Vorremmo ringraziare per i contributi di Dörthe Jülich, Jennifer e Andrew rotonda Mara nello sviluppo di questo protocollo. Sostenere la ricerca fornita dal NICHD, l&#39;American Cancer Society e la March of Dimes.

1. FISSAGGIO <ol><li> Fissare gli embrioni notte a 4 ° C con il 4% paraformaldeide (PFA) in PBS. </li><li> Rimuovere e lavare fissare 2 x PBS, 5 minuti ciascuno a temperatura ambiente (RT). </li><li> Embrioni manualmente dechorionate in PBS in un piatto depressione vetro con pinze orologiaio. Dopo dechorionation, gli embrioni vengono trasferiti utilizzando un incendio lucidato vetro pipetta Pasteur in quanto possono attenersi a pipette polipropilene. </li><li> Trasferimento di embrioni attraverso una serie di 25%, 50% e 75% di metanolo in PBS per 5 minuti ciascuno. </li><li> Sostituire il liquido con il 100% di metanolo, incubare 5 minuti e poi sostituirlo con metanolo fresco. </li><li> Luogo embrioni a -20 ° C per un minimo di un&#39;ora. (Noi di solito incubare gli embrioni durante la notte. Standard di ibridazione in situ lavorerà sugli embrioni conservati per più di un anno, ma non abbiamo esaminato se l&#39;alta risoluzione di ibridazione in situ fluorescente si perde di un immagazzinamento prolungato.) </li><li> Lavare gli embrioni per 5 minuti ciascuno nel 75%, 50%, 25% di metanolo in PBST a temperatura ambiente. Lavare due volte per 5 minuti ciascuno in PBST a temperatura ambiente. </li><li> Fissare di nuovo per 20 minuti in PFA 4% in PBS a temperatura ambiente. </li><li> Lavare due volte per 5 minuti ciascuno in PBST a temperatura ambiente. </li></ol> NOTE SUL PFA: Noi memorizzare 4% PFA in aliquote a -20 º C (vedi tabella dei reagenti). Per la fissazione iniziale, usiamo solo PFA che non è mai stato precedentemente scongelati. Per le fissazioni successive, che spesso utilizza PFA che è stato scongelato in precedenza. La fissazione iniziale sembra essere critica per la colorazione di successo con questo protocollo. 2. Proteinasi E POSTFIXATION <ol><li> Digerire con proteinasi K (5μg/ml in PBST) a temperatura ambiente per 3 a 12 minuti per permeabilize gli embrioni. (Il tempo di incubazione dipende dall&#39;età degli embrioni più giovani stadi sono più sensibili. Dipende anche il lotto di enzima.) Per gli embrioni fase somitogenesis, di solito permeabilize per 3-4 minuti. Durante questa incubazione, poniamo la provetta su un fianco. </li><li> Sciacquare brevemente in PBST e lavare una volta per 5 minuti in PBST. </li><li> Fissare di nuovo per 20 minuti in PFA 4% in PBS a temperatura ambiente. </li><li> Lavare due volte, per 5 minuti ciascuno, in PBST a temperatura ambiente. </li></ol> 3. PREHYBRIDIZATION <ol><li> Incubare gli embrioni per 5 minuti a 65 º C in HYB-. </li><li> Prehybridize a 65 ° C per almeno 1 ora in HYB +. </li></ol> 4. IBRIDAZIONE <ol><li> Rimuovere tutti ma il 50 microlitri della preHYB, ma assicurarsi di mantenere gli embrioni completamente sommerso. </li><li> Aggiungere 1-2 ml di ogni riboprobe (digossigenina e marcato con fluoresceina riboprobes) per gli embrioni e mescolare delicatamente sfogliando il tubo. La quantità di sonda è tipicamente 1-2μl da una reazione di 20μl sintesi sonda. </li><li> Dato che la fluoresceina è sensibile alla luce, i tubi dovrebbero essere avvolti in carta stagnola o comunque esposte alla luce minima da questo punto in avanti. </li><li> Incubare gli embrioni notte a 65 ° C. </li></ol> 5. SONDA RIMOZIONE