Wir möchten die Beiträge von Dörthe Jülich, Jennifer Runde und Andrew Mara in der Entwicklung dieses Protokoll bestätigen. Forschungsförderung durch die NICHD, die American Cancer Society und der March of Dimes zur Verfügung gestellt.

1. FIXATION <ol><li> Fix Embryonen über Nacht bei 4 ° C mit 4% Paraformaldehyd (PFA) in PBS. </li><li> Entfernen Sie fix und waschen 2 x PBS, jeweils 5 Minuten bei Raumtemperatur (RT). </li><li> Manuelles dechorionate Embryonen in PBS in einem Glas Depression Platte mit Uhrmacher Pinzette. Nach dechorionation sind Embryonen mit einem Feuer-poliertem Glas Pasteur Pipette, wie sie Polypropylen Pipette kann bleiben. </li><li> -Embryonen durch eine Reihe von 25%, 50% und 75% Methanol in PBS für jeweils 5 Minuten. </li><li> Ersetzen Flüssigkeit mit 100% Methanol, inkubieren 5 Minuten und ersetzen Sie dann mit frischem Methanol. </li><li> Legen Embryonen bei -20 ° C für mindestens eine Stunde. (Wir normalerweise inkubieren die Embryonen über Nacht. Standard in-situ-Hybridisierung wird an Embryonen für mehr als ein Jahr gespeichert, aber wir haben nicht untersucht, ob die hohe Auflösung der Fluoreszenz-in situ-Hybridisierung bei längerer Lagerung verloren geht.) </li><li> Wash Embryonen für jeweils 5 Minuten in 75%, 50%, 25% Methanol in PBST bei RT. Zweimal waschen für jeweils 5 Minuten in PBST bei RT. </li><li> Fix wieder für 20 Minuten in 4% PFA in PBS bei RT. </li><li> Zweimal waschen für jeweils 5 Minuten in PBST bei RT. </li></ol> HINWEIS ZU PFA: Wir speichern 4% PFA in Aliquots bei -20 º C (siehe Tabelle der Reagenzien). Für die erstmalige Fixierung verwenden wir nur PFA, dass noch nie zuvor aufgetaut. Für die anschließende Fixierungen, benutzen wir oft PFA, dass zuvor aufgetaut. Die anfängliche Fixierung zu sein scheint entscheidend für eine erfolgreiche Färbung mit diesem Protokoll. 2. Proteinase und Nachfixierung <ol><li> Digest mit Proteinase K (5μg/ml in PBST) bei RT für 3 bis 12 Minuten, um die Embryonen permeabilisieren. (Die Inkubationszeit ist abhängig vom Alter der Embryonen als jüngere Stadien empfindlicher sind. Es hängt auch von der Charge des Enzyms.) Für Somitogenese Embryonen, wir in der Regel für 3-4 Minuten permeabilisieren. Während dieser Inkubation legen wir den Mikrozentrifugenröhrchen auf seiner Seite. </li><li> Spülen Sie kurz in PBST waschen einmal für 5 Minuten in PBST. </li><li> Fix wieder für 20 Minuten in 4% PFA in PBS bei RT. </li><li> Zweimal waschen, für jeweils 5 Minuten, in PBST bei RT. </li></ol> 3. Prähybridisierung <ol><li> Inkubieren Sie die Embryonen für 5 Minuten bei 65 º C in HYB-. </li><li> Prehybridize bei 65 º C für mindestens 1 Stunde in HYB +. </li></ol> 4. HYBRIDIZATION <ol><li> Entfernen Sie alle 50 ul der preHYB, aber stellen Sie sicher, dass die Embryonen völlig untergetaucht zu halten. </li><li> Fügen Sie 1-2 ul jeder Ribosonde (Digoxigenin-und Fluorescein-markierten Ribosonden), die Embryonen und mischen durch vorsichtiges Schnippen der Röhre. Die Höhe der Sonde ist in der Regel 1-2μl von einem 20 &amp;mgr; l-Sonde Synthesereaktion. </li><li> Da Fluorescein lichtempfindlich ist, sollten die Rohre in Aluminiumfolie eingewickelt werden oder anderweitig zu wenig Licht von diesem Punkt an ausgesetzt. </li><li> Inkubieren Sie die Embryonen über Nacht bei 65 º C. </li></ol> 5. Sondenentnahme Beachten Sie, dass die Lösungen von diesem Punkt an Mangel Reinigungsmittel. Elimination des Waschmittels scheint die Färbung Reaktionen helfen, aber nicht Ursache der Embryonen zu werden und nicht klebrig. <ol><li> Entfernen Sie die Ribosonde. </li><li> Wash 2 x 30 Minuten bei 65 º C in 50% formamide/2xSSC. </li><li> Wash für 15 Minuten bei 65 º C in 2 x SSC. </li><li> Wash für 30 Minuten 65 º C in 0,2 x SSC. </li></ol> 6. Anti-Fluorescein Antikörperinkubation <ol><li> Block für mindestens 1 Stunde bei RT in 500μl einer Lösung von 1x Maleinsäure-Puffer plus 2% Blocking Reagenz (siehe Tabelle der Reagenzien). </li><li> Fügen Sie die Anti-Fluorescein-POD-Antikörper, wie Roche geliefert, bei einer Verdünnung 1:500 in Blocking-Lösung. </li><li> Inkubieren über Nacht bei 4 º C. Während dieser Inkubation legen wir den Mikrozentrifugenröhrchen auf seiner Seite. </li><li> Wash 4 x 20 Minuten in 1x Maleinsäure-Puffer. Zweimal waschen für jeweils 5 Minuten in PBS. </li></ol> 7. DETECTION von Fluorescein-markierten PROBE <ol><li> Inkubieren 30-60 Minuten in TSA Plus-Fluorescein-Lösung. (Down TSA Substrat Spin bevor Färbelösung. Für die Reaktion verdünnter Tyramid Reagenz 1:50 in Perkin Elmer Verstärkung Verdünnungspuffer.) Während dieser Inkubation legen wir den Mikrozentrifugenröhrchen auf seiner Seite. Reaktionszeit muss empirisch für jede Sonde ermittelt werden. Leider kann die Farbreaktion nicht visuell überwacht werden, da das Substrat fluoreszierenden, und man wird überall grüne Fluoreszenz in der Färbung zu sehen. </li><li> Wash für jeweils 10 Minuten in 30%, 50%, 75% und 100% Methanol in PBS. </li><li> Inkubieren in einer