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

1. Sonda Preparazione <ol><li> Ottenere più oligonucleotidi unici in Xist (oligonucleotidi 20-30 nucleotidi di lunghezza, 63-65 temperatura di fusione ºC, tabella 1), con una modifica 5&#39;-amino e sospendere in acqua. </li><li> Pool quantità equimolari di oligonucleotidi con 5&#39;-amino modifica (totale 4,5 mg) e l&#39;etichetta con colorante fluorescente ammina reattiva seguendo le istruzioni del produttore. <ol><li> Sciogliere gli oligonucleotidi pool in finale 5 ml di acqua priva di nucleasi e aggiungere 3 ml di 1 M bicarbonato di sodio alla soluzione oligonucleotide. </li><li> Aggiungere 2 microlitri DMSO al flaconcino di ammina reattiva tintura e vortex brevemente. </li><li> Miscelare la soluzione di oligonucleotide con il colorante reattivo in DMSO e incubare la miscela a temperatura ambiente per 1 ora al buio. </li></ol></li><li> Purificare le sonde oligonucleotidiche fluorescente di G-25 colonna seguita da precipitazione in etanolo. <ol><li&gt; Centrifuga G-25 colonne a 750 g per 1 minuto per rimuovere buffer di stoccaggio in eccesso. </li><li> Aggiungere l&#39;acqua priva di nucleasi 40 microlitri alla miscela etichettatura prodotta nel passaggio 1.2.3 e applicare alla colonna G-25. Centrifugare a 750 g per 2 minuti. </li><li> Aggiungere 50 ml di acqua priva di nucleasi, 10 microlitri 3 M di acetato di sodio (pH 5.2) e 250 ml di etanolo per la sonda purificato dal punto 1.3.2. Conservare a -80 ° C per 30 minuti e centrifugare a 21.000 xg per 15 min a 4 ° C. </li><li> Lavare gli oligonucleotidi precipitati una volta con 1 ml di etanolo al 70%. </li><li> Risospendere in 50 microlitri di acqua priva di nucleasi o TE (10 mM Tris-HCl, pH 7,5, 1 mM EDTA) per una concentrazione finale di circa 10 pM. </li></ol></li><li> Diluire le sonde oligonucleotidiche fluorescente ad una concentrazione finale di 250 nM con tampone di ibridazione (10% formammide, 2X SSC [NaCl 300 mM; citrato di sodio 30 mM]; 2 mg / ml BSA, 10% destrano solfato). NOTA: Con questo protocollo FISH veloce, oligonucleotide paccappatoi in Xist stati progettati ed utilizzati ad una concentrazione superiore al normale protocollo FISH con sonde oligonucleotidiche per abbreviare il tempo di incubazione per l&#39;ibridazione. </li></ol> 2. Far scorrere Preparazione NOTA: Le diapositive possono essere preparati per immuno-FISH utilizzando il mouse staminali embrionali (ES) o il corpo embrioide (EB) differenziando cellule 20, 21 da una loro crescita direttamente su un vetrino da poli-lisina-trattati o con cytospin con sospensione cellulare. Qui, viene mostrata la preparazione diapositiva Cytospin. <ol><li> Aspirare il mezzo di coltura in un piatto da 6 pozzetti. Lavare con PBS (temperatura ambiente), e aspirare a fondo. </li><li> Aggiungere 0,5 ml di tripsina al piatto 6 pozzetti, incubare per 5-10 minuti a 37 ° C. </li><li> Aggiungere 5 ml di terreno e disperdere le cellule pipettando gentilmente su e giù parecchie volte. </li><li> Trasferire le cellule in una provetta da 15 ml, centrifugare per 5 min a 200 xg a temperatura ambiente. </li><li> Eliminare il supporto did delicatamente sospendere le cellule in 2 ml di PBS. </li><li> Contare le cellule utilizzando emocitometro, e diluire sospensione cellulare a 4 x 10 5 cellule / ml di PBS. </li><li> Assemblare una diapositiva, una carta filtro e una camera con una clip e metterli nelle apposite fessure del rotore del cytospin. Aggiungere 250 ml di ogni campione preparato al punto 2.6 in ciascuna camera dei gruppi assemblati in cytospin. Centrifugare a 1500 rpm per 10 min a temperatura ambiente. </li><li> (10 TUBI mM, pH 6,8; NaCl 100 mm; 300 mM saccarosio; 3 mM MgCl 2) Fill 3 Coplin sul ​​ghiaccio con 40 ml ciascuna di tampone PBS, CSK ghiacciata, e tampone CSKT (tampone CSK con 0,5% Triton X-100), rispettivamente. </li><li> Dopo l&#39;cytospin è finito, trasferire rapidamente il vetrino in PBS freddo e incubare