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.

<table><tbody><tr><td>oligonucleotides with 5&amp;rsquo;-amino modification</td><td>IDT</td><td></td><td></td></tr><tr><td>Alexa Fluor 488 Reactive Dye</td><td>Life Technologies</td><td>A32750</td><td></td></tr><tr><td>MicroSpin G-25 columns</td><td>GE Healthcare</td><td>27-5325-01</td><td></td></tr><tr><td>Cytospin 2</td><td>Shandon</td><td>59900102</td><td></td></tr><tr><td>Bovine Serum Albumin, Molecular Biology Grade (for hybridization buffer)</td><td>Roche</td><td>10715859103</td><td></td></tr><tr><td>Histone H3K27me3 antibody</td><td>Active Motif</td><td>61017</td><td></td></tr><tr><td>Alexa Fluor 555 Goat Anti-Mouse, highly cross-adsorbed</td><td>Life Technologies</td><td>A21424</td><td></td></tr><tr><td>ProLong Gold antifade reagent</td><td>Life Technologies</td><td>P36931</td><td></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

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

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

13.9K 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

Using Fluorescence In Situ Hybridization (FISH) to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes

In humans, chromosome segregation errors in oocytes are responsible for the majority of miscarriages and birth defects. Moreover, as women age, their risk of conceiving an aneuploid fetus increases dramatically and this phenomenon is known as the maternal age effect. One requirement for accurate chromosome segregation during the meiotic divisions is maintenance of sister chromatid cohesion during the extended prophase period that oocytes experience. Cytological evidence in both humans and model organisms suggests that meiotic cohesion deteriorates during the aging process. In addition, segregation errors in human oocytes are most prevalent during meiosis I, consistent with premature loss of arm cohesion. The use of model organisms is critical for unraveling the mechanisms that underlie age-dependent loss of cohesion. Drosophila melanogaster offers several advantages for studying the regulation of meiotic cohesion in oocytes. However, until recently, only genetic tests were available to assay for loss of arm cohesion in oocytes of different genotypes or under different experimental conditions. Here, a detailed protocol is provided for using fluorescence in situ hybridization (FISH) to directly visualize defects in arm cohesion in prometaphase I and metaphase I arrested Drosophila oocytes. By generating a FISH probe that hybridizes to the distal arm of the X chromosome and collecting confocal Z stacks, a researcher can visualize the number of individual FISH signals in three dimensions and determine whether sister chromatid arms are separated. The procedure outlined makes it possible to quantify arm cohesion defects in hundreds of Drosophila oocytes. As such, this method provides an important tool for investigating the mechanisms that contribute to cohesion maintenance as well as the factors that lead to its demise during the aging process.

Using Fluorescence In Situ Hybridization FISH to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes

This manuscript presents a detailed method for generating X-chromosome arm probes and performing fluorescence in situ hybridization (FISH) to examine the state of sister chromatid cohesion in prometaphase and metaphase I arrested Drosophila oocytes. This protocol is suitable for determining whether meiotic arm cohesion is intact or disrupted in different genotypes.

Fluoreszenz In Situ Hybridisierung (FISH) verwenden, um den Zustand der Arm Zusammenhalt im Prometaphase und Metaphase überwachen ich Drosophila Eizellen

In humans, chromosome segregation errors in oocytes are responsible for the majority of miscarriages and birth defects. Moreover, as women age, their risk ...

Forschung

Entwicklungsbiologie

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 ...