Lösung von 1% H 2 0 2 in Methanol für 30 Minuten auf den ersten Peroxidase zu inaktivieren. </li><li> Wash jeweils 10 Minuten in 75%, 50% und 30% Methanol in PBS. Dann zweimal waschen für jeweils 10 Minuten in PBS. Es ist wichtig, dass alle erfüllthanol entfernt werden. </li></ol> NOTES ON THE Tyramid Signalverstärkung: Wir haben mit dem Perkin Elmer TSA-Kits. Wir finden, dass der Alexa-Tyramid Substrate gut Fleck mit dem Perkin Elmer Verstärkung Verdünnungspuffer, aber wir haben nicht den Erfolg mit dem Färbepuffer mit dem Invitrogen / Molecular Probes Kits zur Verfügung gestellt. Schließlich haben wir festgestellt, dass Cy5-Fluoreszenz durch nachfolgende Methanol / H 2 O 2-Behandlung eliminiert wird, während Fluorescein und Alexa-647 nicht betroffen sind. Cy3 kann auch negativ durch die Methanol / H 2 O 2-Behandlung, wie es strukturell zu Cy5 Zusammenhang betroffen sein. Aus diesem Grund sollte Cy3 und Cy5 TSA Reaktionen nur für die zweite Farbreaktion in einem doppelten Fluoreszenz-in situ-Protokoll verwendet werden. 8. ANTI-Digoxygenin Antikörperinkubation <ol><li> Blockieren Sie die Embryonen erneut für mindestens 1 Stunde bei RT in einer Lösung aus 1x Maleinsäure-Puffer plus 2% Blocking Reagenz. </li><li> Fügen Sie die Anti-DIG-POD-Antikörper, wie Roche in einer 1:1000 Verdünnung geliefert in obigen Blockierungslösung </li><li> Inkubieren über Nacht bei 4 º C. Während dieser Inkubation legen wir den Mikrozentrifugenröhrchen auf seiner Seite. </li><li> Wash 4 x 20 Minuten in 1x Maleinsäure-Puffer. Zweimal waschen für jeweils 5 Minuten in PBS. </li></ol> 9. NACHWEIS VON Digoxygenin-ETIKETTIERT PROBE <ol><li> Inkubieren 30-60 Minuten in TSA Plus-Cy5-Lösung (Spin down TSA Substrat, bevor Färbelösung. Für die Reaktion verdünnter Tyramid Reagenz 1:50 in Verdünnungspuffer Verstärkung) Während dieser Inkubation legen wir den Mikrozentrifugenröhrchen auf seiner Seite. Reaktionszeit muss empirisch für jede Sonde ermittelt werden. </li><li> Dreimal für jeweils 10 Minuten in PBST. </li></ol> 10. Propidiumiodid-Färbung <ol><li> Zweimal waschen für jeweils 5 Minuten in 2 x SSC. </li><li> Inkubieren Embryonen für 30 Minuten bei 37 ° C in 50 ul 2 x SSCT mit 10 ul RNAse (für eine Endkonzentration von 100μg/ml). </li><li> Wash sechsmal für jeweils 3 Minuten in 2 x SSC bei RT. </li><li> Stain Embryonen für 8 Minuten in eine Lösung von 330μg/ml Propidiumiodid in 2 x SSC. </li><li> Wash sechsmal für jeweils 3 Minuten in 2 x SSC bei RT. </li><li> Fix für 20 Minuten in 4% PFA in PBS bei RT. </li><li> Zweimal waschen, für jeweils 5 Minuten, in PBST bei RT. </li></ol> 11. MONTAGE <ol><li> Inkubieren Sie die Embryonen für jeweils 10 Minuten in 25% und 50% Glycerin in PBST. Klare Nacht in 75% Glycerin bei 4 º C. </li><li> Wir analysieren und deyolk die Embryonen und flach montieren sie auf einen Objektträger. Das Eigelb können erhebliche Hintergrundfluoreszenz. Dissection ist viel leichter, nachdem die Embryonen ovenight in gylcerol gelöscht. </li></ol> SOLUTIONS: <table border="1"><tbody><tr><td> PBST </td><td> PBS plus 0,1% Tween </td><td></td></tr><tr><td> SSCT </td><td> SSC plus 0,1% Tween </td><td></td></tr><tr><td> HYB- </td><td> 50% Formamid 5xSSC 0,1% Tween-20 </td><td></td></tr><tr><td> HYB + </td><td> HYB- 5mg/ml Torula (Hefe) RNA 50μg/ml Heparin </td><td style="width: 300px;"> Die Torula RNA wird durch Proteinase K-Verdau der RNA mit vorbereiteten anschließender Phenol-, Phenol-Chloroform-und Chloroform-extraction.The RNA ausgefällt und gelöst in DEPC-behandeltem Wasser. </td></tr><tr><td> 1 x Maleinsäure-Puffer </td><td> 150 mM Maleinsäure, 100 mM NaCl (pH 7,5) </td><td></td></tr></tbody></table><img src="/files/ftp_upload/1229/Brend_Holley_Fig1.png" alt="Abbildung 1" width="600" height="688" /> Abbildung 1. Repräsentative Ergebnisse der ganze Berg doppelt Fluoreszenz-in situ-Hybridisierung. (AC). Bilder zeigen einen einzigen konfokalen Schnitt durch den hinteren Bereich eines Zebrafisch-Embryos bei den zehn-Somiten-Stadium. Die Aussicht ist dorsal mit vorderen an die Spitze. Zellkerne mit Propidiumiodid sind blau gefärbt. (A) deltaC mRNA wurde unter Verwendung eines Digoxigenin-markierten Ribosonde und TSA-Cy5-Reagenz. (B) mRNA transkribiert aus dem HER 1-Gen wurde mit einem Fluorescein-markierten Ribosonde und TSA-Fluorescein-Reagens nachgewiesen werden. (C) Die Zusammenlegung der grünen und roten Kanälen eindeutig identifiziert Regionen unterschiedliche oder sich überschneidende Genexpression der beiden Gene. (D, E) Detail HER1 Ausdruck zeigt die hohe Auflösung dieses Verfahrens. Subzelluläre Lokalisation der mRNA ist deutlich zu erkennen. Pfeilspitzen weisen auf eine Zelle ausstellenden aktive Transkription, die durch Punkte der Färbung im Zellkern offenbart. Die Pfeile zeigen eine Zelle zeigt zytoplasmatische Lokalisierung von mRNA. (F, G) Doppelfärbung für HER1 und deltaC zeigt Zellen, die Transkription beider Gene (weiße Pfeilspitze), oder auch Gen getrennt (rote und grüne Pfeile) sind.

