Questo lavoro è stato sostenuto da US National Institutes of Health concede HL082798 e HL111580.

1. Renali tessuto Sezioni <ol><li> Fissato in formalina (10%) sezioni di rene in paraffina sono tagliati su vetrini da microscopio a 5 mm di spessore con un Microm HM 355 S. Le slide possono essere conservati per diversi mesi prima analisi. </li></ol> 2. Soluzione Preparazione <ol><li> Preparare 1 L di 1x PBS in DDH 2 O in una bottiglia con tappo a vite autoclavabile. Riempire un&#39;altra bottiglia con 1 L di DDH 2 O. Aggiungere 1 ml di DEPC per ogni bottiglia, coperchio e agitare vigorosamente. Lasciate le soluzioni siedono notte a temperatura ambiente per distruggere RNasi e poi in autoclave. Aggiungere 400 ml di Tween-20-1 L di 1x PBS per rendere PBS-T. </li><li> Preparare proteinasi K tampone (50 mMTris-HCl, pH 8,0 e 1 mM CaCl 2 in DEPC acqua trattata). </li><li> Preparare una soluzione di 0,2% glicina in PBS-T. </li></ol> 3. Tissue Preparazione <ol><li> Preparare un grande volume di tampone di ibridazione (50-100 ml) composta dal 50% formammide, 5x SSC, 0,1% Tween, 9.2acido citrico mM per aggiustamento del pH a 6, 50 ug / ml di eparina, e 500 mg / ml di RNA di lievito in DEPC trattati DDH 2 O. Dividere in 1 ml aliquote e conservare a -80 ° C. </li><li> Rimuovere la paraffina dal tessuto mediante lavaggio 3x in xilene fresco per 5 minuti ciascuna. </li><li> Reidratare le sezioni di tessuto per 5 min incubazioni in concentrazioni decrescenti di etanolo (100%, 75%, 50% e 25%) in DDH 2 O. </li><li> Trattare i vetrini con DEPC sufficiente a coprire completamente le sezioni di tessuto (circa 0,4-0,5 ml) per 1 min. Rendere sicuri i tessuti non si asciugano. </li><li> Sciacquare i vetrini in DEPC trattati PBS-T due volte per 5 min, la raccolta dei rifiuti DEPC contenente per il corretto smaltimento. Tutti i reagenti devono essere DEPC trattati per eliminare RNasi da questo punto in poi. </li><li> Preriscaldare proteinasi K tampone e inserirlo proteinasi K a una concentrazione finale di 10 pg / ml. Coprire sezioni di tessuto con una soluzione di proteinasi K e incubare a 37 ° C per 5 min. </li><li> Rimuovere rapidamente i proteinassoluzione e K e aggiungere 0,2% glicina in PBS-T per 30 sec. </li><li> Sciacquare i vetrini in DEPC trattati PBS-T due volte per 30 secondi ciascuna. </li><li> Fissare sezioni appena fatto paraformaldeide 4% per 10 min. </li></ol> 4. Ibridazione <ol><li> Aggiungere 50 ml di tampone di ibridazione (formammide 50%, 5x SSC, 0,1% Tween, acido citrico 9,2 mm per l&#39;aggiustamento a pH 6, 50 mg / ml di eparina, 500 mg / ml di RNA di lievito) per sezione di tessuto e coprire con RNAsi HybriSlips gratis tagliare appena più grandi delle sezioni di tessuto. </li><li> Sezioni Prehybridize a umidità controllata ibridazione forno per 2 ore a temperatura di ibridazione determinato per la 3 &#39;della sonda marcata con digossigenina (solitamente Tm DNA della sonda -21 ° C (cioè 54 ° C per miR-382, sonda di DNA Tm = 75 ° C ), utilizzando salviette di laboratorio tessuti imbevuti nel 50% formamide/50% 5x SSC per mantenere la camera umidificata. </li><li> Diluire la sonda di miRNA, controllo positivo (U6, piccolo RNA nucleare) e il controllo negativo (strapazzatesequenza) a 40 nM in tampone di ibridazione (75 microlitri di tampone è necessaria per sezione). </li><li> Riscaldare la sonda nel tampone di ibridazione a 65 ° C per 5 min. </li><li> Togliere i vetrini dalla ibridazione forno e rimuovere coprioggetto e tampone di ibridazione in eccesso da prehybridization. </li><li> Aggiungere 75 ml di sonda contenente tampone per sezione di tessuto, direttamente al tessuto. Coprire con tessuto HybriSlips appena sufficiente a coprire le sezioni di tessuto. </li><li> Mantenere il livello di supporto di diapositive, tornare a ibridazione forno per notte di incubazione a temperatura dal punto 4.2 (circa 16 ore). </li></ol> 5. Stringenza Wash <ol><li> Aggiungere 200 microlitri 2x SSC, riscaldato alla temperatura di ibridazione, poco meno di un bordo del coprioggetto per favorire il rilascio coprioggetto dal tessuto. Rimuovere il vetrino inclinando il vetrino. </li><li> Lavare i vetrini in 200 ml di 50% formammide, 50% 2x SSC a ibridazione 3x temperatura per 30 minuti ciascuno. </li><li> Sciacquarei vetrini 5x in PBS-T a temperatura ambiente per 5 min ciascuno su agitatore orbitale a bassa velocità. </li></ol> 6. Rivelazione <ol><li> Incubare il vetrino in tampone di bloccaggio (2% capra o siero di cavallo, 2 mg / ml di BSA in PBS-T) per 1 ora a temperatura ambiente su agitatore orbitale a bassa velocità camera. </li><li> Diluire frammenti anti-DIG-AP Fab in tampone di bloccaggio a 1:100. Aggiungere 100 microlitri per ogni sezione di tessuto e coprire con HybriSlips tagliato appena sufficiente a coprire la sezione. </li><li> Incubare anticorpo in una camera umidificata a 4 ° C per una notte (circa 16 ore). </li><li> Lavare i vetrini in PBS-T 7x, per 5 min ciascuno su agitatore orbitale a bassa velocità. </li><li> Lavare i vetrini in tampone AP (100 mM TrisHCl pH 9,5, 50 mM MgCl 2, 100 mM NaCl, 0,1% Tween 20) 3x, per 5 minuti ciascuno. </li><li> Aggiungere la soluzione NBT / BCIP al buffer di AP (200 μl/10 tampone ml) </li><li> Aggiungere 400-500 ml di soluzione diluita di NBT / BCIP per ogni diapositiva a coprire completamente tutte le sezioni di tessuto. Sviluppare in un buio, livello, HumidifCamera IED per 4-5 ore. </li></ol> 7. Slides di montaggio <ol><li> Disidratare sezioni in concentrazioni crescenti di etanolo (25%, 50%, 75%, 100%) per 5 min ciascuno. </li><li> Incubare a 5x xilene per chiarire sezioni. </li><li> Monte scorre in mezzo di montaggio Permount e pernottamento a secco. </li></ol>

