Nous tenons à souligner la contribution de Dörthe Jülich, Jennifer et Andrew ronde Mara dans le développement de ce protocole. Soutien à la recherche fournis par le NICHD, l&#39;American Cancer Society et le Mars of Dimes.

1. FIXATION <ol><li> Fix embryons nuit à 4 ° C avec 4% de paraformaldéhyde (PFA) dans le PBS. </li><li> Retirer fixage et de lavage 2 x PBS, 5 minutes chacun à la température ambiante (RT). </li><li> Embryons manuellement dechorionate en PBS dans une plaque de verre de dépression en utilisant une pince horloger. Après dechorionation, les embryons sont transférés à l&#39;aide d&#39;un incendie poli pipette Pasteur en verre, car ils peuvent coller à Pipettes en polypropylène. </li><li> Transfert des embryons à travers une série de 25%, 50% et 75% de méthanol dans du PBS pendant 5 minutes chacune. </li><li> Remplacer liquide avec du méthanol à 100%, incuber 5 minutes, puis le remplacer par du méthanol frais. </li><li> Placez embryons à -20 ° C pour un minimum d&#39;une heure. (En général, nous incuber les embryons pendant la nuit. Standard d&#39;hybridation in situ travaillera sur des embryons conservés depuis plus d&#39;un an, mais nous n&#39;avons pas examiné si la haute résolution de l&#39;hybridation in situ fluorescente est perdu par un stockage prolongé.) </li><li> Lavez les embryons pendant 5 minutes chacun à 75%, 50%, 25% de méthanol dans du PBST à température ambiante. Laver deux fois pendant 5 minutes chacun dans du PBST à température ambiante. </li><li> Fixer à nouveau pendant 20 minutes dans 4% PFA dans du PBS à température ambiante. </li><li> Laver deux fois pendant 5 minutes chacun dans du PBST à température ambiante. </li></ol> NOTE RELATIVE PFA: Nous stockons PFA 4% en aliquots à -20 º C (voir tableau des réactifs). Pour la fixation initiale, nous n&#39;utilisons que des PFA qui n&#39;a jamais été préalablement décongelées. Pour fixations ultérieures, nous utilisons souvent l&#39;IFP qui a déjà été décongelé. La fixation initiale semble être critique pour la coloration réussie avec ce protocole. 2. Protéinase et POSTFIXATION <ol><li> Digest avec la protéinase K (5μg/ml en PBST) à température ambiante pendant 3 à 12 minutes à perméabiliser les embryons. (Le temps d&#39;incubation dépend de l&#39;âge des embryons plus jeunes stades sont plus sensibles. Elle dépend aussi du lot d&#39;enzyme.) Pour embryons au stade somitogenèse, nous avons généralement perméabiliser pendant 3-4 minutes. Pendant cette incubation, nous posons les microtube sur le côté. </li><li> Rincer brièvement dans PBST et laver une fois pendant 5 minutes dans du PBST. </li><li> Fixer à nouveau pendant 20 minutes dans 4% PFA dans du PBS à température ambiante. </li><li> Laver deux fois, pendant 5 minutes chacun, dans le PBST à température ambiante. </li></ol> 3. Préhybridation <ol><li> Incuber les embryons pendant 5 minutes à 65 º C en HYB-. </li><li> Prehybridize à 65 º C pendant au moins 1 heure dans HYB +. </li></ol> 4. HYBRIDATION <ol><li> Retirez tous, mais 50 pi de la preHYB, mais veillez à conserver les embryons complètement submergé. </li><li> Ajouter 1-2 ul de chaque ribosonde (digoxygénine et marquée à la fluorescéine ribosondes) pour les embryons et mélanger délicatement en effleurant le tube. Le montant de la sonde est généralement 1-2μl d&#39;une réaction de synthèse de sonde 20 pi. </li><li> Étant donné que la fluorescéine est sensible à la lumière, les tubes doivent être enveloppés dans du papier aluminium ou autrement exposé à la lumière minimale de ce point vers l&#39;avant. </li><li> Incuber les embryons nuit à 65 º C. </li></ol> 