Si noti che le soluzioni detergenti in avanti da questo punto mancano. Eliminazione di detersivo sembra aiutare le reazioni colorazione, ma provoca gli embrioni a diventare piuttosto appiccicosa. <ol><li> Rimuovere il riboprobe. </li><li> Lavare 2 x 30 minuti a 65 º C nel 50% formamide/2xSSC. </li><li> Lavare per 15 minuti a 65 º C in 2 x SSC. </li><li> Lavare per 30 minuti 65 ° C a 0,2 x SSC. </li></ol> 6. ANTICORPI ANTI-fluoresceina INCUBAZIONE <ol><li> Blocco per almeno 1 ora a temperatura ambiente in 500μl di una soluzione di 1x tampone acido maleico più reattivo 2% di blocco (vedi tabella dei reagenti). </li><li> Aggiungere l&#39;anti-fluoresceina-POD anticorpi, come fornito da Roche, a una diluizione 1:500 nella soluzione di blocco. </li><li> Incubare una notte a 4 ° C. Durante questa incubazione, poniamo la provetta su un fianco. </li><li> Lavare 4 x 20 minuti in 1x tampone acido maleico. Lavare due volte per 5 minuti ciascuno in PBS. </li></ol> 7. RILEVAZIONE DEI fluoresceina ETICHETTA SONDA <ol><li> Incubare per 30-60 minuti in soluzione TSA Plus. fluoresceina. (Spin giù substrato TSA prima di effettuare soluzione colorante. Per la reazione, reagente tyramide diluire 1:50 nel tampone diluente amplificazione Perkin Elmer.) Durante questa incubazione, poniamo la provetta su un fianco. Tempo di reazione deve essere determinato empiricamente per ogni sonda. Purtroppo, la reazione di colorazione non può essere visivamente monitorato come il substrato è fluorescente, e si vedrà onnipresente fluorescenza verde per tutta la reazione di colorazione. </li><li> Lavare per 10 minuti ciascuno nel 30%, 50%, 75% e 100% metanolo in PBS. </li><li> Incubare in una soluzione di 1% di H 2 0 2 in metanolo per 30 minuti per inattivare la perossidasi prima. </li><li> Lavare 10 minuti ciascuno nel 75%, 50% e 30% di metanolo in PBS. Poi lavare due volte per 10 minuti ciascuno in PBS. E &#39;importante che tutti gli incontratohanol essere rimossi. </li></ol> NOTE SUL amplificazione del segnale TYRAMIDE: Abbiamo utilizzato la Perkin Elmer Kit TSA. Troviamo che l&#39;Alexa-Tyramide substrati macchia e utilizzando l&#39;amplificazione Perkin Elmer tampone diluente, ma non abbiamo avuto successo utilizzando il buffer colorazione fornita con il kit di Invitrogen / sonde molecolari. Infine, abbiamo trovato che Cy5 fluorescenza viene eliminato dai successivi metanolo / H 2 O 2, mentre il trattamento con fluorescina e Alexa-647 non sono influenzati. Cy3 può anche essere influenzato negativamente dal metanolo / H 2 O 2 trattamento in quanto è strutturalmente correlata alla Cy5. Per questo motivo, Cy3 e Cy5 reazioni TSA deve essere utilizzato solo per la reazione di colorazione fluorescente secondo in una doppia protocollo in situ. 8. ANTICORPI ANTI-digossigenina INCUBAZIONE <ol><li> Bloccare gli embrioni ancora per almeno 1 ora a temperatura ambiente in una soluzione di 1x tampone acido maleico più 2% reagente di blocco. </li><li> Aggiungere l&#39;anticorpo anti-DIG POD come fornito dalla Roche a una diluizione 1:1000 sopra il blocco soluzione </li><li> Incubare una notte a 4 ° C. Durante questa incubazione, poniamo la provetta su un fianco. </li><li> Lavare 4 x 20 minuti in 1x tampone acido maleico. Lavare due volte per 5 minuti ciascuno in PBS. </li></ol> 9. RILEVAZIONE DEI digossigenina-MARCATO SONDA <ol><li> Incubare per 30-60 minuti in TSA Plus. Cy5 Solution (Spin giù substrato TSA prima