<ol>
	<li>Schulte-Merker, S., Ho, R. K., Herrmann, B. G., N&#252;sslein-Volhard, 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+protein+product+of+the+zebrafish+homologue+of+the+mouse+T-gene+is+expressed+in+nuclie+of+the+germ+ring+and+the+notochord+of+the+early+embryo.">The protein product of the zebrafish homologue of the mouse T-gene is expressed in nuclie of the germ ring and the notochord of the early embryo.</a> Development. 116, 1021-1027 (1992).</li><li>Jowett, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double+in+situ+hybridization+techniques+in+zebrafish.">Double in situ hybridization techniques in zebrafish.</a> Methods. 23, 345-358 (2001).</li><li>J&#252;lich, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=beamter/deltaC+and+the+role+of+Notch+ligands+in+the+zebrafish+somite+segmentation,+hindbrain+neurogenesis+and+hypochord+differentiation.">beamter/deltaC and the role of Notch ligands in the zebrafish somite segmentation, hindbrain neurogenesis and hypochord differentiation.</a> Dev Biol. 286, 391-404 (2005).</li><li>Mara, A., Schroeder, J., Chalouni, C., Holley, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Priming,+Initiation+and+Synchronization+of+the+Segmentation+Clock+by+deltaD+and+deltaC.">Priming, Initiation and Synchronization of the Segmentation Clock by deltaD and deltaC.</a> Nat Cell Biol. 9, 523-530 (2007).</li><li>Lecuyer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+analysis+of+mRNA+localization+reveals+a+prominent+role+in+organizing+cellular+architecture+and+function.">Global analysis of mRNA localization reveals a prominent role in organizing cellular architecture and function.</a> Cell. 131, 174-187 (2007).</li></ol>