5. Sonde démontable Notez que les solutions de ce détergent manque point de l&#39;avant. L&#39;élimination de détergent apparaît pour aider les réactions de coloration, mais ne causent des embryons à devenir plutôt collante. <ol><li> Retirez la ribosonde. </li><li> Lavez 2 x 30 minutes à 65 º C en formamide/2xSSC 50%. </li><li> Laver pendant 15 minutes à 65 º C en 2 x SSC. </li><li> Laver pendant 30 minutes à 65 ° C dans 0,2 x SSC. </li></ol> 6. INCUBATION anticorps anti-fluorescéine <ol><li> Bloc pour au moins 1 heure à température ambiante dans 500μl d&#39;une solution de 1x tampon acide maléique en plus réactif 2% de blocage (voir tableau des réactifs). </li><li> Ajouter l&#39;anticorps anti-fluorescéine-POD, tel que fourni par Roche, à une dilution 1:500 dans la solution de blocage. </li><li> Incuber une nuit à 4 ° C. Pendant cette incubation, nous posons les microtube sur le côté. </li><li> Laver 4 x 20 minutes dans le tampon d&#39;acide maléique 1x. Laver deux fois pendant 5 minutes chacun dans du PBS. </li></ol> 7. DÉTECTION DE SONDE fluorescéine ÉTIQUETÉES <ol><li> Incuber 30-60 minutes dans le CST plus fluorescéine solution. (Spin bas substrat TSA avant de faire une solution de coloration. Pour la réaction, diluer réactif tyramide 1:50 dans Perkin Elmer tampon diluant d&#39;amplification.) Pendant cette incubation, nous posons les microtube sur le côté. Le temps de réaction doit être déterminée empiriquement pour chaque sonde. Malheureusement, la réaction de coloration ne peut pas être contrôlé visuellement que le substrat est fluorescent, et on verra omniprésents fluorescence verte à travers la réaction de coloration. </li><li> Laver pendant 10 minutes chacun à 30%, 50%, 75% et 100% de méthanol dans du PBS. </li><li> Incuber dans une solution de 1% de H 2 0 2 dans le méthanol pendant 30 minutes pour inactiver la peroxydase premier. </li><li> Laver 10 minutes chacune à 75%, 50% et 30% de méthanol dans du PBS. Puis laver deux fois pendant 10 minutes chacun dans du PBS. Il est important que tous les a rencontréHanol être enlevé. </li></ol> NOTES SUR L&#39;amplification du signal tyramide: Nous avons été en utilisant les kits de Perkin Elmer TSA. Nous constatons que les substrats Alexa-tyramide tache bien en utilisant le tampon d&#39;amplification Perkin Elmer diluant, mais nous n&#39;avons pas eu de succès en utilisant la mémoire tampon de coloration fournie avec les kits sondes Invitrogen / moléculaire. Enfin, nous avons constaté que la fluorescence de Cy5 est éliminée par suite de méthanol / H 2 O 2 traitements pendant la fluorescéine et Alexa-647 ne sont pas affectés. Cy3 peut également être affectée par le méthanol / H 2 O 2 traitement car il est structurellement liée à Cy5. Pour cette raison, Cy3 et Cy5 réactions TSA ne devrait être utilisé pour la réaction de coloration fluorescente dans une seconde double dans le protocole in situ. 8. INCUBATION ANTICORPS ANTI-digoxygénine <ol><li> Bloquer les embryons encore pour au moins 1 heure à température ambiante dans une solution de 1x tampon acide maléique majoré de 2% réactif de blocage. </li><li> Ajouter l&#39;anticorps anti-DIG POD tel que fourni par Roche à une dilution 1:1000 dans une solution de blocage ci-dessus </li><li> Incuber une nuit à 4 ° C. Pendant cette incubation, nous posons les microtube sur le côté. </li><li> Laver 4 x 20 minutes dans le tampon d&#39;acide maléique 1x. Laver deux fois pendant 5 minutes chacun dans du PBS. </li></ol> 9. DÉTECTION DE SONDE digoxygénine-ÉTIQUETÉES <ol><li> Incuber 30-60 minutes dans le CST plus Cy5 Solution (Spin bas substrat TSA avant