We thank Hongjae Sunwoo for helpful advice for oligonucleotide design in RNA FISH. We also thank Serenity Curtis for editing the manuscript. N.Y. was supported by a Postdoctoral Fellowship for Research Abroad of the Japan Society for the Promotion of Science (JSPS). This work was supported by the NIH (RO1-GM102184), the March of Dimes Research Foundation (#6-FY12-337), and the Developmental Fund and Trustee Grant at Cincinnati Children's Hospital Medical Center to Y.O.

1. Probe Vorbereitung <ol><li> Erhalten mehrere einzigartige Oligonukleotide in Xist (20-30 Nukleotide Länge Oligonukleotide, 63-65 ° C Schmelztemperatur, Tabelle 1) mit einem 5&#39;-Amino Änderung und Aussetzung im Wasser. </li><li> Pool äquimolare Mengen von Oligonukleotiden mit 5&#39;-Amino-Modifikation (insgesamt 4,5 ug) und Etikett mit aminreaktiven fluoreszierenden Farbstoff nach den Anweisungen des Herstellers. <ol><li> Lösen Sie den gepoolten Oligonukleotide in letzten 5 ul Nuklease-freies Wasser und fügen Sie 3 ul 1 M Natriumbicarbonat mit dem Oligonukleotid-Lösung. </li><li> Add 2 ul DMSO in das Fläschchen mit aminreaktiven Farbstoff und kurz vortexen. </li><li> Mischen Sie die Oligonucleotidlösung mit dem Reaktivfarbstoff in DMSO und Inkubation der Mischung bei Raumtemperatur für 1 Stunde im Dunkeln. </li></ol></li><li> Reinige den fluoreszenzmarkierten Oligonukleotidsonden durch G-25-Säule, gefolgt von Ethanolfällung. <ol><li&gt; Centrifuge G-25-Säule bei 750 g für 1 min, um überschüssige Pufferspeicher zu entfernen. </li><li> In 40 ul Nuklease-freies Wasser auf die in Schritt 1.2.3 hergestellten Markierungsmischung und gelten für die Spalte G-25. Zentrifuge bei 750 × g für 2 min. </li><li> Dann werden 50 &amp; mgr; l Nuklease-freies Wasser, 10 &amp; mgr; l 3 M Natriumacetat (pH 5,2) und 250 &amp; mgr; l Ethanol zu der gereinigten Probe aus Schritt 1.3.2. Bei -80 ° C für 30 Minuten und Zentrifugation bei 21.000 xg für 15 min bei 4 ° C. </li><li> Einmal mit 1 ml 70% Ethanol waschen fällt Oligonukleotide. </li><li> Re-suspendieren in 50 &amp; mgr; l Nuklease-freies Wasser oder TE (10 mM Tris-HCl, pH 7,5; 1 mM EDTA) bei einer Endkonzentration von etwa 10 uM. </li></ol></li><li> Verdünne die fluoreszenzmarkierten Oligonukleotidsonden zu einer Endkonzentration von 250 nM mit Hybridisierungspuffer (10% Formamid; 2 × SSC [300 mM NaCl, 30 mM Natriumcitrat]; 2 mg / ml BSA; 10% Dextransulfat). HINWEIS: Mit dieser schnellen FISH-Protokoll, Oligonukleotid-pRoben Xist wurden entworfen und in einer höheren Konzentration als die normale FISH Protokoll unter Verwendung von Oligonucleotidsonden, um die Inkubationszeit für die Hybridisierung verwendet verkürzen. </li></ol> 2. Schieben