per 5 min. </li><li> Trasferire il vetrino nel buffer CSK ghiacciato e incubare per 1 min. </li><li> Trasferire il vetrino nel buffer CSKT ghiacciato e incubare per 5 min. </li><li> Trasferire il vetrino di nuovo in ghiacciata CSK buffer e incubare per 1 min. </li><li> Trasferire il vetrino in 4% paraformaldeide in PBS e incubare per 10 min a temperatura ambiente. </li></ol> 3. immunofluorescenza <ol><li> Porre i vetrini in una vaschetta Coplin con PBST (1x PBS con 0.1% Tween-20) per diversi secondi. </li><li> Porre i vetrini in un taglio che permette di buffer residuo a scorrere giù la diapositiva. Pulire con cura il liquido in eccesso dal vetrino e metterlo in una scatola pipetta punta vuota con acqua. Sovrapporre la slitta con 40 ml di soluzione bloccante (1x PBS, 1% BSA, 0,1% Tween-20), inserire delicatamente un coprioggetto e incubare per 10 minuti a temperatura ambiente. </li><li> Rimuovere il vetrino, rimuovere la soluzione di blocco, e aggiungere 40 ml di anticorpo primario Histone H3K27me3 diluito ad una concentrazione di 1: 500 in soluzione bloccante. Posizionare il coprioggetto posteriore della diapositiva e incubare per 30 min a temperatura ambiente. NOTA: Un inibitore RNase nella soluzione di blocco potrebbe essere utile per preservare RNA in questo RNA FISProtocollo H quando si utilizzano anticorpi policlonali. </li><li> Lavare il vetrino 2 volte in vasi pieni di Coplin PBST, per 5 minuti ciascuna, scuotendo delicatamente durante il lavaggio. </li><li> Porre i vetrini in un taglio che permette di buffer residuo a scorrere giù la diapositiva. Pulire con cura il liquido in eccesso e posizionare il vetrino nella scatola punta. Overlay con 40 ml di anticorpo secondario (1: 500) diluito in soluzione bloccante, posizionare un coprioggetto e incubare per 15 minuti al buio. Per le seguenti operazioni, mantenere la slitta al buio in ogni momento. </li><li> Lavare 2 volte in Coplin vasi con PBST come al punto 3.4. </li></ol> 4. RNA FISH con sonde oligonucleotidi <ol><li> Pre-riscaldare una scatola pipetta punta vuota con acqua sul fondo per evitare che scivoli di essiccazione a 37 ° C per l&#39;RNA FISH al punto 4.3. </li><li> Porre i vetrini in un taglio che permette di buffer residuo a scorrere giù la diapositiva. Pulire con cura il liquido in eccesso dal vetrino dal punto 3.6 e posizionare il vetrino nella casella pipetta-tip con water in basso. Aggiungere 500 pl di tampone di lavaggio FISH (10% formammide in 2x SSC) della diapositiva e incubare per 5 minuti a temperatura ambiente. </li><li> Rimuovere la soluzione di lavaggio FISH, aggiungere 8 ml di oligonucleotidi sul vetrino, inserire un vetrino, e trasferire la diapositiva di una scatola pipetta punta preriscaldata con acqua sul fondo; incubare a 37 ° C per 1-2 ore. </li><li> Mettere 3 vasetti pre-riscaldato Coplin in un bagno d&#39;acqua a 37 ° C con il tampone di lavaggio FISH, FISH tampone di lavaggio con DAPI, e 2x SSC. </li><li> Dopo 1-2 ore di incubazione per l&#39;RNA FISH, mettere il vetrino nel buffer di lavaggio FISH pre-riscaldato. </li><li> Incubare il vetrino per alcuni minuti fino a quando il vetrino cade della diapositiva. </li><li> Posizionare il vetrino nuovo nel tampone di lavaggio, e continuare incubazione per 5 min. </li><li> Trasferire i vetrini in un&#39;altra vaschetta Coplin con tampone di lavaggio FISH preriscaldata con 0,5 ng / ml DAPI e incubare per 5 min. </li><li> Trasferire i vetrini in un&#39;altra vaschetta Coplin con pre-riscaldato 2x SSC e incubare per 5 kmn. </li><li> Porre i vetrini in un taglio che permette di buffer residuo a scorrere giù la diapositiva. Pulire con cura il liquido in eccesso e posizionare il vetrino di nuovo in una scatola di punta secca. Aggiungere 4 ml di antifade con DAPI sul vetrino e mettere un coprioggetto. </li><li> Sigillare il coprioggetto con smalto e osservare al microscopio. La slitta può essere conservato a 4 ° C al buio per alcuni mesi. </li></ol>