Das Protokoll hier vorgestellten Arbeiten gut mit Sonden, die eine saubere starkes Signal nach Färbung für 30-45 Minuten in einem typischen alkalischen Phosphatase-vermittelte Reaktion zu geben. Vor Durchführung der Fluoreszenz-in situ-Hybridisierung Protokoll haben wir immer prüfen unsere Sonden mit dem Standard nicht-fluoreszierenden Protokoll (sofern in ergänzendes Material zusammen mit der Sonde Synthese-Protokoll). Wir haben weniger Erfolg mit der Fluoreszenz-in situ-Hybridisierung Protokoll hatte bei der Verw...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>Paraformaldehyde 16% solution, EM Grade</td>
<td>&nbsp;</td>
<td>Electron Microscopy Services</td>
<td>15710</td>
<td>Dilute to 4% in 1.33x PBS (final 1x). Store in 2ml aliquots at &#x2013;20&deg;C</td>
</tr>
<tr>
<td>Proteinase K, recombinant PCR grade</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>03115879001</td>
<td>Make a 20mg/ml stock solution in water. Store in 1ml aliquots at &#x2013;20&deg;C</td>
</tr>
<tr>
<td>Blocking Reagent</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11096176001</td>
<td>Make a 10% stock solution in 1x maleic acid buffer. Store in 50ml aliquots at &#x2013;20&deg;C. Stable over multiple freeze/thaws.</td>
</tr>
<tr>
<td>Anti-Fluorescein-POD, Fab fragments</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11426346910</td>
<td>Aliquot and store at -20&deg;C. Keep one working aliquot at 4&deg;C.</td>
</tr>
<tr>
<td>Anti-Digoxigenin-POD, Fab fragments</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11207739910</td>
<td>As above.</td>
</tr>
<tr>
<td>TSA Plus Fluorescein system</td>
<td>&nbsp;</td>
<td>Perkin Elmer</td>
<td>NEL741001KT</td>
<td>Prepare each vial of TSA reagent only when needed. Dissolve in 60&mu;l DMSO. Store at 4&deg;C.</td>
</tr>
<tr>
<td>TSA Plus Cyanine5 system</td>
<td>&nbsp;</td>
<td>Perkin Elmer</td>
<td>NEL745001KT</td>
<td>As above</td>
</tr>
<tr>
<td>TSA Plus Cyanine3 system</td>
<td>&nbsp;</td>
<td>Perkin Elmer</td>
<td>NEL744001KT</td>
<td>As above</td>
</tr>
<tr>
<td>TSA Kit #16 AlexaFluor647 tyramide (plus HRP-goat-anti-rabbit IgG)</td>
<td>&nbsp;</td>
<td>Molecular Probes /Invitrogen</td>
<td>T20926</td>
<td>Dissolve tyramide reagent in 150&mu;l DMSO, aliquot and store at &#x2013;20&deg;C.</td>
</tr>
<tr>
<td>RNase, DNase free</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11119915001</td>
<td>Supplied as 500&mu;g/ml solution</td>
</tr>
<tr>
<td>Propidium Iodide</td>
<td>&nbsp;</td>
<td>Molecular Probes /Invitrogen</td>
<td>P-3566</td>
<td>Supplied as 1mg/ml solution in water.</td>
</tr>		</tbody>
		</table>
		</div>