Vorbereitung HINWEIS: Die Objektträger für Immun FISH mit embryonalen Stammzellen der Maus (ES) oder Embryoidkörper (EB) vorbereitet werden differenzierenden Zellen 20, 21 entweder durch den Anbau von ihnen direkt auf einem Polylysin behandelten Folie oder mit Cytospin mit Zellsuspension. Hier wird durch Cytospin-Präparat gezeigt. <ol><li> Absaugen Kulturmedium in einer 6-Napf-Schale. Mit PBS (Raumtemperatur), und saugen Sie gründlich waschen. </li><li> 0,5 ml Trypsin in den 6-Well-Platte, Inkubation für 5-10 min bei 37 ° C. </li><li> 5 ml Medium und zerstreuen Zellen durch vorsichtiges Auf- und Abpipettieren mehrmals. </li><li> Übertragungszellen in ein 15 ml-Röhrchen, Zentrifuge für 5 Minuten bei 200 × g bei Raumtemperatur. </li><li> Entsorgen Sie das Medium eind sanft die Zellen in 2 ml PBS. </li><li> Zählen von Zellen unter Verwendung eines Hämocytometers und verdünnte Zellsuspension bei 4 x 10 5 Zellen / ml mit PBS. </li><li> Bauen Sie eine Diashow, einen Filter-Karte und eine Kammer mit einem Clip und legen Sie sie in die entsprechenden Schlitze in dem Rotor der Cytospin. Fügen Sie 250 ul jeder Probe in Schritt 2.6 in jede Kammer der Baugruppen in der Cytospin vorbereitet. Zentrifuge bei 1.500 Upm für 10 min bei Raumtemperatur. </li><li> Füllen 3 Coplin Gläser auf Eis mit 40 ml eiskaltem PBS, CSK-Puffer (10 mM PIPES, pH 6,8, 100 mM NaCl, 300 mM Saccharose; 3 mM MgCl 2) und CSKT Puffer (CSK-Puffer mit 0,5% Triton X-100), jeweils. </li><li> Nachdem der Cytospin beendet ist, die Folie schnell in eiskaltem PBS übertragen und Inkubation für 5 min. </li><li> Die Objektträger in eiskaltes CSK Puffer und Inkubation für 1 min. </li><li> Die Objektträger in eiskaltes CSKT Puffer und Inkubation für 5 min. </li><li> Die Objektträger wieder in eiskaltes CSK buffer und Inkubation für 1 min. </li><li> Die Objektträger in 4% Paraformaldehyd in PBS und Inkubation für 10 min bei Raumtemperatur. </li></ol> 3. Immunfluoreszenz <ol><li> Objektträger in eine Coplin-Gefäß mit PBST (1x PBS mit 0,1% Tween-20) für mehrere Sekunden. </li><li> Legen Sie die Dias schräg so dass Restpuffer zu fließen auf der Rutsche. Wischen Sie vorsichtig die überschüssige Flüssigkeit aus der Folie und legen Sie sie in einem leeren Pipettenspitzen-Box mit Wasser. Überlagern Sie die Folie mit 40 ul Blockierungslösung (1x PBS, 1% BSA, 0,1% Tween-20), sanft legen Sie eine Deckglas, und Inkubation für 10 min bei Raumtemperatur. </li><li> Entfernen Sie das Deckglas, entfernen Sie die Blockierungslösung, und fügen Sie 40 ul der Histone H3K27me3 primären Antikörpers zu einer Konzentration von 1: 500 verdünnt in Blockierungslösung. Legen Sie das Deckglas wieder auf der Folie und Inkubation für 30 min