Diese Arbeit wurde vom US-amerikanischen National Institutes of Health und gewährt HL082798 HL111580.

1. Nierengewebeschnitten <ol><li> Formalin-fixierte (10%) und in Paraffin eingebetteten Nierenschnitte auf Objektträger mit 5 mm Dicke mit einem Microm HM 355 S. Die Folien können mehrere Monate vor der Analyse gelagert werden geschnitten. </li></ol> 2. Herstellung der Lösung <ol><li> Bereiten 1 l 1x PBS in ddH 2 O in einer autoklavierbar Schraubverschluss der Flasche. Füllen Sie eine Flasche mit 1 l ddH 2 O. 1 ml DEPC zu jeder Flasche, Deckel und kräftig schütteln. Lassen Sie die Lösungen sitzen über Nacht bei Raumtemperatur auf RNAsen zu zerstören und dann im Autoklaven. 400 ml Tween-20 zu 1 l 1x PBS auf PBS-T zu machen. </li><li> Vorbereitung Proteinase K Puffer (50 mM Tris-HCl, pH 8,0 und 1 mM CaCl 2 in DEPC-behandeltem Wasser). </li><li> Eine Lösung aus 0,2% Glycin in PBS-T. </li></ol> 3. Gewebepräparation <ol><li> Vorbereitung einer großen Anzahl von Hybridisierungspuffer (50-100 ml), 50% Formamid, 5x SSC, 0,1% Tween, 9,2mM Zitronensäure zur Einstellung auf pH 6, 50 ug / ml Heparin und 500 ug / ml Hefe-RNA in DEPC-behandeltem ddH 2 O. Teilen Sie sich in 1 ml Aliquots und bei -80 º C. </li><li> Entfernen Sie das Paraffin aus dem Gewebe durch Waschen 3x in frischem Xylol für jeweils 5 min. </li><li> Rehydrierung der Gewebeschnitte von 5 min Inkubation in abnehmenden Konzentrationen von Ethanol (100%, 75%, 50% und 25%) in ddH 2 O. </li><li> Behandeln Sie die Folien mit DEPC genug, um die Gewebeschnitte (etwa 0,4-0,5 ml) für 1 min vollständig zu bedecken. Stellen Sie sicher, Gewebe trocknen nicht aus. </li><li> Spülen Sie die Folien in DEPC behandeltem PBS-T zweimal 5 min, das Sammeln der DEPC haltige Abfälle zur ordnungsgemäßen Entsorgung. Alle Reagenzien sollten DEPC behandelt werden, um RNAsen von diesem Zeitpunkt an zu beseitigen. </li><li> Vorwärmen Proteinase K Puffer und fügen Proteinase K bis zu einer Endkonzentration von 10 ug / ml. Bedeckung Gewebeschnitte mit Proteinase K-Lösung und Inkubation bei 37 º C für 5 min. </li><li> Entfernen Sie schnell die proteinasE-K-Lösung und mit 0,2% Glycin in PBS-T für 30 sek. </li><li> Rinse Folien in DEPC behandeltem PBS-T zweimal für jeweils 30 Sekunden. </li><li> Fix Abschnitte in frisch zubereiteten 4% Paraformaldehyd für 10 min. </li></ol> 4. Kreuzung <ol><li> In 50 ul Hybridisierungspuffer (50% Formamid, 5x SSC, 0,1% Tween, 9,2 mM Zitronensäure zur Einstellung auf pH 6, 50 ug / ml Heparin, 500 ug / ml Hefe-RNA) pro Gewebeschnitt und decken mit RNAse frei HybriSlips schneiden nur größer als die Gewebeschnitten. </li><li> Prehybridize Abschnitte der Luftfeuchtigkeit gesteuert Hybridisierungsofen für 2 Stunden bei Hybridisierungstemperatur