zebrafish whole mount high-resolution double fluorescent in situ hybridization

Whole mount<em&gt; In situ</em&gt; Hybridisierung ist eine der am häufigsten verwendeten Techniken in der Entwicklungsbiologie. Hier präsentieren wir ein hochauflösendes Doppel-Fluoreszenz-<em&gt; In situ</em&gt; Hybridisierung Protokoll zur Analyse der genauen Expressionsmuster eines einzelnen Gens und zur Bestimmung der Überlappung der Ausdruck Domains von zwei Genen. Das Protokoll ist eine modifizierte Version der Standard-<em&gt; In situ</em&gt; Hybridisierung mit alkalischer Phosphatase und Substraten wie NBT / BCIP und Fast Red<sup&gt; 1,2</sup&gt;. Dieses Protokoll nutzt Standard Digoxygenin und Fluorescein markierten Sonden mit Tyramid Signalverstärkung (TSA)<sup&gt; 3</sup&gt;. Die im Handel erhältlichen TSA-Kits ermöglichen eine flexible experimentellen Design als Fluoreszenzemission von grün auf rot weit in Kombination mit verschiedenen nukleären Flecken, wie Propidiumiodid oder Fluoreszenz Immunhistochemie für Proteine ​​verwendet werden. TSA erzeugt einen reaktiven fluoreszierenden Substrat, das schnell kovalent bindet an Einheiten, die typischerweise Tyrosinreste, in unmittelbarer Nähe des markierten antisense Ribosonde. Die daraus resultierende Färbungsmuster sind hochauflösende, dass subzelluläre Lokalisation der mRNA beobachtet mit konfokaler werden<sup&gt; 3,4</sup&gt;. Man kann werdenden Transkripte in der chromosomalen Loci zu beobachten, zu unterscheiden nukleare und zytoplasmatische Färbung und visualisieren anderen Mustern wie kortikale Lokalisation der mRNA. Studies in<em&gt; Drosophila</em&gt; Zeigen, dass rund 70% der mRNAs spezifische Muster der subzellulären Lokalisation, die häufig korrelieren mit der Funktion des kodierten Proteins aufweisen<sup&gt; 5</sup&gt;. In Verbindung mit Computer-Aided Rekonstruktion von 3D-konfokale Datensätze kombiniert, ermöglicht es unseren Protokoll der detaillierten Analyse der mRNA-Verteilung mit subzellulärer Auflösung in ganzen Wirbeltier-Embryonen.

Watch this Scientific Journal Video about Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization at JoVE.com

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ Hybridisierung ist eine der am häufigsten verwendeten Techniken in der Entwicklungsbiologie. Hier präsentieren wir ein hochauflösendes Doppel-Fluoreszenz-in situ-Hybridisierung Protokoll zur Analyse der genauen Expressionsmuster eines einzelnen Gens und zur Bestimmung der Überlappung der Ausdruck Domains von zwei Genen. Wir gehören eine Propidiumiodid nukleare Gegenfärbung des Gewebes Organisation hervorzuheben.

Zebrafisch Whole Mount High-Resolution Doppel Fluorescent In-situ Hybridisierung

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

zebrafish-whole-mount-high-resolution-double-fluorescent-situ

Research

JoVE Journal

Biology

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

23.1K Aufrufe.  Yale University. Whole mount in situ Hybridisierung ist eine der am häufigsten verwendeten Techniken in der Entwicklungsbiologie. Hier präsentieren wir ein hochauflösendes Doppel-Fluoreszenz-in situ-Hybridisierung Protokoll zur Analyse der genauen Expressionsmuster eines einzelnen Gens und zur Bestimmung der Überlappung der Ausdruck Domains von zwei Genen. Wir gehören eine Propidiumiodid nukleare Gegenfärbung des Gewebes Organisation hervorzuheben.