bei Raumtemperatur. HINWEIS: Ein RNase-Inhibitor in der Blockierungslösung könnte hilfreich sein, um RNA in dieser RNA FIS erhaltenH-Protokoll bei polyklonalen Antikörpern eingesetzt werden. </li><li> Wash schieben 2 mal in Coplin Gläser mit PBST gefüllt, je 5 Minuten, leichtes Schütteln beim Waschen. </li><li> Legen Sie die Dias schräg so dass Restpuffer zu fließen auf der Rutsche. Wischen Sie vorsichtig die überschüssige Flüssigkeit und legen Sie die Folie wieder in die Spitze ein. Overlay mit 40 ul des sekundären Antikörpers (1: 500) in Blocking-Lösung verdünnt, setzen Sie ein Deckglas, und Inkubation für 15 min im Dunkeln. Für die folgenden Schritte, halten Sie die Folie in der Dunkelheit zu allen Zeiten. </li><li> In Färbetrog mit PBST, wie in Schritt 3.4 Waschen Sie 2 mal. </li></ol> 4. RNA-FISH mit Oligonukleotidsonden <ol><li> Pre-Aufwärmen eine leere Pipettenspitzen-Box mit Wasser auf dem Boden, um Folien aus dem Trocknen bei 37 ° C für die RNA-FISH in Schritt 4.3 zu verhindern. </li><li> Legen Sie die Dias schräg so dass Restpuffer zu fließen auf der Rutsche. Wischen Sie vorsichtig die überschüssige Flüssigkeit aus der Folie aus Schritt 3.6 und platzieren Sie die Folie in der Pipettenspitzen-Box mit water an der Unterseite. Gib 500 ul FISH Waschpuffer (10% Formamid in 2 × SSC) auf dem Objektträger, Inkubation 5 min bei Raumtemperatur. </li><li> Entfernen FISH Waschlösung, fügen 8 ul von Oligonukleotid-Sonden auf dem Objektträger, legen Sie ein Deckglas, und übertragen Sie die Folie in eine vorgewärmte Pipettenspitzen-Box mit Wasser auf dem Boden; Inkubieren bei 37 ° C für 1-2 Std. </li><li> Platz 3 vorgewärmt Coplin-Glaszylinder in einem Wasserbad bei 37 ° C mit FISH Waschpuffer, FISH Waschpuffer mit DAPI und 2 × SSC. </li><li> Nach 1-2 h Inkubation für RNA FISH, setzen Sie den Schieber in die vorgewärmte FISH Waschpuffer. </li><li> Inkubieren für ein paar Minuten, bis die Deck fällt der Folie. </li><li> Den Objektträger wieder in die Waschpuffer, und weiter Inkubation für 5 min. </li><li> Die Objektträger in einem anderen Coplin-Gefäß mit vorgewärmter FISH Waschpuffer mit 0,5 ng / ml DAPI und Inkubation für 5 min. </li><li> Die Objektträger in einem anderen Coplin Glas mit vorgewärmten 2x SSC und Inkubation für 5 min. </li><li> Legen Sie die Dias schräg so dass Restpuffer zu fließen auf der Rutsche. Wischen Sie vorsichtig die überschüssige Flüssigkeit und legen Sie den Rückfall in einem trockenen Spitze Box. In 4 ul Antifade mit DAPI auf den Objektträger und setzen Sie ein Deckglas. </li><li> Deckglasränder mit Nagellack und beobachten unter dem Mikroskop. Der Schieber kann bei 4 ° C im Dunkeln für einige Monate gelagert werden. </li></ol>