für die 3 &#39;mit Digoxigenin markierten Sonde (in der Regel DNA Tm der Sonde bestimmt -21 ° C (dh, 54 ° C für miR-382, DNA-Sonde Tm = 75 ° C ), unter Verwendung von Gewebelabor Tücher in 50% formamide/50% 5x SSC eingeweicht, um die Kammer zu halten befeuchtet. </li><li> Verdünnen Sie die miRNA-Sonde, positive Kontrolle (U6, small nuclear RNA) und Negativkontrolle (RühreiSequenz) bis 40 nm in Hybridisierungspuffer (75 ul Puffer pro Sektion erforderlich). </li><li> Erwärmen die Sonde in Hybridisierungspuffer bei 65 º C für 5 min. </li><li> Objektträger aus der Hybridisierungsofen und Deckgläser und überschüssige Hybridisierungspuffer entfernen Prähybridisierung. </li><li> In 75 ul der Sonde, die Puffer pro Gewebeschnitt, direkt auf Gewebe. Decken Gewebe mit HybriSlips gerade groß genug, um die Gewebeschnitte zu decken. </li><li> Keeping Diahalter Ebene zurück zu Hybridisierungsofen für Inkubation über Nacht bei einer Temperatur von Schritt 4.2 (ca. 16 h). </li></ol> 5. Die Stringenz Wash <ol><li> In 200 ul 2x SSC, erhitzt, um die Hybridisierungstemperatur, knapp eine Kante der Deckgläser zu helfen, das Deckglas aus dem Gewebe. Entfernen Sie das Deckglas durch Kippen der Folie. </li><li> Waschen der Objektträger in 200 ul 50% Formamid, 50% 2 × SSC bei Hybridisierungstemperatur 3x je 30 min. </li><li> Spülendie Schieber 5x in PBS-T bei Raumtemperatur für jeweils 5 min auf Schüttler bei niedriger Geschwindigkeit. </li></ol> 6. Entdeckung <ol><li> Inkubieren Folie in Blockierungspuffer (2% Ziegen-oder Pferdeserum, 2 mg / ml BSA in PBS-T) 1 Stunde lang bei Raumtemperatur auf Schüttler bei niedriger Geschwindigkeit. </li><li> Verdünnter Anti-DIG-AP Fab-Fragmenten in Blockierungspuffer bei 1:100. 100 l pro Gewebeschnitt und decken mit HybriSlips schneiden gerade groß genug, um den Abschnitt zu decken. </li><li> Inkubieren-Antikörper in einer befeuchteten Kammer bei 4 ° C über Nacht (ca. 16 h). </li><li> In PBS-T 7x Waschen der Objektträger für 5 Minuten auf jeder Orbitalschüttler bei niedriger Geschwindigkeit. </li><li> Waschen der Objektträger in AP-Puffer (100 mM TrisHCl pH 9,5, 50 mM MgCl 2, 100 mM NaCl, 0,1% Tween 20) 3x für jeweils 5 min. </li><li> In NBT / BCIP Lösung AP-Puffer (200 ml μl/10 Puffer) </li><li> In 400 bis 500 ml verdünnt NBT / BCIP-Lösung zu jeder Folie, um alle Gewebeschnitte vollständig zu bedecken. Entwickeln Sie in einem dunklen, Niveau, humidified Kammer für 4-5 Std. </li></ol> 7. Montageschienen <ol><li> Dehydratisieren Abschnitte in steigenden Konzentrationen von Ethanol (25%, 50%, 75%, 100%) für jeweils 5 min. </li><li> Inkubation in Xylol 5x, um Abschnitte zu löschen. </li><li> Berg rutscht in Permount Montagemedium und über Nacht trocknen. </li></ol>