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Non c&#39;è nulla da rivelare.

L&#39;obiettivo di questo articolo è quello di descrivere un protocollo per miRNA ibridazione in situ che funziona bene nei tessuti renali fissati in formalina. Mentre si lavora su questo protocollo sono stati identificati alcuni importanti fonti di artefatti di colorazione. Una grande attenzione a questi punti può aiutare a evitare di macchiare artefatto e aumentare la probabilità di una corsa ISH successo. Una delle cause più evitabili di artefatti di colorazione può verifica...

MicroRNA sono piccoli (lunghi circa 22 nucleotidi) RNA non codificanti che vengono prodotti in modo endogeno. In genere funzionano per sopprimere l&#39;espressione della proteina attraverso la repressione traslazionale o degradazione mRNA. miRNA si legano agli obiettivi mRNA con la complementarità incompleta, rendendo possibile per un singolo miRNA per sopprimere bersagli multipli. Capire quali tipi e strutture cellulari esprimono miRNA è una parte importante di comprendere i meccanismi attraverso i quali alterazioni di espressione miRNA influenza delle cellule e dei tessuti fenotipi. Mentre i metodi quali miRNA sequenziamento, qPCR e Northern blotting possono essere usate per il rilevamento dei miRNA nei tessuti interi, questo approccio non permette di determinare il tipo specifico di cellule venivano dall&#39;interno di un dato tessuto. Dissezione di componenti cellulari e strutturali prima dell&#39;analisi con questi metodi può essere molto difficile e le condizioni necessarie per raggiungere isolamenti adeguati può portare ad alterations nell&#39;espressione genica o la degradazione di RNA. microRNA ibridazione in situ è un metodo utilizzato per visualizzare la posizione microRNA e livelli di espressione nei tessuti. Questa tecnica è particolarmente utile in tessuti composti di strutture eterogenee, come il rene. I microRNA hanno dimostrato di svolgere un ruolo di regolamentazione in 2,3 sviluppo del rene e la fisiologia 4. alterazioni di espressione microRNA hanno anche dimostrato di essere coinvolti con patologie renali come la fibrosi 5-10, nefropatia diabetica 7, carcinoma renale 11,12, e danno renale acuto 13. Nella nostra ricerca, abbiamo scoperto che l&#39;ottimizzazione microRNA ibridazione in situ per i tessuti renali è stata preziosa per determinare le esatte posizioni strutturali di espressione miRNA sia in salute e malattia 14. Determinazione dell&#39;espressione tubolare e cellulare di diversi microRNA è importante perché la loro regolazione di targets possono dipendere funzioni cellulari. Negli stati malati è importante anche per determinare in che modo le alterazioni dell&#39;espressione dei miRNA possono essere funzione incidono. L&#39;obiettivo del metodo qui descritto è stato quello di sviluppare metodologie ISH esistenti predisposti da Kloosterman et al. 15, altri ricercatori 16,17, e quelle suggerite dalla Exiqon 1 e ottimizzare il metodo di formalina tessuti renali fissi. Abbiamo utilizzato con successo questo metodo per identificare le diverse differenze regionali nella renale espressione di microRNA miR-382 con unilaterale ostruzione ureterale 18. Questo approccio può essere utilizzato con altri tessuti e, con ulteriore ottimizzazione.

<table border="0" cellpadding="0" cellspacing="0" width="481">
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		<col />
		<col />
		<col />
		<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 questo articolo descriviamo un metodo di rilevazione colorimetrico del miRNA nel rene mediante ibridazione in situ con sonde digossigenina taggato microRNA. Questo protocollo, sviluppato originariamente da Kloosterman e colleghi per un uso ampio con sonde Exiqon miRNA 1, è stato modificato per superare le sfide insite nell&#39;analisi miRNA nei tessuti renali. Si tratta di problemi quali l&#39;identificazione struttura e difficile da rimuovere la sonda residuo e anticorpi. L&#39;uso di relativamente sottile, spessore 5 mm, sezioni di tessuto consentiti per una chiara visualizzazione delle strutture renali, mentre un forte segnale della sonda è stato trattenuto nelle cellule. Inoltre, le condizioni di concentrazione sonda e di incubazione sono stati ottimizzati per facilitare la visualizzazione di espressione microRNA con basso background e segnale aspecifico. Qui, il protocollo ottimizzato è descritto, di captazione tessuto iniziale e preparazione attraverso il montaggio di diapositive alla fine della procedura. La compone di basenti di questo protocollo possono essere modificati per applicazione ad altri tessuti e modelli di coltura cellulare.

Le aree di una sezione di tessuto che diventano asciugato durante le ibridazioni, incubazioni o lavare gradini generalmente finiscono colorazione più scura durante lo sviluppo NBT / BCIP. Figura 1 mostra una porzione di una sezione in cui il rene HybriSlip scivolato del bordo il tessuto, consentendole di diventare parzialmente disidratato. Nonostante reidratazione e la copertura nei passaggi rimanenti, il segnale nella porzione disidratata è artificialmente elevato. L&#39;...

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

Questo articolo viene descritto un protocollo in situ ottimizzato per il rilevamento colorimetrico di espressione microRNA nelle sezioni di rene fisse formalina.

MicroRNA<em&gt; In situ</em&gt; Ibridazione per tessuti renali fissati in formalina

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 Views.  Medical College of Wisconsin.  Questo articolo viene descritto un protocollo in situ ottimizzato per il rilevamento colorimetrico di espressione microRNA nelle sezioni di rene fisse formalina. 

Video: 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.

Doppia Monte intero ibridazione in situ di embrioni di pollo primi

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

Ricerca

Biologia

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.

Zebrafish intero monte ad alta risoluzione doppia fluorescente In Situ Ibridazione

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

Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

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

Fluorescence in situ hybridization FISH Protocol in Human Sperm

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

Fluorescenza In situ</emIbridazione> (FISH) Protocollo di sperma umano

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.

Ibridazione in situ per la localizzazione precisa delle Trascrizioni nelle piante

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.

Radioattivo In situ Ibridazione per il rilevamento Diversi schemi di espressione genica nei tessuti

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.

Rilevamento di RNA virale per fluorescenza<em&gt; In situ</em&gt; Hybridization (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.

Analisi di Single-cell trascrizione del gene per l&#39;RNA fluorescente<em&gt; In Situ</em&gt; Ibridazione (FISH)

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

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

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

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

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

Cromosoma Timing Replica combinazione con fluorescente<em&gt; In situ</em&gt; Ibridazione

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

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

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

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

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

Tutta la Monte RNA fluorescente<em&gt; In situ</em&gt; L&#39;ibridazione di<em&gt; Drosophila</em&gt; Embrioni

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

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.

Visualizzazione e l&#39;analisi di molecole di mRNA mediante fluorescenza<em&gt; In Situ</em&gt; Ibridazione 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 ...