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No conflicts of interest declared.

In diesem Papier haben wir eine schnelle Immun FISH-Protokoll, das weniger als 5 Stunden dauert, Präparat, Xist-RNA FISH und Immunfluoreszenz für H3K27me3 abzuschließen präsentiert. Im Vergleich mit der allgemeinen Immun FISH Ansatz für Xist-RNA-Erkennung, die in der Regel braucht Inkubation über Nacht für die RNA-FISH, dieses Protokoll nicht nur deutlich reduziert Zeitaufwand, sondern verbessert auch die Empfindlichkeit der Immun-FISH mit Oligonukleotid-Sonden. Seit Xist ist hoch wä...

Säugetier X-Chromosom Inaktivierung (XCI) ist eine Strategie, um die Unwucht in X-verknüpften Gens Dosis zwischen XX und XY, wobei eines der beiden X-Chromosomen in Weibchen transkriptionell inaktivierten 1 kompensieren. X-Chromosom-Inaktivierung ist eine große Modellsystem für lange nicht-kodierende RNA (lncRNA) Forschung. X-Chromosom-Inaktivierung von mehreren lncRNAs geregelt und wurde ausgiebig in den letzten Jahrzehnten untersucht, um die Übersprechmechanismen zwischen lncRNAs, Transkription, Chromatinstruktur und Kernorganisation 2, 3 aufzudecken. Die X-Inaktivierung Zentrum (XIC) auf dem X-Chromosom lokalisiert ist eine komplexe genetische Locus einer Reihe von Genen, die Herstellung nicht-kodierenden RNAs 4 besteht. X-inaktiv spezifische Transkript (Xist) lncRNA in eutherian Säugetieren ist eine solche lncRNA, die eine entscheidende Rolle bei der X-Chromosom-Inaktivierung 5,6 spielt. Xist Transkripte umgeben den Ort der futur Xi bis X-Chromosom-Inaktivierung zu initiieren, und erscheinen wie eine Wolke, wenn visualisiert mittels RNA FISH; Diese Formation wird als &quot;Xist Cloud&quot; 7 bezeichnet. Seit Xist-RNA interagiert mit verschiedenen Chromatin-modifizierende Enzyme, wird Co-Lokalisation der Xist Wolken mit verschiedenen epigenetische Modifikationen für stille Chromatin und repressive Transkription während X-Chromosom Inaktivierung 8 beobachtet. Beispielsweise interagiert Xist-RNA mit polycomb repressive komplexen 2 (PRC2), die für H3K27me3 verantwortlich ist und induziert eine repressive Chromatin Zustand 9. Das Auftreten der Xist Wolke am Xi und seine Co-Lokalisierung mit der intensiven H3K27me3 Änderung stellt eine fakultative Heterochromatin Landschaft des Xi 10,11. Zytogenetischen Techniken, wie DNA / RNA-FISH haben einen langen Weg von der traditionellen Methode mit radioaktiv markierten Sonden 12 auf die jüngsten und modernsten Techniken mit verbesserter Empfindlichkeit und kommenFluoreszenz-Bildgebung mit mehreren Oligonukleotidsonden 13,14. DNA / RNA-FISH in Verbindung mit Immunfluoreszenz wurde routinemäßig als zytologische Werkzeug benutzt, um Raum-Zeit-Atom-Organisation, RNA-Lokalisierung, Chromatinstruktur und Modifikationen zu verstehen. Die Standardsonde Vorbereitung RNA FISH beinhaltet die Verwendung von Plasmid oder bakteriellen künstlichen Chromosoms (BAC) Klonen und deren anschließende Markierung entweder mit Nick-Translation oder Random-Priming-15. Fast 70% der Gene in Mäusen und 40% der Gene in Menschen zeigen jedoch eine Überlappung von Sense- und Antisense-Transkripte 16, daher um Sense- und Antisense-Transkripte unterscheidet die eine strangspezifische FISH-Verfahren. In vitro transkribierte RNA-Sonden (Ribosonde ) werden oft für strangspezifischen RNA-FISH 17,18 verwendet; Dies ist jedoch mit Herstellung eines Plasmid-Klon oder PCR-Produkt mit T7, SP6 oder T3-Promotor und Synthetisieren Ribosonden. Außerdem Ribosonden von genom abgeleitetic DNA oder cDNA enthalten oft unspezifische Regionen und repetitiven Elementen, was zu hohen Hintergrundgeräuschen führen. Ein weiteres Problem ist, dass Ribosonden, die einige hundert Nukleotide lang sind, und enthalten mehrere Fluorophore, nicht effizient in den Zellkern einzudringen. Um dies zu umgehen, wurden mehrere kürzere Oligonukleotidsonden mit einem einzigen Fluorophor am Ende markierten entwickelt worden, die eine gute Empfindlichkeit, einheitliche Signalstärke und der Leichtigkeit der Reinigung und Handhabung von 14 haben. Darüber hinaus sind DNA-Oligonukleotide in der Regel stabiler als RNA. Wir verwendeten eine ähnliche Strategie unter Verwendung von Oligonukleotiden in Xist RNA FISH 19 mit Immunfluoreszenz die epigenetische Dynamik des Xi durch Xist lncRNA während des Prozesses der X-Chromosom Inaktivierung induziert verstehen. Dieses Protokoll beschreibt die Erstellung von Oligonukleotid-Sonden und die richtige Vorbereitung der Zellen, sowie die Verwertung von Immunfluoreszenz und RNA-FISH. Xist-RNA-FISH mit mehreren oligonucleotides ist kostensparenden Lösung in den langfristigen, wenn Xist RNA FISH wird routinemäßig in einem Labor durchgeführt wird. Diese Technik kann verwendet werden, um lncRNAs in Zellen zu identifizieren und gleichzeitig die Abbildung ihrer Co-Lokalisierung mit epigenetische Modifikationen oder Faktoren werden. Ein großer Vorteil des Protokolls ist die Fähigkeit, leicht zu modifizieren, seine Forschungsinteressen gerecht zu werden.