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Es gibt nichts zu offenbaren.

Das Ziel dieses Artikels war es, ein Protokoll für die miRNA-in-situ-Hybridisierung, die gut funktioniert in Formalin fixiert Nierengewebe zu beschreiben. Während der Erarbeitung dieses Protokoll mehrere wichtige Quellen der Färbung Artefakt identifiziert worden. Besondere Aufmerksamkeit auf diese Punkte können zur Vermeidung Färbung Artefakt und erhöhen die Wahrscheinlichkeit einer erfolgreichen ISH Sicht. Eines der am meisten vermeidbaren Ursachen der Färbung Artefakt kann ...

MicroRNA sind kleine (ca. 22 Nukleotide lange) nicht-kodierenden RNAs, die endogen produziert werden. Sie funktionieren in der Regel um die Proteinexpression durch translationale Repression oder mRNA-Abbau zu unterdrücken. miRNAs binden an mRNA Ziele mit unvollständigen Komplementarität, die es ermöglicht, eine einzelne miRNA auf mehrere Ziele zu unterdrücken. Zu verstehen, welche Zelltypen und Strukturen auszudrücken miRNAs ist ein wichtiger Teil der das Verständnis der Mechanismen, durch die Veränderungen in der miRNA-Expression Einfluss Zell-und Gewebe Phänotypen. Während Methoden wie miRNA-Sequenzierung, qPCR und Northern Blot zur Detektion von miRNAs in ganzen Gewebe verwendet werden, ist dieser Ansatz nicht erlauben es, festzustellen, welche spezifischen Zelltyp sie innerhalb eines bestimmten Gewebes kam. Dissection von zellulären und strukturellen Komponenten vor der Analyse mit Hilfe dieser Methoden kann sehr schwierig sein, und die benötigt wird, um angemessene Bedingungen Isolierungen erreichen können, um al führenterations in der Genexpression oder den Abbau von RNAs. microRNA in-situ-Hybridisierung ist eine Methode verwendet, um Standort-und microRNA-Expressionsniveaus im Gewebe sichtbar zu machen. Diese Technik ist besonders in Geweben von heterogenen Strukturen, wie z. B. der Niere zusammen wertvoll. MicroRNAs sind gezeigt worden, um eine regulatorische Rolle bei der Nierenentwicklung 2,3 und Physiologie 4 zu spielen. microRNA Expression Änderungen haben auch gezeigt, dass mit Nierenpathologie wie Fibrose 5-10, diabetische Nephropathie 7, Nierenkarzinom 11,12 und akutem Nierenversagen 13 einbezogen werden. In unserer Forschung haben wir festgestellt, dass die Optimierung microRNA in-situ-Hybridisierung für Nierengewebe war wertvoll für die Ermittlung der genauen Struktur Standorte der miRNA-Expression sowohl in Gesundheit und Krankheit 14. Bestimmung der Rohr und zelluläre Expression verschiedener microRNAs ist wichtig, weil die Regulierung der targets kann von zellulären Funktionen sein. In Krankheitszuständen ist es auch wichtig, um zu bestimmen, wie Veränderungen in der miRNA-Expression beeinflussen werden Funktion. Das Ziel der hier beschriebenen Methode war, auf den bestehenden Methoden, die von ISH Kloosterman et al. 15. andere Forscher entwickelt 16,17 zu bauen, und die von Exiqon 1 vorgeschlagen und Optimierung der Methode zur Formalin fixiert Nierengewebe. Wir haben erfolgreich diese Methode verwendet, um regionale Unterschiede in der Nieren microRNA miR-382 Expression mit einseitiger Ureterobstruktion 18 zu identifizieren. Dieser Ansatz kann mit anderen Geweben als auch verwendet werden, mit zusätzlichen Optimierung.

<table border="0" cellpadding="0" cellspacing="0" width="481">
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		<col />
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		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Paraformaldehyde</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">P6148</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">PBS</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:137px;">70011044</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Diethyl Pyrocarbonate</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">D5758</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Tween-20</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">P1379</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Xylenes</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">534056</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Ethanol</td>
			<td style="width:71px;">Ultra Pure</td>
			<td style="width:137px;">200CSPTP</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:195px;">Tris-HCl</td>
			<td style="width:71px;">Life Technologies</td>
			<td style="width:137px;">15506-017</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">CaCl2</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">C2536</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Glycine</td>
			<td style="width:71px;">J.T. Baker</td>
			<td style="width:137px;">4059-00</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Citric Acid</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">C2404</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Formamide</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">F9037</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:195px;">20x SSC Buffer</td>
			<td style="width:71px;">Life Technologies</td>
			<td style="width:137px;">15557-044</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Heparin</td>
			<td style="width:71px;">SAGENT</td>
			<td style="width:137px;">25021-400-30</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:195px;">Yeast RNA</td>
			<td style="width:71px;">Life Technologies</td>
			<td style="width:137px;">AM7118</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">MgCl2</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">M8266-1004</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">NaCl</td>
			<td style="width:71px;">J.T. Baker</td>
			<td style="width:137px;">4058-01</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Proteinase K</td>
			<td style="width:71px;">Fermentas</td>
			<td style="width:137px;">EO0491</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">3&#8217; DIG- miRNA probe</td>
			<td style="width:71px;">Exiqon</td>
			<td style="width:137px;">miR dependent-05</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">3&#8217; DIG- Scrambled miR probe</td>
			<td style="width:71px;">Exiqon</td>
			<td style="width:137px;">99004-05</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">3&#8217; DIG- U6 miR probe</td>
			<td style="width:71px;">Exiqon</td>
			<td style="width:137px;">99002-05</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Anti-Digoxigenin-Ab Fab</td>
			<td rowspan="2" style="width:71px;">Roche</td>
			<td rowspan="2" style="width:137px;">11093274910</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Fragments</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">NBT/BCIP</td>
			<td style="width:71px;">Roche</td>
			<td style="width:137px;">11681451001</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Permount</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:137px;">SP15-500</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Coverslips</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:137px;">Fit to tissue size</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:195px;">Microtom HM 355 S</td>
			<td style="width:71px;">Thermo Scientific</td>
			<td style="width:137px;">23-900-672</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">In-slide Out Hybridization Oven</td>
			<td style="width:71px;">Boekel</td>
			<td style="width:137px;">241000</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Aluminum Tray Assembly</td>
			<td style="width:71px;">Boekel</td>
			<td style="width:137px;">C2403973</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Stainless Steel Rack Insert</td>
			<td style="width:71px;">Boekel</td>
			<td style="width:137px;">C2403754</td>
			<td style="width:79px;"></td>
		</tr>
	</tbody>
</table>