Serie: Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Double Whole Mount in situ Hybridization of Early Chick Embryos

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  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

Doppel Whole Mount in situ Hybridisierung von Anfang Hühnerembryonen

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

Forschung

Biologie

Method for Whole Mount Antibody Staining in Chick

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 antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

Verfahren zur Whole Mount Antikörper-Färbung in Küken

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of specific mRNA transcripts within whole mount specimen. This technique (adapted from Albertson and Yelick, 2005) was used in an upper level undergraduate Comparative Vertebrate Biology laboratory classroom at Syracuse University. The first two thirds of the Comparative Vertebrate Biology lab course gave students the opportunity to study the embryology and gross anatomy of several organisms representing various chordate taxa primarily via traditional dissections and the use of models. The final portion of the course involved an innovative approach to teaching anatomy through observation of vertebrate development employing molecular techniques in which WISH was performed on zebrafish embryos. A heterozygous fibroblast growth factor 8 a (fgf8a) mutant line, ace, was used. Due to Mendelian inheritance, ace intercrosses produced wild type, heterozygous, and homozygous ace/fgf8a mutants in a 1:2:1 ratio. RNA probes with known expression patterns in the midline and in developing anatomical structures such as the heart, somites, tailbud, myotome, and brain were used. WISH was performed using zebrafish at the 13 somite and prim-6 stages, with students performing the staining reaction in class. The study of zebrafish embryos at different stages of development gave students the ability to observe how these anatomical structures changed over ontogeny. In addition, some ace/fgf8a mutants displayed improper heart looping, and defects in somite and brain development. The students in this lab observed the normal development of various organ systems using both external anatomy as well as gene expression patterns. They also identified and described embryos displaying improper anatomical development and gene expression (i.e., putative mutants).
For instructors at institutions that do not already own the necessary equipment or where funds for lab and curricular innovation are limited, the financial cost of the reagents and apparatus may be a factor to consider, as will the time and effort required on the part of the instructor regardless of the setting. Nevertheless, we contend that the use of WISH in this type of classroom laboratory setting can provide an important link between developmental genetics and anatomy. As technology advances and the ability to study organismal development at the molecular level becomes easier, cheaper, and increasingly popular, many evolutionary biologists, ecologists, and physiologists are turning to research strategies in the field of molecular biology. Using WISH in a Comparative Vertebrate Biology laboratory classroom is one example of how molecules and anatomy can converge within a single course. This gives upper level college students the opportunity to practice modern biological research techniques, leading to a more diversified education and the promotion of future interdisciplinary scientific research.

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Whole mount in situ hybridization (WISH) was used in an upper level undergraduate Comparative Vertebrate Biology course in addition to vertebrate dissections. This gave students the opportunity to study gene expression patterns as well as gross anatomy, linking the study of molecular and organismal biology within one course.

Mit Whole Berg In situ Hybridisierung zu Molecular Biology und Organismal Link-

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of ...

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures are lengthy and technically demanding with multiple important steps that collectively contribute to the quality of the final result. This protocol describes in detail several key quality control steps for optimizing probe labeling and performance. Overall, our protocol provides a detailed description of the critical steps necessary to reproducibly obtain high quality results. First, we describe the generation of digoxygenin (DIG) labeled RNA probes via in vitro transcription of DNA templates generated by PCR. We describe three critical quality control assays to determine the amount, integrity and specific activity of the DIG-labeled probes. These steps are important for generating a probe of sufficient sensitivity to detect endogenous mRNAs in a whole mouse embryo. In addition, we describe methods for the fixation and storage of E8.5-E11.5 day old mouse embryos for in situ hybridization. Then, we describe detailed methods for limited proteinase K digestion of the rehydrated embryos followed by the details of the hybridization conditions, post-hybridization washes and RNase treatment to remove non-specific probe hybridization. An AP-conjugated antibody is used to visualize the labeled probe and reveal the expression pattern of the endogenous transcript. Representative results are shown from successful experiments and typical suboptimal experiments.

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

This whole mount in situ hybridization protocol discusses critical steps that ensure reproducible high quality results for gene expression studies in E8.5-E11.5 day old mouse embryos.