de faire une solution de coloration. Pour la réaction, diluer réactif tyramide 1:50 dans le tampon d&#39;amplification de diluant) Pendant cette incubation, nous posons les microtube sur le côté. Le temps de réaction doit être déterminée empiriquement pour chaque sonde. </li><li> Laver trois fois pendant 10 minutes chacun dans du PBST. </li></ol> 10. COLORATION iodure de propidium <ol><li> Laver deux fois pendant 5 minutes chacun dans 2 x SSC. </li><li> Incuber embryons pendant 30 minutes à 37 ° C dans 50 ul SSCT 2 x avec 10 pl RNAse (pour une concentration finale de 100μg/ml). </li><li> Laver à six reprises de 3 minutes chacun dans 2 x SSC à température ambiante. </li><li> Tache embryons pendant 8 minutes dans une solution d&#39;iodure de propidium 330μg/ml dans 2 x SSC. </li><li> Laver à six reprises de 3 minutes chacun dans 2 x SSC à température ambiante. </li><li> Fix pendant 20 minutes dans 4% PFA dans du PBS à température ambiante. </li><li> Laver deux fois, pendant 5 minutes chacun, dans le PBST à température ambiante. </li></ol> 11. MONTAGE <ol><li> Incuber les embryons pendant 10 minutes chacun à 25% et 50% de glycérol dans le PBST. Effacer une nuit dans du glycérol 75% à 4 º C. </li><li> Nous disséquer et deyolk les embryons et les plats les monter sur une lame de microscope. Le jaune peut produire la fluorescence de fond important. La dissection est beaucoup plus facile après les embryons ont effacé ovenight dans gylcerol. </li></ol> SOLUTIONS: <table border="1"><tbody><tr><td> PBST </td><td> PBS plus Tween 0,1% </td><td></td></tr><tr><td> SSCT </td><td> SSC ainsi Tween 0,1% </td><td></td></tr><tr><td> HYB- </td><td> 50% de formamide 5xSSC 0,1% de Tween-20 </td><td></td></tr><tr><td> HYB + </td><td> HYB- 5mg/ml torula (levure) ARN 50μg/ml héparine </td><td style="width: 300px;"> L&#39;ARN est préparé torula par la protéinase K digestion des ARN avec subséquentes phénol, phénol-chloroforme-, et le chloroforme-extraction.The ARN est précipité et dissous dans l&#39;eau traitée au DEPC. </td></tr><tr><td> Une mémoire tampon x acide maléique </td><td> 150 mM d&#39;acide maléique, 100mm de NaCl (pH 7,5) </td><td></td></tr></tbody></table><img src="/files/ftp_upload/1229/Brend_Holley_Fig1.png" alt="Figure 1" width="600" height="688" /> Figure 1. Les résultats représentatifs de l&#39;ensemble du montage à double hybridation in situ fluorescente. (AC). Les images montrent une seule section confocale à travers la région postérieure d&#39;un embryon de poisson zèbre au stade de dix somites. La vue est dorsale antérieure vers le haut. Noyaux colorés avec de l&#39;iodure de propidium sont de couleur bleue. (A) ARNm deltaC été détectée en utilisant une ribosonde digoxygénine-étiquetés et TSA-Cy5 réactif. (B) ARNm transcrit à partir du gène HER1 a été détecté avec une ribosonde marquée à la fluorescéine et TSA-fluorescéine réactif. (C) La fusion des canaux vert et rouge identifie sans ambiguïté de l&#39;expression génique des régions distinctes ou de chevauchement des deux gènes. (D, E) Détail de l&#39;expression HER1 démontrant la haute résolution de cette procédure. Localisation subcellulaire des ARNm peut être clairement discernées. Pointes de flèches indiquent une cellule présentant transcription active, a révélé par des points de coloration dans le noyau. Les flèches indiquent une cellule montrant la localisation cytoplasmique des ARNm. (F, G) pour la double coloration HER1 et deltaC révèle les cellules qui sont à la fois la transcription des gènes (tête de flèche blanche), ou soit des gènes séparément (pointes de flèche rouge et vert).