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			<td height="21" style="height:21px;width:267px;">oligonucleotides with 5&#8217;-amine modification</td>
			<td style="width:187px;">IDT</td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Alexa Fluor 488 Reactive Dye</td>
			<td style="width:187px;">Life Technologies</td>
			<td style="width:119px;">A32750</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">MicroSpin G-25 columns</td>
			<td style="width:187px;">GE Healthcare</td>
			<td style="width:119px;">27-5325-01</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Cytospin 2</td>
			<td style="width:187px;">Shandon</td>
			<td align="right" style="width:119px;">59900102</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Bovine Serum Albumin, Molecular Biology Grade (for hybridization buffer)</td>
			<td style="width:187px;">Roche</td>
			<td style="width:119px;">10715859103</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Histone H3K27me3 antibody</td>
			<td style="width:187px;">Active Motif</td>
			<td style="width:119px;">61017</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Accutase</td>
			<td style="width:187px;">Innovative Cell Technologies</td>
			<td style="width:119px;">AT104</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Alexa Fluor 555 Goat Anti-Mouse, highly cross-adsorbed</td>
			<td style="width:187px;">Life Technologies</td>
			<td style="width:119px;">A21424</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">ProLong Gold antifade reagent</td>
			<td style="width:187px;">Life Technologies</td>
			<td style="width:119px;">P36931</td>
		</tr>
	</tbody>
</table>

quick fluorescent in situ hybridization protocol for xist rna combined with immunofluorescence of histone modification in x-chromosome inactivation

Kombinieren RNA Fluoreszenz in situ Hybridisierung (FISH) mit Immunfluoreszenz (Immuno-FISH) schafft eine Technik, die auf der Ebene einzelner Zellen verwendet werden können, um die räumliche Dynamik RNA Lokalisierung mit gleichzeitige Einblick in die Lokalisierung von Proteinen, epigenetische Modifikationen und andere Details zu detektieren die durch Immunfluoreszenz markiert werden kann. X-Chromosom-Inaktivierung ist ein Paradigma für lange nicht-kodierende RNA (lncRNA) vermittelte Gen-Silencing. X-inaktiven spezifischen Transkripts (Xist) lncRNA Ansammlung (genannt Xist Wolke) an einem der beiden X-Chromosomen in weiblichen Säugern ist ein kritischer Schritt, um X-Chromosom Inaktivierung initiieren. Xist-RNA direkt oder indirekt in Wechselwirkung mit verschiedenen Chromatin-modifizierende Enzyme und stellt verschiedene epigenetischen Landschaften in den inaktiven X-Chromosom (Xi). Eine bekannte epigenetischen Kennzeichen der Xi ist der Histone H3 trimethyl-Lysin-27 (H3K27me3) Änderung. Hier haben wir eine einfache und schnelle Immun FISH beschreibenProtokoll zum Nachweis von Xist-RNA mit RNA-FISH mit mehreren Oligonukleotid-Sonden in Verbindung mit Immunfluoreszenz von H3K27me3, um die Lokalisierung von Xist-RNA und die damit verbundenen epigenetische Veränderungen zu untersuchen. Verwendung von Oligonucleotidsonden zu einer kürzeren Inkubationszeit und empfindlicher Nachweis von Xist RNA im Vergleich zu in vitro transkribierte RNA-Sonden (Ribosonden). Dieses Protokoll stellt ein leistungsfähiges Werkzeug für das Verständnis der Dynamik von lncRNAs und die damit verbundenen epigenetische Modifikation der Chromatinstruktur, Kern Organisation und Transkriptionsregulation.