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No conflicts of interest declared.

In questo lavoro, abbiamo presentato una rapida protocollo immuno-FISH, che richiede meno di 5 ore per completare la preparazione vetrino, Xist RNA FISH, e immunofluorescenza per H3K27me3. In confronto con l&#39;approccio generale immuno-FISH per Xist RNA rilevazione, che di solito ha bisogno di incubazione durante la notte per l&#39;RNA FISH, questo protocollo non solo riduce significativamente il tempo trascorso, ma migliora anche la sensibilità di immuno-FISH con sonde oligonucleotidi. P...

Mammalian cromosoma X inattivazione (XCI) è una strategia per compensare lo squilibrio nel dosaggio del gene X-linked tra XX e XY, in cui uno dei due cromosomi X nelle femmine è trascrizionalmente inattivato 1. Inattivazione del cromosoma X è un ottimo sistema modello per lungo RNA non codificante (lncRNA) di ricerca. Inattivazione del cromosoma X è regolata da più lncRNAs, ed è stato ampiamente studiato negli ultimi decenni per scoprire i meccanismi di crosstalk tra lncRNAs, la trascrizione, la struttura della cromatina e l&#39;organizzazione nucleare 2, 3. Il centro X inattivazione (XIC) situato sul cromosoma X è un locus genetico complesso formato da un certo numero di geni che producono RNA non codificanti 4. X-inattiva trascrizione specifico (Xist) lncRNA nei mammiferi eutherian è uno di questi lncRNA che svolge un ruolo cruciale nel cromosoma X inattivazione 5,6. Trascrizioni Xist circondano la posizione del future Xi per avviare inattivazione del cromosoma X, e appaiono come una nuvola quando visualizzato utilizzando RNA FISH; questa formazione è denominato &quot;Xist Cloud&quot; 7. Dal Xist RNA interagisce con vari enzimi che modificano la cromatina, co-localizzazione delle nuvole Xist con diverse modificazioni epigenetiche per cromatina silente e trascrizione repressiva si osserva durante cromosoma X inattivazione 8. Ad esempio, Xist RNA interagisce con Polycomb complesso repressione 2 (PRC2) che è responsabile per H3K27me3 e induce uno stato cromatina repressione 9. Il verificarsi della nuvola Xist sul Xi e la sua co-localizzazione con la modifica intensiva H3K27me3 rappresenta un paesaggio eterocromatina facoltativa del Xi 10,11. Tecniche di citogenetica, come il DNA / RNA FISH hanno percorso una lunga strada dal metodo tradizionale utilizzando sonde radiomarcate 12 alle tecniche più recenti e avanzate con maggiore sensibilità eimmagini fluorescenti utilizzando più sonde oligonucleotidiche 13,14. DNA / RNA FISH accoppiato con immunofluorescenza è stato regolarmente utilizzato come strumento citologico per comprendere l&#39;organizzazione spazio-temporale nucleare, localizzazione RNA, la struttura della cromatina e modifiche. La preparazione sonda più standard per RNA FISH implica l&#39;uso di plasmide o cromosomiche artificiale (BAC) cloni batterici e la loro successiva etichettatura o con traduzione nick o adescamento casuali 15. Tuttavia, quasi il 70% dei geni nei topi e il 40% dei geni nell&#39;uomo mostrano una sovrapposizione di senso e antisenso trascrizioni 16, quindi richiede un metodo FISH specifico strand per distinguere senso e antisenso trascrizioni. In vitro sonde RNA trascritti (riboprobe ) sono spesso utilizzati per specifici filamento di RNA FISH 17,18; Tuttavia, ciò comporta la preparazione di un