Ganze Berge In-situ Hybridisierung von E8.5 bis E11.5 Maus Embryonen

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures ...

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes.
The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE1.
Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System2 (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription and regulation of a variety of genes expressed during the different stages of the P. falciparum life cycle and is adaptable to other malaria parasite species and other organisms and cell types.

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH

Fluorescent in situ hybridization (FISH) to identify mRNA transcripts in individual cells allows analysis of polygenic activity such as the simultaneous transcription of more than one member of the var multigene family in Plasmodium falciparum infected erythrocytes 1. The technique is adaptable and can be used on different types of genes, cells and organisms.

Analyse von Einzel-Zell-Gene Transkription durch RNA-Fluorescent<em&gt; In Situ</em&gt; Hybridisierung (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 ...

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The specification of replication timing is thought to be a dynamic process regulated by tissue-specific and developmental cues that are responsive to epigenetic modifications. However, the mechanisms regulating where and when DNA replication initiates along chromosomes remains poorly understood. Homologous chromosomes usually replicate synchronously, however there are notable exceptions to this rule. For example, in female mammalian cells one of the two X chromosomes becomes late replicating through a process known as X inactivation1. Along with this delay in replication timing, estimated to be 2-3 hr, the majority of genes become transcriptionally silenced on one X chromosome. In addition, a discrete cis-acting locus, known as the X inactivation center, regulates this X inactivation process, including the induction of delayed replication timing on the entire inactive X chromosome. In addition, certain chromosome rearrangements found in cancer cells and in cells exposed to ionizing radiation display a significant delay in replication timing of &gt;3 hours that affects the entire chromosome2,3. Recent work from our lab indicates that disruption of discrete cis-acting autosomal loci result in an extremely late replicating phenotype that affects the entire chromosome4. Additional 'chromosome engineering' studies indicate that certain chromosome rearrangements affecting many different chromosomes result in this abnormal replication-timing phenotype, suggesting that all mammalian chromosomes contain discrete cis-acting loci that control proper replication timing of individual chromosomes5.
Here, we present a method for the quantitative analysis of chromosome replication timing combined with fluorescent in situ hybridization. This method allows for a direct comparison of replication timing between homologous chromosomes within the same cell, and was adapted from6. In addition, this method allows for the unambiguous identification of chromosomal rearrangements that correlate with changes in replication timing that affect the entire chromosome. This method has advantages over recently developed high throughput micro-array or sequencing protocols that cannot distinguish between homologous alleles present on rearranged and un-rearranged chromosomes. In addition, because the method described here evaluates single cells, it can detect changes in chromosome replication timing on chromosomal rearrangements that are present in only a fraction of the cells in a population.

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

A quantitative method for the analysis of chromosome replication timing is described. The method utilizes BrdU incorporation in combination with fluorescent in situ hybridization (FISH) to assess replication timing of mammalian chromosomes. This technique allows for the direct comparison of rearranged and un-rearranged chromosomes within the same cell.

Chromosome Replizieren Timing mit Fluorescent Kombiniert<em&gt; In situ</em&gt; Hybridisierung

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The ...

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels 1. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest 2. As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens 3. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources 4-11. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure 12. Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.

Whole Berg RNA Fluorescent<em&gt; In situ</em&gt; Hybridisierung<em&gt; Drosophila</em&gt; Embryonen

Assessing  the expression pattern of a gene, as well as the subcellular localization  properties of its transcribed RNA, are key features for ...