Repräsentative Bilder der schnellen Immun FISH in 1A gezeigt. Co-Lokalisation der Xist RNA Wolke und H3K27me3 Signal auf dem Xi in differen weiblichen Zellen nachgewiesen. Am Tag 12 nach Differenzierung, mehr als 90% des EB-Zellen hatte eine Xist Wolke (1B). (; 150 97%, n = 1C) kurze Oligonucleotidsonden effizient in den Zellkern durchdrungen wird, was zu einer Visualisierung mit Xist RNA fast alle H3K27me3 Signale kolokalisiert. <p class="jove_content" fo:keep-tog...

Watch this Scientific Journal Video about Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation at JoVE.co

Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation

We developed an easily customized strand-specific fluorescent in situ hybridization (FISH) protocol combined with immunofluorescence. This allows for a detailed examination of RNA dynamics with simultaneous insight into the chromatin structure, nuclear organization, and transcriptional regulation at the single cell level.

Schnell Fluorescent<em&gt; In-situ-</em&gt; Hybridisierung Protokoll für Xist-RNA in Kombination mit Immunfluoreszenz von Histonmodifikationen in X-Chromosom-Inaktivierung

Combining RNA fluorescent in situ hybridization (FISH) with immunofluorescence (immuno-FISH) creates a technique that can be employed at the single cell ...

quick-fluorescent-situ-hybridization-protocol-for-xist-rna-combined

Research

JoVE Journal

Biology

Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation

13.8K Aufrufe.  Cincinnati Children's Hospital Medical Center. We developed an easily customized strand-specific fluorescent in situ hybridization (FISH) protocol combined with immunofluorescence. This allows for a detailed examination of RNA dynamics with simultaneous insight into the chromatin structure, nuclear organization, and transcriptional regulation at the single cell level. 

Serie: Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation

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

Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species

The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of tissue specific expression is limited because of problems in dissecting individual tissues. Expression data needs to be confirmed and complemented with expression patterns using e.g. in situ hybridization, a technique used to localize cell specific mRNA expression. The in situ hybridization method is laborious, time-consuming and often requires extensive optimization depending on species and tissue. In situ experiments are relatively more difficult to perform in woody species such as the conifer Norway spruce (Picea abies). Here we present a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including P. abies. With just a few adjustments, including altered RNase treatment and proteinase K concentration, we could use the protocol to study tissue specific expression of homologous genes in male reproductive organs of one gymnosperm and two angiosperm species; P. abies, Arabidopsis thaliana and Brassica napus. The protocol worked equally well for the species and genes studied. AtAP3 and BnAP3 were observed in second and third whorl floral organs in A. thaliana and B. napus and DAL13 in microsporophylls of male cones from P. abies. For P. abies the proteinase K concentration, used to permeablize the tissues, had to be increased to 3 g/ml instead of 1 g/ml, possibly due to more compact tissues and higher levels of phenolics and polysaccharides. For all species the RNase treatment was removed due to reduced signal strength without a corresponding increase in specificity. By comparing tissue specific expression patterns of homologous genes from both flowering plants and a coniferous tree we demonstrate that the DIG in situ protocol presented here, with only minute adjustments, can be applied to a wide range of plant species. Hence, the protocol avoids both extensive species specific optimization and the laborious use of radioactively labeled probes in favor of DIG labeled probes. We have chosen to illustrate the technically demanding steps of the protocol in our film.
Anna Karlgren and Jenny Carlsson contributed equally to this study.
Corresponding authors: Anna Karlgren at Anna.Karlgren@ebc.uu.se and Jens F. Sundstr&#246;m at Jens.Sundstrom@vbsg.slu.se

Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species

We describe a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including Norway spruce. With just a few adjustments, including altered RNase treatment and proteinase K concentration, the protocol may be used in studies of different tissues and species.