clone plasmidico o prodotti di PCR con T7, SP6, T3 o promotore e riboprobes sintetizzare. Inoltre, ribosonde derivato da genomic DNA o cDNA contengono spesso regioni non-specifici e gli elementi ripetitivi, che si traducono in alto rumore di fondo. Un altro problema è che riboprobes, che sono a poche centinaia di nucleotidi di lunghezza e contengono più fluorofori, non possono penetrare efficacemente nel nucleo. Per aggirare questo, più sonde oligonucleotidiche marcate con brevi un unico fluoroforo alla fine sono stati sviluppati che hanno una buona sensibilità, potenza del segnale uniforme, e la facilità di purificazione e movimentazione 14. Inoltre, gli oligonucleotidi di DNA sono generalmente più stabile di RNA. Abbiamo applicato una simile strategia di utilizzo di oligonucleotidi in Xist RNA FISH 19 con immunofluorescenza per comprendere le dinamiche epigenetici del Xi indotte da Xist lncRNA durante il processo di inattivazione del cromosoma X. Questo protocollo descrive la creazione di sonde oligonucleotidiche e corretta preparazione di cellule, così come l&#39;utilizzo di immunofluorescenza e RNA FISH. Xist RNA FISH utilizzando oligonucle multiplaotides è economicamente efficiente nel lungo termine se Xist RNA FISH viene eseguita di routine nel proprio laboratorio. Questa tecnica può essere utilizzata per identificare lncRNAs nelle cellule e contemporaneamente mappatura sua co-localizzazione con modificazioni epigenetiche o fattori. Uno dei principali vantaggi del protocollo è la capacità di modificare facilmente per soddisfare i propri interessi di ricerca.

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

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

Combinando RNA ibridazione in situ fluorescente (FISH) con immunofluorescenza (immuno-FISH) crea una tecnica che può essere impiegata a livello di singola cellula per rilevare la dinamica spaziale della localizzazione RNA con visione simultanea nella localizzazione delle proteine, modificazioni epigenetiche e altri dettagli che può essere evidenziato mediante immunofluorescenza. Inattivazione del cromosoma X è un paradigma per lungo RNA non codificante (lncRNA) mediata silenziamento genico. Trascrizione specifico (Xist) accumulo lncRNA X-inattiva (chiamato nuvola Xist) su uno dei due cromosomi X nelle femmine di mammifero è un passo fondamentale per avviare l&#39;inattivazione del cromosoma X. Xist RNA direttamente o indirettamente interagisce con vari enzimi che modificano la cromatina e introduce distinti paesaggi epigenetici alla inattiva cromosoma X (Xi). Una nota caratteristica epigenetica del Xi è l&#39;istone H3 trimetil-lisina 27 (H3K27me3) modifica. Qui, descriviamo un immuno-FISH semplice e veloceprotocollo per la rilevazione Xist RNA utilizzando RNA FISH con sonde multiple oligonucleotide accoppiato con immunofluorescenza di H3K27me3 per esaminare la localizzazione di Xist RNA e modificazioni epigenetiche associate. Utilizzando sonde oligonucleotidiche risultati in un tempo di incubazione più breve e la rilevazione più sensibile di Xist RNA rispetto alle sonde di RNA trascritto in vitro (ribosonde). Questo protocollo fornisce un potente strumento per comprendere le dinamiche di lncRNAs e la sua modificazione epigenetica associato, la struttura della cromatina, l&#39;organizzazione nucleare e regolazione trascrizionale.