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

Marine elasmobranchs are valued animal models for biomedical and genomic studies as they are the most primitive vertebrates to have adaptive immunity and have unique mechanisms for osmoregulation 1-3. As the most primitive living jawed-vertebrates with paired appendages, elasmobranchs are an evolutionarily important model, especially for studies in evolution and development. Marine elasmobranchs have also been used to study aquatic toxicology and stress physiology in relationship to climate change 4. Thus, development and adaptation of methodologies is needed to facilitate and expand the use of these primitive vertebrates to multiple biological disciplines. Here I present the successful adaptation of RNA whole mount in situ hybridization and histological techniques to study gene expression and cell histology in elasmobranchs.
Monitoring gene expression is a hallmark tool of developmental biologists, and is widely used to investigate developmental processes 5. RNA whole mount in situ hybridization allows for the visualization and localization of specific gene transcripts in tissues of the developing embryo. The expression pattern of a gene's message can provide insight into what developmental processes and cell fate decisions a gene may control. By comparing the expression pattern of a gene at different developmental stages, insight can be gained into how the role of a gene changes during development.
While whole mount in situ's provides a means to localize gene expression to tissue, histological techniques allow for the identification of differentiated cell types and tissues. Histological stains have varied functions. General stains are used to highlight cell morphology, for example hematoxylin and eosin for general staining of nuclei and cytoplasm, respectively. Other stains can highlight specific cell types. For example, the alcian blue stain reported in this paper is a widely used cationic stain to identify mucosaccharides. Staining of the digestive tract with alcian blue can identify the distribution of goblet cells that produce mucosaccharides. Variations in mucosaccharide constituents on short peptides distinguish goblet cells by function within the digestive tract 6. By using RNA whole mount in situ's and histochemical methods concurrently, cell fate decisions can be linked to gene-specific expression.
Although RNA in situ's and histochemistry are widely used by researchers, their adaptation and use in marine elasmobranchs have met limited and varied success. Here I present protocols developed for elasmobranchs and used on a regular basis in my laboratory. Although further modification of the RNA in situ's hybridization method may be needed to adapt to different species, the protocols described here provide a strong starting point for researchers wanting to adapt the use of marine elasmobranchs to their scientific inquiries.

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

By combining methods for RNA whole mount in situ hybridization and histology, gene expression can be linked with cell fate decisions in the developing embryo. These methods have been adapted to marine elasmobranchs and facilitate the use of these animals as model organisms for biomedical, toxicology and comparative studies.

RNA<em&gt; In situ</em&gt; Hybridisierung in whole mount Embryonen und Histologie für Marine Elasmobranchs Angepasst

Marine  elasmobranchs are valued animal models for biomedical and genomic studies as  they are the most primitive vertebrates to have adaptive immunity ...

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression both in terms of tissue distribution and developmental stage. Protocols are described for the use of WISH of zebrafish embryos using antisense RNA probes labeled with digoxigenin. Probes are generated by incorporating digoxigenin-linked nucleotides through in vitro transcription of gene templates that have been cloned and linearized. The chorions of embryos harvested at defined developmental stages are removed before incubation with specific probes. Following a washing procedure to remove excess probe, embryos are incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase. By employing a chromogenic substrate for alkaline phosphatase, specific gene expression can be assessed. Depending on the level of gene expression the entire procedure can be completed within 2-3 days.

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

The zebrafish, a small tropical fish, has become a popular model for studying gene function during vertebrate development and disease. The temporal and spatial expression of target genes can be determined by in situ hybridization. Our improved protocol allows for the detection of low abundant transcripts with low non-specific background signal.

Hohe Auflösung Ganze Berge<em&gt; In Situ</em&gt; Hybridisierung innerhalb Zebrafischembryonen Gene Expression und Funktion zu untersuchen

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression ...

Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. The whole-mount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning and/or colabeling is ultimately required to pinpoint the exact cell types undergoing apoptosis. The whole-mount Casp3 assay is optimized for analysis of fixed embryos between the 4-cell stage and 32 hr-post-fertilization and is useful for a number of applications, including analysis of zebrafish mutants and morphants, overexpression of mutant and wild-type mRNAs, and exposure to chemicals. Compared to acridine orange staining, which can identify apoptotic cells in live embryos in a matter of hours, Casp3 and TUNEL assays take considerably longer to complete (2-4 days). However, because of the dynamic nature of apoptotic cell formation and clearance, analysis of fixed embryos ensures accurate comparison of apoptotic cells across multiple samples at specific time points. We have also found the Casp3 assay to be superior to analysis of apoptotic cells by the whole-mount TUNEL assay in regard to cost and reliability. Overall, the Casp3 assay represents a robust, highly reproducible assay in which to analyze apoptotic cells in early zebrafish embryos.

Certain genetic perturbations or exposure to toxins can disrupt normal developmental processes leading to death of specific cell types. The analysis of activated Caspase 3 by whole-mount immunofluorescence in zebrafish embryos reveals stage- and tissue-specific localization of cells specifically undergoing apoptosis.

Die Analyse der Apoptose in Zebrafischembryonen von Whole-mount Immunfluoreszenz an aktivierte Caspase 3 Detect

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis ...