Seegurken-Darmzellen

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