microrna in situ hybridization for formalin fixed kidney tissues

In diesem Artikel beschreiben wir eine Methode zur kolorimetrischen Nachweis von miRNA in der Niere durch in-situ-Hybridisierung mit Digoxigenin markierten Sonden microRNA. Dieses Protokoll, das ursprünglich von Kloosterman und Kollegen für eine breite Verwendung mit Exiqon miRNA Sonden 1 entwickelt, wurde modifiziert, um Herausforderungen in der miRNA-Analyse im Nierengewebe zu überwinden. Dazu gehören Themen wie Identifikation und Struktur schwer zu Rest Sonde und Antikörper zu entfernen. Die Verwendung von relativ dünnen, 5 mm dick, Gewebeschnitte erlaubt für klare Visualisierung von Nieren Strukturen, während eine starke Probe-Signal wurde in den Zellen zurückgehalten. Zusätzlich wurden Sondenkonzentration und Inkubationszeit Bedingungen optimiert, um die Visualisierung von microRNA Expression mit niedrigen Hintergrund und unspezifische Signal zu erleichtern. Hier wird die optimierte Protokoll beschrieben, im Anfangsgewebeentnahme und Vorbereitung durch die Montage der Schieber am Ende des Verfahrens. Die Grund components dieses Protokoll kann für die Anwendung auf anderen Geweben und Zellkulturmodelle verändert werden.

Die Bereiche von einem Gewebeschnitt, die während der Hybridisierungen, Inkubationen getrocknet werden oder Waschschritte der Regel am Ende dunkler Färbung während der NBT / BCIP Entwicklung. Abbildung 1 zeigt einen Teil einer Niere Abschnitt, in dem die HybriSlip rutschte der Kante das Gewebe, so dass sie teilweise dehydriert. Trotz Rehydrierung und Abdeckung in den verbleibenden Schritten wird das Signal in dem entwässerten Teil künstlich hoch. Die Bedeutung der Steue...

Watch this Scientific Journal Video about MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues at JoVE.com

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

Dieser Artikel beschreibt ein in situ-Hybridisierungsprotokoll für die kolorimetrische Detektion der microRNA Expression in Formalin fixiert Nierenschnitten optimiert.

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 ...

microrna-in-situ-hybridization-for-formalin-fixed-kidney-tissues

Research

JoVE Journal

Biology

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

14.9K Aufrufe.  Medical College of Wisconsin.  Dieser Artikel beschreibt ein in situ-Hybridisierungsprotokoll für die kolorimetrische Detektion der microRNA Expression in Formalin fixiert Nierenschnitten optimiert. 

Serie: MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

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

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 ...