di effettuare soluzione colorante. Per la reazione, reagente tyramide diluire 1:50 nel tampone di amplificazione diluente) Durante questa incubazione, poniamo le provetta su un fianco. Tempo di reazione deve essere determinato empiricamente per ogni sonda. </li><li> Lavare tre volte per 10 minuti ciascuno in PBST. </li></ol> 10. Ioduro di propidio COLORAZIONE <ol><li> Lavare due volte per 5 minuti ciascuno in 2 x SSC. </li><li> Incubare gli embrioni per 30 minuti a 37 ° C in 50 microlitri 2 x SSCT con 10 microlitri RNAse (per una concentrazione finale di 100μg/ml). </li><li> Lavare sei volte per 3 minuti ciascuno in 2 x SSC a temperatura ambiente. </li><li> Macchia embrioni per 8 minuti in una soluzione di ioduro di propidio 330μg/ml in 2 x SSC. </li><li> Lavare sei volte per 3 minuti ciascuno in 2 x SSC a temperatura ambiente. </li><li> Fix per 20 minuti in PFA 4% in PBS a temperatura ambiente. </li><li> Lavare due volte, per 5 minuti ciascuno, in PBST a temperatura ambiente. </li></ol> 11. MONTAGGIO <ol><li> Incubare gli embrioni per 10 minuti ciascuno nel 25% e 50% glicerolo in PBST. Chiaro durante la notte nel 75% glicerolo a 4 ° C. </li><li> Noi sezionare e deyolk gli embrioni e piatta montarle su un vetrino da microscopio. Il tuorlo può produrre significativi fluorescenza di fondo. Dissezione è molto più facile dopo che gli embrioni hanno eliminato ovenight in gylcerol. </li></ol> SOLUZIONI: <table border="1"><tbody><tr><td> PBST </td><td> PBS addizionato con lo 0,1% Tween </td><td></td></tr><tr><td> SSCT </td><td> SSC più Tween 0,1% </td><td></td></tr><tr><td> HYB- </td><td> 50% formamide 5xSSC 0,1% Tween-20 </td><td></td></tr><tr><td> HYB + </td><td> HYB- 5mg/ml torula (lievito) RNA 50μg/ml eparina </td><td style="width: 300px;"> L&#39;RNA torula si prepara digestione proteinasi K di RNA con conseguente-fenolo, fenolo-cloroformio, cloroformio e-extraction.The RNA è precipitato e disciolto in DEPC trattati con acqua. </td></tr><tr><td> 1 x tampone acido maleico </td><td> 150mm acido maleico, 100mM NaCl (pH 7,5) </td><td></td></tr></tbody></table><img src="/files/ftp_upload/1229/Brend_Holley_Fig1.png" alt="Figura 1" width="600" height="688" /> Figura 1. Risultati rappresentante del monte intero doppio ibridazione in situ fluorescente. (AC). Le immagini mostrano una singola sezione confocale attraverso la regione posteriore di un embrione di pesce zebra a dieci somite palco. La vista è dorsale con anteriore verso l&#39;alto. Macchiato nuclei con ioduro di propidio sono di colore blu. (A) deltaC mRNA è stato rilevato utilizzando un riboprobe digossigenina-etichettati e TSA-Cy5 reagente. (B) mRNA trascritto dal gene HER1 è stato rilevato con un riboprobe marcato con fluoresceina e TSA-fluoresceina reagente. (C) Unire i canali verde e rosso identifica in modo univoco le regioni di espressione genica distinto o sovrapposizione dei due geni. (D, E) Particolare HER1 espressione che dimostrano l&#39;alta risoluzione di questa procedura. Localizzazione subcellulare di mRNA può essere chiaramente discernere. Frecce indicano una cella espositrici trascrizione attivo, rivelato da punti di colorazione nel nucleo. Le frecce indicano una cella che mostra la localizzazione citoplasmatica di mRNA. (F, G) per doppia colorazione HER1 e deltaC rivela cellule che sono la trascrizione entrambi i geni (bianco punta di freccia), o sia gene separatamente (punte di freccia rossa e verde).

<ol>
	<li>Schulte-Merker, S., Ho, R. K., Herrmann, B. G., N&#252;sslein-Volhard, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+protein+product+of+the+zebrafish+homologue+of+the+mouse+T-gene+is+expressed+in+nuclie+of+the+germ+ring+and+the+notochord+of+the+early+embryo.">The protein product of the zebrafish homologue of the mouse T-gene is expressed in nuclie of the germ ring and the notochord of the early embryo.</a> Development. 116, 1021-1027 (1992).</li><li>Jowett, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double+in+situ+hybridization+techniques+in+zebrafish.">Double in situ hybridization techniques in zebrafish.</a> Methods. 23, 345-358 (2001).</li><li>J&#252;lich, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=beamter/deltaC+and+the+role+of+Notch+ligands+in+the+zebrafish+somite+segmentation,+hindbrain+neurogenesis+and+hypochord+differentiation.">beamter/deltaC and the role of Notch ligands in the zebrafish somite segmentation, hindbrain neurogenesis and hypochord differentiation.</a> Dev Biol. 286, 391-404 (2005).</li><li>Mara, A., Schroeder, J., Chalouni, C., Holley, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Priming,+Initiation+and+Synchronization+of+the+Segmentation+Clock+by+deltaD+and+deltaC.">Priming, Initiation and Synchronization of the Segmentation Clock by deltaD and deltaC.</a> Nat Cell Biol. 9, 523-530 (2007).</li><li>Lecuyer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+analysis+of+mRNA+localization+reveals+a+prominent+role+in+organizing+cellular+architecture+and+function.">Global analysis of mRNA localization reveals a prominent role in organizing cellular architecture and function.</a> Cell. 131, 174-187 (2007).</li></ol>

