Diese Arbeit wurde von der Generalitat de Catalunya (2005-SGR00437) unterstützt. Die Autoren danken J. Blanco und F. Vidal für ihre Überwachung und Ermutigung, und J. Lucas für seine technische Unterstützung.

I. Aufbereitung der Probe und Zellfixierung <ol><li> Verlassen Sie die Samenprobe in sterilen Behältern bei Raumtemperatur für 20 Minuten, bis Verflüssigung. </li><li> Übertragen Sie die Probe in ein Zentrifugenröhrchen und spinnen sie bei 1000g für 5 Minuten. </li><li> Entfernen Sie vorsichtig und den Überstand verwerfen mit einer Pasteurpipette. </li><li> Add hypotonischen Lösung (KCl, 0,075 M) vorgewärmt auf 37 ° C, Tropfen für Tropfen, beim Mischen auf einem Vortex auf ein Endvolumen von 10 ml zu erhalten. </li><li> Das Röhrchen wird in ein Wasserbad bei 37 ° C für 30 Minuten. </li><li> Zentrifugation bei 1000g für 5 Minuten. Vorsichtig den Überstand verwerfen durch Dekantieren, ohne das Pellet. </li><li> Nach Resuspendieren des Pellets, fügen Sie frisch zubereitete Carnoy Fixativ (3:1 Methanol: Essigsäure) tropfenweise unter Mischen auf einem Vortex auf ein Endvolumen von 8 ml zu erhalten. </li><li> Wiederholen Sie die Punkte 6 und 7 so oft wie notwendig, um ein weißes Pellet zu erhalten. </li><li> Fügen Sie frisch zubereitete Methanol: Essigsäure (3:1) tropfenweise auf das Endvolumen auf die zelluläre Konzentration für den Erhalt von guten Zellausbreitung (in diesen Fällen mit einem kleinen Pellets erforderlich einstellen, werden nur wenige Tropfen genügen, wohingegen Proben mit einem hohen Anteil von Spermien erholt, kann das Endvolumen erreicht 1-2 ml). Ein Test gleiten kann, indem er die resuspendierten Probe auf eine ungefettete Rutsche und prüft es unter einem Phasenkontrast-Mikroskop vorgenommen werden. Auf diese Weise können die zelluläre Dispersion überprüfen und würde es korrigieren, wenn es notwendig ist, in weiteren Dias. </li><li> Um die Erweiterungen, fallen aus einer Höhe von mindestens 40 cm zwei oder drei Tropfen der Zellsuspension in die Mitte des unfrosted Dias (zuvor in Methanol bei -20 ° C gelagert, um sie zu entfetten). Machen So aktivieren Sie den Ort der Probe in das Protokoll, markieren Sie den Bereich mit den Spermien-Erweiterung auf der gegenüberliegenden Seite der Folie mit einem Diamant-Bleistift. </li><li> Bewahren Sie die Objektträger bei -20 ° C für mindestens 24 Stunden. Hinweis: Die Zugabe von Methanol: Essigsäure (3:1) tropfenweise unter Mischen ist ein sehr wichtiger Schritt, um die Bildung von Spermien Aggregate zu vermeiden. </li></ol> II. Dekondensation <ol><li> Die Objektträger bei -20 ° C gelagert muss abgetaut, um mit dem Protokoll fortgesetzt werden (lassen Sie sie bei Raumtemperatur). </li><li> Legen Sie die Folie in zwei aufeinander folgenden Färbetrog mit 2x Kochsalzlösung-Natriumcitrat-Lösung (2 × SSC) für jeweils 3 Minuten. </li><li> Die Objektträger durch eine Reihe von Ethanol wäscht für 2 Minuten in jede Coplin Glas. Beginnen Sie mit 70% Ethanol, folgen um 90% und endet mit 100%. Dry die Rutsche verlassen Sie es bei Raumtemperatur. </li><li> Inkubieren Sie die Folie in Dithiothreitol-Lösung (1,4-Dithiothreitol 5mm, 1% Triton X-100, 2-Amino-2-(hydroxymethyl) -1,3-propandiol 50 mM) bei 37 ° C in den Brutschrank, um das Chromatin decondense. Dieses Produkt wirkt durch Aufbrechen der Disulfidbrücken der Protamine, dass die Spule der DNA in den Spermien Zellkern. Dies ist ein sehr wichtiger Schritt, weil die DNA in den Spermien Zellkern stark verdichtet wird. Die Inkubationszeit der Folien in Dithiothreitol-Lösung (DTT) muss nach dem Muster der Reaktivität zu diesem Artikel eingestellt werden. Normalerweise ist diese Zeit ca. 8 Minuten, obwohl es 2 bis 15 Minuten variieren. </li><li> Unmittelbar, übertragen Sie die Folie an zwei aufeinander folgenden Färbetrog mit 2 × SSC für 3 Minuten in jeder Coplin. </li><li> Weiter, indem die Folie durch eine Reihe von Ethanol wäscht für 2 Minuten in jede Coplin Glas (70%, 90% und 100% Ethanol). Lassen Sie die Folie trocken bei Raumtemperatur. Hinweis: Übermäßige DTT wäre in disperser FISH-Signale am Ende des Verfahrens führen, während eine zu kurze Belichtung in einem Mangel an bestimmten Signalen führen würde. </li></ol> III. Hybridisierung <ol><li> Denaturieren Spermien-DNA durch Inkubation der Folie in einer Coplin Glas mit Formamid-Lösung (70% Formamide/2xSSC) bei 73 ° C für 5 Minuten. </li><li> Die Objektträger durch Ethanol-Lösungen für 1 Minute pro Coplin jar (Beginn mit 70% Ethanol, 85% und Finish mit 100%). Lassen Sie die Folie trocken bei Raumtemperatur. </li><li> Fügen Sie direkt 5 l des entsprechenden ready-to-use-Sonde Mischung zu einem 15x15 Deckglas (AneuVysion Assay Multi-Farb-Probe Panel: CEP 18/X/Y oder LSI 13/21). </li><li> Wie es in dem Video zu sehen ist, legen Sie vorsichtig die Folie auf das Deckglas, um gemeinsam das Zielgebiet und die Sonde Mischung. Seal es mit Gummi-Zement. </li><li> Legen Sie die Folie in einen vorgewärmten 37 ° C Hybridisierung Kammer (HYBrite ™) und inkubieren bei 37 ° C für 6-24 Stunden. Hinweis: Während der Denaturierung der Spermaprobe ist zwingend erforderlich, die Notwendigkeit der Denaturierung der Sonden wird vom Hersteller bestimmt. Die Sonde Mischungen im Sinne dieses Protokolls sind gebrauchsfertig und in diesem Fall Sonde Denaturierung ist nicht erforderlich. Das Volumen der Sonde Mischung wird je nach Größe der hybridisierten Bereich variieren. </li></ol> IV. WäschtPost-Hybridisierung <ol><li> Nehmen Sie die Folie aus der Hybridisierung Kammer. </li><li> Entfernen Sie die Gummi-Zement und vorsichtig das Deckglas Schiebetüren sanft zur Seite. </li><li> Zur Beseitigung der unspezifischen Hybridisierungssignale Ort der Schieber für 2 Minuten in einem Coplin Glas mit 0.4xSSC/0.3% NP-40 vorgewärmt auf 73 ° C in einem Wasserbad. </li><li> Übertragen Sie die Folie an eine 2xSSC/0.1% NP-40-Waschlösung bei Raumtemperatur für 1 Minute. Lassen Sie die Folie trocken bei Raumtemperatur. </li><li> Fügen Sie eine Gegenfärbung Produkt der Zielregion (8 l für ein 18x18 Deckglas). Die häufigste Produkt verwendet wird 4 &#39;,6-Diamino-2-phenylindole (DAPI, Vysis Inc.). </li><li> Wie es in dem Video zu sehen ist, decken die hybridisierte Region mit einem Deckglas (die Hybridisierung des Deckglases mit Nagellack versiegelt werden können erhalten). </li><li> Shop hybridisierten Objektträger bei -20 ° C im Dunkeln, bis ihre Aussicht Analyse. Unter diesen Bedingungen die Folien können bis zu 12 Monate ohne in Fluoreszenzsignalintensität signifikante verloren gespeichert werden. Hinweis: Eine höhere Temperatur oder eine übermäßige Waschzeit kann bei der Beseitigung von einigen Signalen führen, während das Gegenteil nicht waschen würde aus unspezifischen Sonde Hybridisierungen. Das Volumen der Gegenfärbung Produkt zugesetzt werden nach der Größe der Hybridisierung Bereich zur Deckung variieren. </li></ol> V. Visualisierung Die Präparate können unter einem Epifluoreszenzmikroskop mit einem Triple-Band-Filter für DAPI / Texas Red / FITC und Single-Band-Filter für Aqua, FITC und Texas Red ausgestattet ausgewertet werden. Standard-Bewertungskriterien muss für die korrekte Auswertung der Spermakerne 1 gefolgt werden. Die Vorbereitung mit Sonden für die Chromosomen X, Y und 18 hybridisiert sollten Signale für diese drei Chromosomen-Display. Jeder normale Spermien müssen zeigen eine blaue Signal (entspricht dem Chromosom 18) und ein grünes Signal (X-tragenden Spermien) oder ein rotes Signal (Y-Spermien). In der Vorbereitung mit Sonden für die Chromosomen 13 und 21, ein grünes Signal für das Chromosom 13 und ein rotes Signal für Chromosom 21 hybridisiert sollte in jedem normalen Spermien unterschieden werden.