Immagini rappresentative della rapida immuno-FISH sono mostrati nella Figura 1A. Co-localizzazione del segnale Xist RNA nuvola e H3K27me3 sul Xi è stato rilevato nel differenziare cellule femminili. Al giorno 12 sulla differenziazione, più del 90% delle cellule EB aveva una nuvola Xist (Figura 1B). Sonde oligonucleotidiche Breve efficientemente penetrati nei nuclei, portando ad una visualizzazione di quasi tutti H3K27me3 segnali co-localizzato con Xist RNA (Figura 1C;...

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

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

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

Veloce fluorescente<em&gt; In Situ</em&gt; Protocollo di ibridazione per Xist RNA combinata con immunofluorescenza di istone modifica in cromosoma X inattivazione

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

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

Research

JoVE Journal

Biology

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

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

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

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

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

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.

Non radioattivo In situ Protocollo di ibridazione applicabile per la Norvegia Abete e una serie di specie di piante

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

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

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

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

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

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 ...

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 ...

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

Marine elasmobranchs are valued animal models for biomedical and genomic studies as they are the most primitive vertebrates to have adaptive immunity and have unique mechanisms for osmoregulation 1-3. As the most primitive living jawed-vertebrates with paired appendages, elasmobranchs are an evolutionarily important model, especially for studies in evolution and development. Marine elasmobranchs have also been used to study aquatic toxicology and stress physiology in relationship to climate change 4. Thus, development and adaptation of methodologies is needed to facilitate and expand the use of these primitive vertebrates to multiple biological disciplines. Here I present the successful adaptation of RNA whole mount in situ hybridization and histological techniques to study gene expression and cell histology in elasmobranchs.
Monitoring gene expression is a hallmark tool of developmental biologists, and is widely used to investigate developmental processes 5. RNA whole mount in situ hybridization allows for the visualization and localization of specific gene transcripts in tissues of the developing embryo. The expression pattern of a gene's message can provide insight into what developmental processes and cell fate decisions a gene may control. By comparing the expression pattern of a gene at different developmental stages, insight can be gained into how the role of a gene changes during development.
While whole mount in situ's provides a means to localize gene expression to tissue, histological techniques allow for the identification of differentiated cell types and tissues. Histological stains have varied functions. General stains are used to highlight cell morphology, for example hematoxylin and eosin for general staining of nuclei and cytoplasm, respectively. Other stains can highlight specific cell types. For example, the alcian blue stain reported in this paper is a widely used cationic stain to identify mucosaccharides. Staining of the digestive tract with alcian blue can identify the distribution of goblet cells that produce mucosaccharides. Variations in mucosaccharide constituents on short peptides distinguish goblet cells by function within the digestive tract 6. By using RNA whole mount in situ's and histochemical methods concurrently, cell fate decisions can be linked to gene-specific expression.
Although RNA in situ's and histochemistry are widely used by researchers, their adaptation and use in marine elasmobranchs have met limited and varied success. Here I present protocols developed for elasmobranchs and used on a regular basis in my laboratory. Although further modification of the RNA in situ's hybridization method may be needed to adapt to different species, the protocols described here provide a strong starting point for researchers wanting to adapt the use of marine elasmobranchs to their scientific inquiries.

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

By combining methods for RNA whole mount in situ hybridization and histology, gene expression can be linked with cell fate decisions in the developing embryo. These methods have been adapted to marine elasmobranchs and facilitate the use of these animals as model organisms for biomedical, toxicology and comparative studies.

RNA<em&gt; In situ</emIbridazione&gt; in embrioni Mount interi e istologia Adattato per Elasmobranchi Marine

Marine  elasmobranchs are valued animal models for biomedical and genomic studies as  they are the most primitive vertebrates to have adaptive immunity ...