Biologie

Exploring X Chromosomal Aberrations in Ovarian Cells by Using Fluorescence In Situ Hybridization

Millions of people worldwide deal with issues concerning fertility. Reduced fertility, or even infertility, may be due to many different causes, including genetic disorders, of which chromosomal abnormalities are the most common. Fluorescence in situ hybridization (FISH) is a well-known and frequently used method to detect chromosomal aberrations in humans. FISH is mainly used for the analysis of chromosomal abnormalities in the spermatozoa of males with numerical or structural chromosomal aberrations. Furthermore, this technique is also frequently applied in females to detect X chromosomal aberrations that are known to cause ovarian dysgenesis. However, information on the X chromosomal content of ovarian cells from females with X chromosomal aberrations in lymphocytes and/or buccal cells is still lacking.
The aim of this study is to advance basic research regarding X chromosomal aberrations in females, by presenting two methods based on FISH to identify the X chromosomal content of ovarian cells. First, a method is described to determine the X chromosomal content of isolated ovarian cells (oocytes, granulosa cells, and stromal cells) in non-grafted ovarian cortex tissue from females with X chromosomal aberrations. The second method is directed at evaluating the effect of chromosomal aberrations on folliculogenesis by determining the X chromosomal content of ovarian cells of newly formed secondary and antral follicles in ovarian tissue, from females with X chromosomal aberrations after long-term grafting into immunocompromised mice. Both methods could be helpful in future research to gain insight into the reproductive potential of females with X chromosomal aberrations.

Exploring X Chromosomal Aberrations in Ovarian Cells by Using Fluorescence In Situ Hybridization

This article presents two methods based on fluorescence in situ hybridization to determine the X chromosomal content of ovarian cells in non-grafted and grafted ovarian cortex tissue from females with X chromosomal aberrations.

Erforschung von X-Chromosomen-Aberrationen in Ovarialzellen durch Fluoreszenz-in-situ-Hybridisierung

Millions of people worldwide deal with issues concerning fertility. Reduced fertility, or even infertility, may be due to many different causes, including ...

Genetik

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 ...

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological processes. In particular, appropriate regulation of calcium activity patterns during embryogenesis is necessary for many aspects of vertebrate neural development, including proper neural tube closure, synaptogenesis, and neurotransmitter phenotype specification. While the observation that calcium activity patterns can differ in both frequency and amplitude suggests a compelling mechanism by which these fluxes might transmit encoded signals to downstream effectors and regulate gene expression, existing population-level approaches have lacked the precision necessary to further explore this possibility. Furthermore, these approaches limit studies of the role of cell-cell interactions by precluding the ability to assay the state of neuronal determination in the absence of cell-cell contact. Therefore, we have established an experimental workflow that pairs time-lapse calcium imaging of dissociated neuronal explants with a fluorescence in situ hybridization assay, allowing the unambiguous correlation of calcium activity pattern with molecular phenotype on a single-cell level. We were successfully able to use this approach to distinguish and characterize specific calcium activity patterns associated with differentiating neural cells and neural progenitor cells, respectively; beyond this, however, the experimental framework described in this article could be readily adapted to investigate correlations between any time-series activity profile and expression of a gene or genes of interest.

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

We present a two-part protocol that combines fluorescent calcium imaging with in situ hybridization, allowing the experimenter to correlate patterns of calcium activity with gene expression profiles on a single-cell level.

Fluoreszierende Calcium-Bildgebung und anschließende In-Situ-Hybridisierung zur neuronalen Vorläufercharakterisierung bei Xenopus laevis

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological ...

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can be hampered by the lack of specific primary or secondary antibodies for immunostaining. Here we describe a protocol to detect the expression of three different genes in the same brain section using double fluorescence in situ hybridization with two gene-specific probes followed by immunostaining with an antibody of high specificity directed against the protein encoded by a third gene. The Aspartoacyclase (ASPA) gene, mutations of which can lead to a rare human white matter disease - Canavan disease - is thought to be expressed in oligodendrocytes and microglia but not in astrocytes and neurons. However, the precise expression pattern of ASPA in the brain has yet to be established. This protocol has allowed us to determine that ASPA is expressed in a subset of mature oligodendrocytes and it can be generally applied to a wide range of gene expression pattern studies.

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Localizing gene expression to specific cell types can be challenging due to the lack of specific antibodies. Here we describe a protocol for simultaneous triple detection of gene expression by combining double fluorescence RNA in situ hybridization with immunostaining.

Die Kombination von Doppel-Fluoreszenz<em&gt; In Situ</em&gt; Die Hybridisierung mit Immunmarkierung für Nachweis der Expression von drei Gene in Mäusehirnschnitten

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can ...

Neurowissenschaften

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 ...

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

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

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

Kombiniert Immunfluoreszenz und DNA FISH auf 3D erhaltene Interphase Nuclei, um Änderungen in 3D Nuclear Organization Study

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

Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

Fluorescent in situ hybridization (FISH) is a molecular technique that identifies the presence and spatial distribution of specific RNA transcripts within cells. Neurochemical phenotyping of functionally identified neurons usually requires concurrent labelling with multiple antibodies (targeting protein) using immunohistochemistry (IHC) and optimization of in situ hybridization (targeting RNA), in tandem. A &#34;neurochemical signature&#34; to characterize particular neurons may be achieved however complicating factors include the need to verify FISH and IHC targets before combining the methods, and the limited number of RNAs and proteins that may be targeted simultaneously within the same tissue section.
Here we describe a protocol, using both fresh frozen and fixed mouse brain preparations, which detects multiple mRNAs and proteins in the same brain section using RNAscope FISH followed by fluorescence immunostaining, respectively. We use the combined method to describe the expression pattern of low abundance mRNAs (e.g., galanin receptor 1) and high abundance mRNAs (e.g., glycine transporter 2), in immunohistochemically identified brainstem nuclei.
Key considerations for protein labelling downstream of the FISH assay extend beyond tissue preparation and optimization of FISH probe labelling. For example, we found that antibody binding and labelling specificity can be detrimentally affected by the protease step within the FISH probe assay. Proteases catalyze hydrolytic cleavage of peptide bonds, facilitating FISH probe entry into cells, however they may also digest the protein targeted by the subsequent IHC assay, producing off target binding. The subcellular location of the targeted protein is another factor contributing to IHC success following FISH probe assay. We observed IHC specificity to be retained when the targeted protein is membrane bound, whereas IHC targeting cytoplasmic protein required extensive troubleshooting. Finally, we found handling of slide-mounted fixed frozen tissue more challenging than fresh frozen tissue, however IHC quality was overall better with fixed frozen tissue, when combined with RNAscope.

Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

This protocol describes a method for combining fluorescence in situ hybridization (FISH) and fluorescence immunohistochemistry (IHC) in both fresh frozen and fixed mouse brain sections, with the goal of achieving multilabel FISH and fluorescence IHC signal. IHC targeted cytoplasmic and membrane attached proteins.

Kombination von Multiplex-Fluoreszenz-In-situ-Hybridisierung mit fluoreszierender Immunhistochemie an frischen, gefrorenen oder fixierten Mäusehirnschnitten

Fluorescent in situ hybridization (FISH) is a molecular technique that identifies the presence and spatial distribution of specific RNA transcripts within ...

Combined DNA-RNA Fluorescent In situ Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in cytogenetics and cancer diagnostics, and can detect aberrations of the genome, which often has important clinical implications. RNA FISH can be used to detect RNA molecules in cells and has provided important insights in regulation of gene expression. Combining DNA and RNA FISH within the same cell is technically challenging, as conditions suitable for DNA FISH might be too harsh for fragile, single stranded RNA molecules. We here present an easily applicable protocol which enables the combined, simultaneous detection of Xist RNA and DNA encoded by the X chromosomes. This combined DNA-RNA FISH protocol can likely be applied to other systems where both RNA and DNA need to be detected.

Combined DNA-RNA Fluorescent In situ Hybridization FISH to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) allows the detection of nucleic acids in their native environment within cells. We here describe a protocol for the combined, simultaneous detection of RNA and DNA by means of FISH, which can be used to study X chromosome inactivation in mouse embryonic stem cells.

Kombinierte DNA-RNA-Leuchtstoff<em&gt; In situ</em&gt; Hybridisierung (FISH) zu X-Chromosom-Inaktivierung in Differenzierte Weiblich embryonalen Stammzellen der Maus Studieren

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