<ol>
	<li>Blanco, J., Egozcue, J., Vidal, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence+of+chromosome+21+disomy+in+human+spermatozoa+as+determined+by+fluorescent+in-situ+hybridization.">Incidence of chromosome 21 disomy in human spermatozoa as determined by fluorescent in-situ hybridization.</a> Hum. Reprod. 11, 722-726 (1996).</li></ol>

The authors have nothing to disclose.

Dieses Protokoll beschreibt, wie die menschliche Samenproben für den Erhalt von Spermien FISH Präparate Prozess. Mit diesem Protokoll ist es möglich, chromosomale Anomalien bei männlichen Keimzellen zu analysieren. Spermatozoen Aneuploidietestung hat Anwendungen entweder im Rahmen der Grundlagenforschung und in der reproduktiven Beratung der unfruchtbaren Männer. Obwohl in der klinischen Diagnostik der am häufigsten untersuchten Chromosomen X, Y, 13, 18 und 21, andere kommerzielle Sonden für eine breite Palette v...

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>1,4-Dithiothreitol (DTT)</td>
<td>Material</td>
<td>Roche</td>
<td>10197777001</td>
<td>&nbsp;</td>
<tr>
<td>2-Amino-2-(hydroxymethyl)-1,3-propanediol</td>
<td>Material</td>
<td>Roche</td>
<td>10708976001</td>
<td>&nbsp;</td>
<tr>
<td>Acetic acid</td>
<td>Material</td>
<td>Merck</td>
<td>100.063</td>
<td>&nbsp;</td>
<tr>
<td>AneuVysion&reg; Multicolor DNA Probe Kit<ul style="padding:2px;"><li>20xSaline-Sodium Citrate</li><li>4,6-diamidino-2-phenylindole (DAPI II)</li><li>CEP 18/X/Y DNA probe</li><li>NP-40</li><li>LSI 13/21 DNA probe</li></ul></td>
<td>Material</td>
<td>Vysis Inc.</td>
<td>32-161075 <ul style="padding:2px;"><li>30-805850</li><li>30-804941 </li><li>30-171077</li><li>30-804820</li><li>30-171078</li></ul></td>
<td>&nbsp;</td>
<tr>
<td>Centrifuge 5804R</td>
<td>Tool</td>
<td>Eppendorf</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Centrifuge tube</td>
<td>Material</td>
<td>Nunc</td>
<td>347856</td>
<td>&nbsp;</td>
<tr>
<td>Coplin jar (glass)</td>
<td>Material</td>
<td>Barloworld Scientific</td>
<td>Hellendahl (ZCT278)</td>
<td>&nbsp;</td>
<tr>
<td>Coplin jar (plastic)</td>
<td>Material</td>
<td>Deltalab</td>
<td>191087</td>
<td>&nbsp;</td>
<tr>
<td>Cover slips (15x15)</td>
<td>Material</td>
<td>Knittel gl&auml;ser</td>
<td>4600115</td>
<td>&nbsp;</td>
<tr>
<td>Cover slips (18x18)</td>
<td>Material</td>
<td>Knittel gl&auml;ser</td>
<td>4600118</td>
<td>&nbsp;</td>
<tr>
<td>Diamond pencil</td>
<td>Material</td>
<td>Hammacher</td>
<td>335117010</td>
<td>&nbsp;</td>
<tr>
<td>Ethanol</td>
<td>Material</td>
<td>Merck</td>
<td>100.983</td>
<td>&nbsp;</td>
<tr>
<td>Formamide</td>
<td>Material</td>
<td>Roche</td>
<td>11814320001</td>
<td>&nbsp;</td>
<tr>
<td>Freezer</td>
<td>Tool</td>
<td>Zanussi</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Gloves</td>