Nicht radioaktiv In situ Hybridisierung Protocol Einsetzbar für Fichte und einer Reihe von Pflanzenarten

The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of ...

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

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 ...

Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic process in the parents. In human males, these errors can lead to the production of spermatozoa with numerical chromosome abnormalities which represent an increased risk of transmitting these anomalies to the offspring.
For this reason, the technique of fluorescence in situ hybridization (FISH) on sperm nuclei has become a protocol widely incorporated in the context of clinical diagnosis. This practice provides an estimate of the frequencies of numerical chromosome abnormalities in the gametes of the patients that seek for genetic reproductive advice.
To date, the chromosomes most frequently included in sperm FISH analysis are chromosomes X, Y, 13, 18 and 21.
This video-article describes, step by step, how to process and fix a human semen sample, how to decondense and denature the sperm chromatin, how to proceed to obtain sperm FISH preparations, and how to visualize the results at the microscope. Special remarks of the most relevant steps are given to achieve the best results.

Fluorescence in situ hybridization FISH Protocol in Human Sperm

This video-article describes, step by step, how to process a semen sample to achieve good-quality fluorescence in situ hybridization on human spermatozoa. Preparations obtained can be used for aneuploidy screening in the context of clinical diagnosis.

Fluoreszenz In situ Hybridisierung (FISH)-Protokoll in menschlichen Spermien

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic ...

In Situ Hybridization for the Precise Localization of Transcripts in Plants

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene expression. Microarray and next generation sequencing approaches are being used to investigate developmental, physiological and stress response processes, dissect epigenetic and small RNA pathways, and build large gene regulatory networks1-3. While these techniques facilitate the simultaneous analysis of large gene sets, they typically provide a very limited spatiotemporal resolution of gene expression changes. This limitation can be partially overcome by using either profiling method in conjunction with lasermicrodissection or fluorescence-activated cell sorting4-7. However, to fully understand the biological role of a gene, knowledge of its spatiotemporal pattern of expression at a cellular resolution is essential. Particularly, when studying development or the effects of environmental stimuli and mutants can the detailed analysis of a gene's expression pattern become essential. For instance, subtle quantitative differences in the expression levels of key regulatory genes can lead to dramatic phenotypes when associated with the loss or gain of expression in specific cell types. 
Several methods are routinely used for the detailed examination of gene expression patterns. One is through analysis of transgenic reporter lines. Such analysis can, however, become time-consuming when analyzing multiple genes or working in plants recalcitrant to transformation. Moreover, an independent validation to ensure that the transgene expression pattern mimics that of the endogenous gene is typically required. Immunohistochemical protein localization or mRNA in situ hybridization present relatively fast alternatives for the direct visualization of gene expression within cells and tissues. The latter has the distinct advantage that it can be readily used on any gene of interest. In situ hybridization allows detection of target mRNAs in cells by hybridization with a labeled anti-sense RNA probe obtained by in vitro transcription of the gene of interest. 
Here we outline a protocol for the in situ localization of gene expression in plants that is highly sensitivity and specific. It is optimized for use with paraformaldehyde fixed, paraffin-embedded sections, which give excellent preservation of histology, and DIG-labeled probes that are visualized by immuno-detection and alkaline-phosphatase colorimetric reaction. This protocol has been successfully applied to a number of tissues from a wide range of plant species, and can be used to analyze expression of mRNAs as well as small RNAs8-14.

In Situ Hybridization for the Precise Localization of Transcripts in Plants

The in situ hybridization protocol described here allows a direct localization of mRNA and small RNA expression at the cellular level with high sensitivity and specificity. The procedure is optimized for paraffin-embedded plant tissue sections, is applicable to a wide range of plants and tissues, and can be completed within ten days.

In-situ-Hybridisierung für die genaue Lokalisierung der Transkripte in Pflanzen

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

Nachweis viraler RNA durch Fluoreszenz<em&gt; In-situ-</em&gt; Hybridisierung (FISH)

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

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 ...