<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>

Le protocole présenté ici fonctionne bien avec des sondes qui donnent un signal propre forte après coloration pendant 30-45 minutes dans une phosphatase alcaline médiée typiques de réaction. Avant de procéder à l&#39;fluorescente protocole d&#39;hybridation in situ, nous examinons toujours nos sondes en utilisant la norme non fluorescent protocole (fourni dans le matériel supplémentaire avec le protocole de synthèse de la sonde). Nous avons eu moins de succès avec les fluorescents protocole d&#39;hybridation...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<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>
		</table>
		</div>

zebrafish whole mount high-resolution double fluorescent in situ hybridization

Whole montage<em&gt; In situ</emL&#39;hybridation&gt; est l&#39;une des techniques les plus utilisées en biologie du développement. Ici, nous présentons une haute résolution à double fluorescentes<em&gt; In situ</emProtocole d&#39;hybridation&gt; pour analyser le profil d&#39;expression précise d&#39;un seul gène et de déterminer le chevauchement des domaines d&#39;expression de deux gènes. Le protocole est une version modifiée de la norme<em&gt; In situ</emL&#39;hybridation&gt; en utilisant la phosphatase alcaline et de substrats tels que NBT / BCIP et Fast-Rouge<sup&gt; 1,2</sup&gt;. Ce protocole utilise la norme sondes digoxygénine et marqué à la fluorescéine ainsi que l&#39;amplification du signal tyramide (TSA)<sup&gt; 3</sup&gt;. Les kits disponibles dans le commerce TSA permettent flexibles expérimentale de conception telle émission de fluorescence du vert au rouge lointain peut être utilisé en combinaison avec divers colorants nucléaires, comme l&#39;iodure de propidium, ou immunohistochimie de fluorescence pour les protéines. TSA produit un substrat réactif fluorescent rapidement covalente lie à des fragments, des résidus de tyrosine généralement, dans le voisinage immédiat de la ribosonde antisens étiquetés. L&#39;intensité de la coloration qui en résultent sont à haute résolution dans cette localisation subcellulaire des ARNm peut être observé en utilisant la microscopie confocale à balayage laser<sup&gt; 3,4</sup&gt;. On peut observer des transcriptions naissant au loci chromosomiques, de distinguer la coloration nucléaire et cytoplasmique et visualiser d&#39;autres modèles tels que la localisation corticale de l&#39;ARNm. Studies in<em&gt; Drosophile</em&gt; Indiquent qu&#39;environ 70% des ARNm présentent des modèles spécifiques de localisation subcellulaire qui souvent en corrélation avec la fonction de la protéine codée<sup&gt; 5</sup&gt;. Lorsqu&#39;il est combiné avec assistée par ordinateur reconstruction des ensembles de données 3D confocale, notre protocole permet l&#39;analyse détaillée de la distribution de l&#39;ARNm avec des sub-cellulaire de la résolution embryons de vertébrés ensemble.

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

Monter toute l&#39;hybridation in situ est l&#39;une des techniques les plus utilisées en biologie du développement. Ici, nous présentons une haute résolution double fluorescentes dans le protocole de l&#39;hybridation in situ pour analyser le profil d&#39;expression précise d&#39;un seul gène et de déterminer le chevauchement des domaines d&#39;expression de deux gènes. Nous incluons une iodure de propidium nucléaires contre-coloration pour mettre en évidence l&#39;organisation tissulaire.

Zebrafish entier Mont haute résolution double fluorescent In Situ Hybridation

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

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Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

23.1K vues.  Yale University. Monter toute l'hybridation in situ est l'une des techniques les plus utilisées en biologie du développement. Ici, nous présentons une haute résolution double fluorescentes dans le protocole de l'hybridation in situ pour analyser le profil d'expression précise d'un seul gène et de déterminer le chevauchement des domaines d'expression de deux gènes. Nous incluons une iodure de propidium nucléaires contre-coloration pour mettre en évidence l...

Vidéo: 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.

Double Mont entier hybridation in situ d&#39;embryons de poulet début

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

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Biologie

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.

Méthode de coloration Mont anticorps entier de 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.

Utilisation Mont entier<em&gt; In situ</emHybridation&gt; Lien vers biologie moléculaire et 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.

Whole mont In Situ</emHybridation> de E8.5 à E11.5 embryons de souris

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.

Analyse de la transcription génique unique cellule par l&#39;ARN fluorescent<em&gt; In Situ</emHybridation&gt; (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.

Timing chromosome Réplication Combiné avec fluorescent<em&gt; In situ</em&gt; Hybridation

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.

Toute Mont ARN fluorescent<em&gt; In situ</emHybridation&gt; d&#39;<em&gt; Drosophila</em&gt; Embryons

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.

ARN<em&gt; In situ</emHybridation&gt; en embryons entiers de montage et histologie Adapté pour les élasmobranches marins

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.

Haute Résolution Mont entier<em&gt; In Situ</em&gt; Hybridation dans les embryons de poisson zèbre pour étudier l&#39;expression des gènes et la fonction

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.

Analyse de l&#39;apoptose dans embryons de poissons zèbres par Whole montage immunofluorescence pour détecter caspase 3

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