<td>Material</td>
<td>Sanyc</td>
<td>101/3300</td>
<td>&nbsp;</td>
<tr>
<td>HYBrite&trade;</td>
<td>Tool</td>
<td>Vysis Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Immersion Oil</td>
<td>Material</td>
<td>Olympus</td>
<td>35505</td>
<td>&nbsp;</td>
<tr>
<td>Incubator</td>
<td>Tool</td>
<td>Heraeus</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Methanol</td>
<td>Material</td>
<td>Merck</td>
<td>106.009</td>
<td>&nbsp;</td>
<tr>
<td>Micropipette</td>
<td>Material</td>
<td>Labsystems</td>
<td>Finnpipette (4500000)</td>
<td>&nbsp;</td>
<tr>
<td>Micropipette replacement tip</td>
<td>Material</td>
<td>Daslab</td>
<td>16-2001</td>
<td>&nbsp;</td>
<tr>
<td>Nail varnish</td>
<td>Material</td>
<td>Quo-cosmetics</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Olympus BX60 epifluorescence microscope</td>
<td>Tool</td>
<td>Olympus</td>
<td>&nbsp;</td>
<td>Equipped with a triple-band filter for DAPI/Texas Red/FITC and single-band filters for Aqua, FITC and Texas Red.</td>
</tr>
<tr>
<td>Pasteur Pipette</td>
<td>Material</td>
<td>Rubilabor</td>
<td>211.0230</td>
<td>&nbsp;</td>
<tr>
<td>Phase contrast microscope</td>
<td>Tool</td>
<td>Nikon Diaphot</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Plastic pipette (3ml)</td>
<td>Material</td>
<td>Deltalab</td>
<td>200007</td>
<td>&nbsp;</td>
<tr>
<td>Potassium chloride (KCl)</td>
<td>Material</td>
<td>Fluka</td>
<td>60128</td>
<td>&nbsp;</td>
<tr>
<td>Rubber cement</td>
<td>Material</td>
<td>Best-test</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Slides</td>
<td>Material</td>
<td>Knittel gl&auml;ser</td>
<td>4520022</td>
<td>&nbsp;</td>
<tr>
<td>Slide Saver Boxes</td>
<td>Material</td>
<td>Deltalab</td>
<td>19276.1</td>
<td>&nbsp;</td>
<tr>
<td>Sterile container</td>
<td>Material</td>
<td>Deltalab</td>
<td>409726</td>
<td>&nbsp;</td>
<tr>
<td>Syringe</td>
<td>Material</td>
<td>PentaFerte</td>
<td>002022300</td>
<td>&nbsp;</td>
<tr>
<td>Thermometer</td>
<td>Material</td>
<td>Comark Instruments, Inc</td>
<td>KM12</td>
<td>&nbsp;</td>
<tr>
<td>Tri-distilled water</td>
<td>Material</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>Material</td>
<td>Sigma</td>
<td>9002-93-1</td>
<td>&nbsp;</td>
<tr>
<td>Tweezers</td>
<td>Material</td>
<td>B/Braun</td>
<td>Aesculap (BD224R)</td>
<td>&nbsp;</td>
<tr>
<td>Vertical laminar flow hood</td>
<td>Tool</td>
<td>Burdinola</td>
<td>OR-ST 1500</td>
<td>&nbsp;</td>
<tr>
<td>Vortex mixer</td>
<td>Tool</td>
<td>Fisher Scientific</td>
<td>FB15012</td>
<td>&nbsp;</td>
<tr>
<td>Water bath</td>
<td>Tool</td>
<td>Raypa&reg;</td>
<td>&nbsp;</td>
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fluorescence in situ hybridization (fish) protocol in human sperm

Aneuploidien sind die häufigsten Chromosomenstörungen beim Menschen. Die meisten dieser Veränderungen ergeben sich aus meiotischen Fehler bei der gametogenic Prozess bei den Eltern. In der menschlichen Männchen, können diese Fehler auf die Produktion von Spermien mit numerischen Chromosomenanomalien, die eine erhöhte Gefahr der Übertragung dieser Anomalien auf die Nachkommen vertreten führen. Aus diesem Grund hat die Technik der Fluoreszenz in situ Hybridisierung (FISH) auf Spermakerne zu einem Protokoll weithin im Zusammenhang mit der klinischen Diagnose integriert. Diese Praxis liefert eine Schätzung der Frequenzen von numerischen Chromosomenanomalien in den Keimzellen der Patienten, die zur genetischen Reproduktion Rat zu suchen. Bis heute sind die Chromosomen am häufigsten in Spermien FISH Analyse mit einbezogen Chromosomen X, Y, 13, 18 und 21. Dieses Video-Artikel beschreibt Schritt für Schritt, wie zu verarbeiten und zu befestigen ein menschliches Sperma Probe, wie man decondense und denaturieren die Spermien Chromatin, wie es weitergehen soll, um Spermien FISH Präparate zu erhalten, und wie die Ergebnisse am Mikroskop sichtbar zu machen. Besondere Hinweise über die wichtigsten Schritte gegeben werden, um optimale Ergebnisse zu erzielen.

Watch this Scientific Journal Video about Fluorescence in situ hybridization FISH Protocol in Human Sperm at JoVE.com

Fluorescence in situ hybridization FISH Protocol in Human Sperm

Dieses Video-Artikel beschreibt Schritt für Schritt, wie man eine Samenprobe Prozess zu einer qualitativ hochwertigen Fluoreszenz zu erreichen<em&gt; In situ</em&gt; Hybridisierung auf menschliche Spermien. Die Vorbereitungen erhalten können Aneuploidietestung im Zusammenhang mit der klinischen Diagnose verwendet werden.

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 ...

fluorescence-in-situ-hybridization-fish-protocol-in-human-sperm

Research

JoVE Journal

Biology

Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

30.0K Aufrufe.  Universitat Autónoma de Barcelona. Dieses Video-Artikel beschreibt Schritt für Schritt, wie man eine Samenprobe Prozess zu einer qualitativ hochwertigen Fluoreszenz zu erreichen In situ Hybridisierung auf menschliche Spermien. Die Vorbereitungen erhalten können Aneuploidietestung im Zusammenhang mit der klinischen Diagnose verwendet werden.

Serie: Fluorescence in situ hybridization FISH Protocol in Human Sperm

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 ...

Forschung

Biologie

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 ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

Visualisierung und Analyse von mRNA Moleküle mittels Fluoreszenz<em&gt; In Situ</em&gt; Hybridisierung in<em&gt; Saccharomyces cerevisiae</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA probes. This protocol, originally developed by Kloosterman and colleagues for broad use with Exiqon miRNA probes1, has been modified to overcome challenges inherent in miRNA analysis in kidney tissues. These include issues such as structure identification and hard to remove residual probe and antibody. Use of relatively thin, 5 mm thick, tissue sections allowed for clear visualization of kidney structures, while a strong probe signal was retained in cells. Additionally, probe concentration and incubation conditions were optimized to facilitate visualization of microRNA expression with low background and nonspecific signal. Here, the optimized protocol is described, covering the initial tissue collection and preparation through the mounting of slides at the end of the procedure. The basic components of this protocol can be altered for application to other tissues and cell culture models.

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

This article describes an in situ hybridization protocol optimized for colormetric detection of microRNA expression in formalin fixed kidney sections.

MicroRNA<em&gt; In situ</em&gt; Hybridisierung für Formalin fixiert Nierengewebe

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA ...

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

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in ...

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

Combining RNA fluorescent in situ hybridization (FISH) with immunofluorescence (immuno-FISH) creates a technique that can be employed at the single cell level to detect the spatial dynamics of RNA localization with simultaneous insight into the localization of proteins, epigenetic modifications and other details which can be highlighted by immunofluorescence. X-chromosome inactivation is a paradigm for long non-coding RNA (lncRNA)-mediated gene silencing. X-inactive specific transcript (Xist) lncRNA accumulation (called an Xist cloud) on one of the two X-chromosomes in mammalian females is a critical step to initiate X-chromosome inactivation. Xist RNA directly or indirectly interacts with various chromatin-modifying enzymes and introduces distinct epigenetic landscapes to the inactive X-chromosome (Xi). One known epigenetic hallmark of the Xi is the Histone H3 trimethyl-lysine 27 (H3K27me3) modification. Here, we describe a simple and quick immuno-FISH protocol for detecting Xist RNA using RNA FISH with multiple oligonucleotide probes coupled with immunofluorescence of H3K27me3 to examine the localization of Xist RNA and associated epigenetic modifications. Using oligonucleotide probes results in a shorter incubation time and more sensitive detection of Xist RNA compared to in vitro transcribed RNA probes (riboprobes). This protocol provides a powerful tool for understanding the dynamics of lncRNAs and its associated epigenetic modification, chromatin structure, nuclear organization and transcriptional regulation.

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 ...

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly effective at mapping highly repetitive sequences to heterochromatic regions, where computational approaches face prohibitive challenges. Here we describe a streamlined protocol for DNA ISH that circumvents formamide washes that are standard steps in other DNA ISH protocols. Our protocol is optimized for hybridization with short single strand DNA probes that carry fluorescent dyes, which effectively mark repetitive DNA sequences within heterochromatic chromosomal regions across a number of different insect tissue types. However, applications may be extended to use with larger probes and visualization of single copy (non-repetitive) DNA sequences. We demonstrate this method by mapping several different repetitive sequences to squashed chromosomes from Drosophila melanogaster neural cells and Nasonia vitripennis spermatocytes. We show hybridization patterns for both small, commercially synthesized probes and for a larger probe for comparison. This procedure uses simple laboratory supplies and reagents, and is ideal for investigators who have little experience with performing DNA ISH.

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

Here, we present a simple method for performing fluorescence DNA in situ hybridization (DNA ISH) to visualize repetitive heterochromatic sequences on slide-mounted chromosomes. The method requires minimal reagents and it is versatile for use with short or long probes, different tissues, and detection with fluorescence or non-fluorescence-based signals.

Einfache Methode zur Fluoreszenz DNA<em&gt; In-situ-</em&gt; Die Hybridisierung an Squashed Chromosomen

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly ...

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization (FISH) with PNA Probes in Caenorhabditis elegans

Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase or an alternative pathway, known as alternative lengthening of telomeres (ALT)1. Recently, C. elegans has emerged as a multicellular model organism for the study of telomere and ALT2. Visualization of repetitive sequences in the genome is critical in understanding the biology of telomeres. While telomere length can be measured by telomere restriction fragment assay or quantitative PCR, these methods only provide the averaged telomere length. On the contrary, fluorescence in situ hybridization (FISH) can provide the information of the individual telomeres in cells. Here, we provide protocols and representative results of the method to determine telomere length of C. elegans by fluorescent in situ hybridization. This method provides a simple, but powerful, in situ procedure that does not cause noticeable damage to morphology. By using fluorescently labeled peptide nucleic acid (PNA) and digoxigenin-dUTP-labeled probe, we were able to visualize two different repetitive sequences: telomere repeats and template of ALT (TALT) in C. elegans embryos and gonads.

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization FISH with PNA Probes in Caenorhabditis elegans

We report a concise procedure of fluorescence in situ hybridization (FISH) in the gonad and embryos of Caenorhabditis elegans for observing and quantifying repetitive sequences. We successfully observed and quantified two different repetitive sequences, telomere repeats and template of alternative lengthening of telomeres (TALT).

Beobachtung und Quantifizierung der Telomere und repetitiver Sequenzen mittels Fluoreszenz<em&gt; In Situ</em&gt; Die Hybridisierung (FISH) mit PNA-Sonden in<em&gt; Caenorhabditis elegans</em

Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase ...

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

A method is described for labeling individual messenger RNA (mRNA) transcripts in fixed bacteria for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH allows the measurement of cell-to-cell variability in mRNA copy number of genes of interest, as well as the subcellular location of the transcripts. The main steps involved are fixation of the bacterial cell culture, permeabilization of cell membranes, and hybridization of the target transcripts with sets of commercially available short fluorescently-labeled oligonucleotide probes. smFISH can allow the imaging of the transcripts of multiple genes in the same cell, with limitations imposed by the spectral overlap between different fluorescent markers. Following completion of the protocol illustrated below, cells can be readily imaged using a microscope coupled with a camera suitable for low-intensity fluorescence. These images, together with cell contours obtained from segmentation of phase contrast frames, or from cell membrane staining, allow the calculation of the mRNA copy number distribution of a sample of cells using open-source or custom-written software. The labeling method described here can also be applied to image transcripts with stochastic optical reconstruction microscopy (STORM).

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

This manuscript describes a method for labeling individual messenger RNA (mRNA) transcripts with fluorescently-labeled DNA probes, for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH is a visualization method that allows the simultaneous detection, localization, and quantification of single mRNA molecules in fixed individual cells.

Methode zur Kennzeichnung Transkripte in einzelnen Escherichia coli Zellen für Einzelmolekül-Fluoreszenz In Situ Hybridisierung Experimente