Il protocollo presentato qui funziona bene con le sonde che danno un forte segnale pulito dopo la colorazione per 30-45 minuti in una fosfatasi alcalina-mediata tipica reazione. Prima di eseguire il protocollo di ibridazione fluorescente in situ, abbiamo sempre testare le nostre sonde utilizzando lo standard non fluorescente protocollo (fornito in materiale supplementare con il protocollo di sintesi sonda). Abbiamo avuto meno successo con il protocollo di ibridazione fluorescente in situ utilizzando sonde più debole, m...

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		<div>
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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>Paraformaldehyde 16% solution, EM Grade</td>
<td>&nbsp;</td>
<td>Electron Microscopy Services</td>
<td>15710</td>
<td>Dilute to 4% in 1.33x PBS (final 1x). Store in 2ml aliquots at &#x2013;20&deg;C</td>
</tr>
<tr>
<td>Proteinase K, recombinant PCR grade</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>03115879001</td>
<td>Make a 20mg/ml stock solution in water. Store in 1ml aliquots at &#x2013;20&deg;C</td>
</tr>
<tr>
<td>Blocking Reagent</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11096176001</td>
<td>Make a 10% stock solution in 1x maleic acid buffer. Store in 50ml aliquots at &#x2013;20&deg;C. Stable over multiple freeze/thaws.</td>
</tr>
<tr>
<td>Anti-Fluorescein-POD, Fab fragments</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11426346910</td>
<td>Aliquot and store at -20&deg;C. Keep one working aliquot at 4&deg;C.</td>
</tr>
<tr>
<td>Anti-Digoxigenin-POD, Fab fragments</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11207739910</td>
<td>As above.</td>
</tr>
<tr>
<td>TSA Plus Fluorescein system</td>
<td>&nbsp;</td>
<td>Perkin Elmer</td>
<td>NEL741001KT</td>
<td>Prepare each vial of TSA reagent only when needed. Dissolve in 60&mu;l DMSO. Store at 4&deg;C.</td>
</tr>
<tr>
<td>TSA Plus Cyanine5 system</td>
<td>&nbsp;</td>
<td>Perkin Elmer</td>
<td>NEL745001KT</td>
<td>As above</td>
</tr>
<tr>
<td>TSA Plus Cyanine3 system</td>
<td>&nbsp;</td>
<td>Perkin Elmer</td>
<td>NEL744001KT</td>
<td>As above</td>
</tr>
<tr>
<td>TSA Kit #16 AlexaFluor647 tyramide (plus HRP-goat-anti-rabbit IgG)</td>
<td>&nbsp;</td>
<td>Molecular Probes /Invitrogen</td>
<td>T20926</td>
<td>Dissolve tyramide reagent in 150&mu;l DMSO, aliquot and store at &#x2013;20&deg;C.</td>
</tr>
<tr>
<td>RNase, DNase free</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11119915001</td>
<td>Supplied as 500&mu;g/ml solution</td>
</tr>
<tr>
<td>Propidium Iodide</td>
<td>&nbsp;</td>
<td>Molecular Probes /Invitrogen</td>
<td>P-3566</td>
<td>Supplied as 1mg/ml solution in water.</td>
</tr>		</tbody>
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zebrafish whole mount high-resolution double fluorescent in situ hybridization

Intero monte<em&gt; In situ</emIbridazione&gt; è una delle tecniche più utilizzate in biologia dello sviluppo. Qui, presentiamo una ad alta risoluzione doppio fluorescente<em&gt; In situ</em&gt; Protocollo di ibridazione per analizzare il modello preciso espressione di un singolo gene e per determinare la sovrapposizione dei domini di espressione di due geni. Il protocollo è una versione modificata dello standard<em&gt; In situ</em&gt; Ibridazione con fosfatasi alcalina e substrati come NBT / BCIP e Fast Red<sup&gt; 1,2</sup&gt;. Questo protocollo utilizza standard di sonde digossigenina e marcato con fluoresceina amplificazione del segnale tyramide (TSA)<sup&gt; 3</sup&gt;. I kit disponibili in commercio permettono TSA flessibili di progettazione sperimentale emissione di fluorescenza da verde a rosso-lontano può essere utilizzato in combinazione con diverse macchie di nucleare, come lo ioduro di propidio, o immunoistochimica fluorescenza per le proteine. TSA produce un substrato reattivo fluorescente che rapidamente si lega covalentemente alla combinazione di sostanze, residui di tirosina di solito, nelle immediate vicinanze del riboprobe antisenso etichettati. Il risultante pattern di colorazione sono ad alta risoluzione in quella localizzazione subcellulare del mRNA può essere osservato con microscopio confocale a scansione laser<sup&gt; 3,4</sup&gt;. Si può osservare trascrizioni nascente presso i loci cromosomici, distinguere colorazione nucleare e citoplasmatica e visualizzare altri modelli come ad esempio la localizzazione corticale di mRNA. Studi in<em&gt; Drosophila</em&gt; Indicano che circa il 70% di mRNA presentano modelli specifici di localizzazione subcellulare che spesso in correlazione con la funzione della proteina codificata<sup&gt; 5</sup&gt;. Quando combinato con il computer-aided ricostruzione di set di dati 3D confocale, il nostro protocollo permette l&#39;analisi dettagliata della distribuzione mRNA con risoluzione sub-cellulare negli embrioni dei vertebrati tutto.