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. Each of these features can be accomplished with in situ hybridization to mRNAs within cells. Here we present a radioactive in situ hybridization method modified from Clayton et al. (1988)1 that has been working successfully in our lab for many years, especially for adult vertebrate brains2-5. The long complementary RNA (cRNA) probes to the target sequence allows for detection of low abundance transcripts6,7. Incorporation of radioactive nucleotides into the cRNA probes allows for further detection sensitivity of low abundance transcripts and quantitative analyses, either by light sensitive x-ray film or emulsion coated over the tissue. These detection methods provide a long-term record of target gene expression. Compared with non-radioactive probe methods, such as DIG-labeling, the radioactive probe hybridization method does not require multiple amplification steps using HRP-antibodies and/or TSA kit to detect low abundance transcripts. Therefore, this method provides a linear relation between signal intensity and targeted mRNA amounts for quantitative analysis. It allows processing 100-200 slides simultaneously. It works well for different developmental stages of embryos. Most developmental studies of gene expression use whole embryos and non-radioactive approaches8,9, in part because embryonic tissue is more fragile than adult tissue, with less cohesion between cells, making it difficult to see boundaries between cell populations with tissue sections. In contrast, our radioactive approach, due to the larger range of sensitivity, is able to obtain higher contrast in resolution of gene expression between tissue regions, making it easier to see boundaries between populations. Using this method, researchers could reveal the possible significance of a newly identified gene, and further predict the function of the gene of interest.

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

This protocol is successfully used to quantitatively detect levels and spatial patterns of mRNA expression in multiple tissue types across vertebrate species. The method can detect low abundance transcripts and allows processing of hundreds of slides simultaneously. We present this protocol using expression profiling of avian embryonic brain formation as an example.

Radioaktiv In-situ- Hybridisierung zum Nachweis Diverse Genexpressionsmuster in Tissue

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the 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 ...

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 ...

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 ...

Proteomic Sample Preparation from Formalin Fixed and Paraffin Embedded Tissue

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale quantitative analysis of formalin fixed and paraffin embedded (FFPE) tissue. The procedure comprises four distinct steps. The first one is the preparation of sections from the FFPE material and microdissection of cells of interest. In the second step the isolated cells are lysed and processed using 'filter aided sample preparation' (FASP) technique. In this step, proteins are depleted from reagents used for the sample lysis and are digested in two-steps using endoproteinase LysC and trypsin. 
After each digestion, the peptides are collected in separate fractions and their content is determined using a highly sensitive fluorescence measurement. Finally, the peptides are fractionated on 'pipette-tip' microcolumns. The LysC-peptides are separated into 4 fractions whereas the tryptic peptides are separated into 2 fractions. In this way prepared samples allow analysis of proteomes from minute amounts of material to a depth of 10,000 proteins. Thus, the described workflow is a powerful technique for studying diseases in a system-wide-fashion as well as for identification of potential biomarkers and drug targets.

Archival formalin fixed and paraffin embedded (FFPE) clinical samples are valuable material for investigation of diseases. Here we demonstrate a sample preparation workflow allowing in-depth proteomic analysis of microdissected FFPE tissue.

Proteomik Probenvorbereitung von Formalin fixiert und in Paraffin eingebetteten Gewebe

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale ...

qPCR Is a Sensitive and Rapid Method for Detection of Cytomegaloviral DNA in Formalin-fixed, Paraffin-embedded Biopsy Tissue

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for developing severe infection. Many laboratory methods for the detection of CMV infection have been developed, including serology, viral culture, and molecular methods. Often, these methods reflect systemic involvement with CMV and do not specifically identify local tissue involvement. Therefore, detection of CMV infection in the GI tract is frequently done by traditional histology of biopsy tissue. Hematoxylin and eosin (H&#38;E) staining in conjunction with immunohistochemistry (IHC) have remained the mainstays of examining these biopsies. H&#38;E and IHC sometimes result in atypical (equivocal) staining patterns, making interpretation difficult. It was shown that quantitative polymerase chain reaction (qPCR) for CMV can successfully be performed on formalin-fixed, paraffin-embedded (FFPE) biopsy tissue for very high sensitivity and specificity. The goal of this protocol is to demonstrate how to perform qPCR testing for the detection of CMV in FFPE biopsy tissue in a clinical laboratory setting. This method is likely to be of great benefit for patients in cases of equivocal staining for CMV in GI biopsies.

This protocol describes qPCR detection of cytomegalovirus in formalin-fixed, paraffin-embedded biopsy tissue, which is rapid, sensitive, specific, and useful for interpreting equivocal hematoxylin and eosin or immunohistochemical staining patterns.

qPCR ist eine empfindliche und schnelle Methode zur Detektion von DNA Zytomegalieviren in Formalin-fixierte, in Paraffin eingebettete Gewebebiopsie

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for ...

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.