Watch this Scientific Journal Video about Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization at JoVE.com

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Montare tutto ibridazione in situ è ​​una delle tecniche più utilizzate in biologia dello sviluppo. Qui, presentiamo una ad alta risoluzione doppio fluorescente in protocollo ibridazione in situ per analizzare il modello preciso espressione di un singolo gene e per determinare la sovrapposizione dei domini di espressione di due geni. Includiamo un propidio ioduro nucleare contro-macchia per evidenziare l&#39;organizzazione dei tessuti.

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 ...

zebrafish-whole-mount-high-resolution-double-fluorescent-situ

Research

JoVE Journal

Biology

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

23.1K Views.  Yale University. Montare tutto ibridazione in situ è ​​una delle tecniche più utilizzate in biologia dello sviluppo. Qui, presentiamo una ad alta risoluzione doppio fluorescente in protocollo ibridazione in situ per analizzare il modello preciso espressione di un singolo gene e per determinare la sovrapposizione dei domini di espressione di due geni. Includiamo un propidio ioduro nucleare contro-macchia per evidenziare l'organizzazione dei tessuti.

Video: Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

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

Method for Whole Mount Antibody Staining in Chick

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 antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

Metodo per la colorazione intero anticorpi Monte a Chick

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of specific mRNA transcripts within whole mount specimen. This technique (adapted from Albertson and Yelick, 2005) was used in an upper level undergraduate Comparative Vertebrate Biology laboratory classroom at Syracuse University. The first two thirds of the Comparative Vertebrate Biology lab course gave students the opportunity to study the embryology and gross anatomy of several organisms representing various chordate taxa primarily via traditional dissections and the use of models. The final portion of the course involved an innovative approach to teaching anatomy through observation of vertebrate development employing molecular techniques in which WISH was performed on zebrafish embryos. A heterozygous fibroblast growth factor 8 a (fgf8a) mutant line, ace, was used. Due to Mendelian inheritance, ace intercrosses produced wild type, heterozygous, and homozygous ace/fgf8a mutants in a 1:2:1 ratio. RNA probes with known expression patterns in the midline and in developing anatomical structures such as the heart, somites, tailbud, myotome, and brain were used. WISH was performed using zebrafish at the 13 somite and prim-6 stages, with students performing the staining reaction in class. The study of zebrafish embryos at different stages of development gave students the ability to observe how these anatomical structures changed over ontogeny. In addition, some ace/fgf8a mutants displayed improper heart looping, and defects in somite and brain development. The students in this lab observed the normal development of various organ systems using both external anatomy as well as gene expression patterns. They also identified and described embryos displaying improper anatomical development and gene expression (i.e., putative mutants).
For instructors at institutions that do not already own the necessary equipment or where funds for lab and curricular innovation are limited, the financial cost of the reagents and apparatus may be a factor to consider, as will the time and effort required on the part of the instructor regardless of the setting. Nevertheless, we contend that the use of WISH in this type of classroom laboratory setting can provide an important link between developmental genetics and anatomy. As technology advances and the ability to study organismal development at the molecular level becomes easier, cheaper, and increasingly popular, many evolutionary biologists, ecologists, and physiologists are turning to research strategies in the field of molecular biology. Using WISH in a Comparative Vertebrate Biology laboratory classroom is one example of how molecules and anatomy can converge within a single course. This gives upper level college students the opportunity to practice modern biological research techniques, leading to a more diversified education and the promotion of future interdisciplinary scientific research.

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Whole mount in situ hybridization (WISH) was used in an upper level undergraduate Comparative Vertebrate Biology course in addition to vertebrate dissections. This gave students the opportunity to study gene expression patterns as well as gross anatomy, linking the study of molecular and organismal biology within one course.

Utilizzando il monte intero In situ Ibridazione a Link Biologia Molecolare e organismal

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of ...

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures are lengthy and technically demanding with multiple important steps that collectively contribute to the quality of the final result. This protocol describes in detail several key quality control steps for optimizing probe labeling and performance. Overall, our protocol provides a detailed description of the critical steps necessary to reproducibly obtain high quality results. First, we describe the generation of digoxygenin (DIG) labeled RNA probes via in vitro transcription of DNA templates generated by PCR. We describe three critical quality control assays to determine the amount, integrity and specific activity of the DIG-labeled probes. These steps are important for generating a probe of sufficient sensitivity to detect endogenous mRNAs in a whole mouse embryo. In addition, we describe methods for the fixation and storage of E8.5-E11.5 day old mouse embryos for in situ hybridization. Then, we describe detailed methods for limited proteinase K digestion of the rehydrated embryos followed by the details of the hybridization conditions, post-hybridization washes and RNase treatment to remove non-specific probe hybridization. An AP-conjugated antibody is used to visualize the labeled probe and reveal the expression pattern of the endogenous transcript. Representative results are shown from successful experiments and typical suboptimal experiments.

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

This whole mount in situ hybridization protocol discusses critical steps that ensure reproducible high quality results for gene expression studies in E8.5-E11.5 day old mouse embryos.

Tutto il monte In Situ</emIbridazione> di E8.5 a E11.5 embrioni di topo

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures ...

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 ...

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression both in terms of tissue distribution and developmental stage. Protocols are described for the use of WISH of zebrafish embryos using antisense RNA probes labeled with digoxigenin. Probes are generated by incorporating digoxigenin-linked nucleotides through in vitro transcription of gene templates that have been cloned and linearized. The chorions of embryos harvested at defined developmental stages are removed before incubation with specific probes. Following a washing procedure to remove excess probe, embryos are incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase. By employing a chromogenic substrate for alkaline phosphatase, specific gene expression can be assessed. Depending on the level of gene expression the entire procedure can be completed within 2-3 days.

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

The zebrafish, a small tropical fish, has become a popular model for studying gene function during vertebrate development and disease. The temporal and spatial expression of target genes can be determined by in situ hybridization. Our improved protocol allows for the detection of low abundant transcripts with low non-specific background signal.

Alta Risoluzione intero monte<em&gt; In Situ</em&gt; Ibridazione in embrioni di zebrafish per studiare l&#39;espressione genica e la funzione

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression ...

Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. The whole-mount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning and/or colabeling is ultimately required to pinpoint the exact cell types undergoing apoptosis. The whole-mount Casp3 assay is optimized for analysis of fixed embryos between the 4-cell stage and 32 hr-post-fertilization and is useful for a number of applications, including analysis of zebrafish mutants and morphants, overexpression of mutant and wild-type mRNAs, and exposure to chemicals. Compared to acridine orange staining, which can identify apoptotic cells in live embryos in a matter of hours, Casp3 and TUNEL assays take considerably longer to complete (2-4 days). However, because of the dynamic nature of apoptotic cell formation and clearance, analysis of fixed embryos ensures accurate comparison of apoptotic cells across multiple samples at specific time points. We have also found the Casp3 assay to be superior to analysis of apoptotic cells by the whole-mount TUNEL assay in regard to cost and reliability. Overall, the Casp3 assay represents a robust, highly reproducible assay in which to analyze apoptotic cells in early zebrafish embryos.

Certain genetic perturbations or exposure to toxins can disrupt normal developmental processes leading to death of specific cell types. The analysis of activated Caspase 3 by whole-mount immunofluorescence in zebrafish embryos reveals stage- and tissue-specific localization of cells specifically undergoing apoptosis.

Analisi di apoptosi in embrioni di zebrafish da Whole-mount immunofluorescenza per rilevare caspasi 3 attivata

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis ...