Biz bu protokolü geliştirme Dörthe Jülich, Jennifer Yuvarlak ve Andrew Mara katkılarını kabul etmek istiyorum. NICHD, Amerikan Kanser Derneği ve Dimes Mart tarafından sağlanan araştırma desteği.

1. FİKSASYON <ol><li> Fix 4 geceleme embriyolar ° C PBS içinde% 4 paraformaldehid (PFA). </li><li> Kaldır düzeltmek ve oda sıcaklığında (RT) 2 x PBS, her 5 dakika yıkayın. </li><li> Saatçi forseps kullanarak bir bardak depresyon plaka PBS Manuel dechorionate embriyolar. Polipropilen pipetler yapışabilir dechorionation sonra, embriyolar bir yangın parlatılmış cam Pasteur pipeti kullanılarak aktarılır. </li><li> Her 5 dakika için bir dizi% 25,% 50 ve PBS içinde% 75 metanol ile embriyo transferine. </li><li> % 100 metanol ile sıvı değiştirin, inkübasyon ve 5 dakika sonra taze metanol ile değiştirin. </li><li> En az bir saat için -20 ° C&#39;de Yeri embriyolar. (Biz genellikle in situ hibridizasyon Standart bir yıldan daha fazla süre saklanan embriyolar üzerinde çalışacak. Gecede embriyoların inkübe, ancak biz değil, uzun süreli depolama floresan in situ hibridizasyon yüksek çözünürlüklü kayıp olup olmadığı incelenir.) </li><li> 5 dakika% 75,% 50,% 25 RT PBST metanol embriyolar yıkayın. RT az 5 dakika PBST içinde her biri için iki kez yıkayın. </li><li> 4 RT PBS içinde% PFA 20 dakika için tekrar sabitleyin. </li><li> RT az 5 dakika PBST içinde her biri için iki kez yıkayın. </li></ol> PFA İLGİLİ NOT: (Reaktiflerin tabloya bakınız), -20 º C&#39;de alikotları% 4 PFA depolar. Ilk sabitleme için, biz sadece daha önceden çözülmüş olmamıştı PFA kullanabilirsiniz. Sonraki tespitler, genellikle daha önceden çözülmüş olduğunu PFA kullanmak. Ilk fiksasyon bu protokol ile başarılı bir şekilde boyanması için kritik görünüyor. 2. Proteinaz ve Postfiksasyon <ol><li> Embriyoların permeabilize 3 ila 12 dakika RT proteinaz K (PBST içinde 5μg/ml) ile Digest. (Inkübasyon süresi, genç aşamalarında daha duyarlı olmaları nedeniyle embriyoların yaşına bağlıdır. Aynı zamanda, enzim toplu bağlıdır.) Somitogenesis dönem embriyoların için, genellikle 3-4 dakika permeabilize. Bu İnkübasyon sırasında, mikrosantrifüj tüp yan yattı. </li><li> PBST kısaca durulayın ve PBST 5 dakika için bir kez yıkayın. </li><li> 4 RT PBS içinde% PFA 20 dakika için tekrar sabitleyin. </li><li> RT PBST, her 5 dakika için, iki kez yıkayın. </li></ol> 3. PREHYBRIDIZATION <ol><li> Embriyoların 65 º C&#39;de 5 dakika inkübe HYB. </li><li> HYB en az 1 saat 65 º C&#39;de Prehybridize. </li></ol> 4. Melezleme <ol><li> Ama preHYB 50 ul çıkarın, ancak tamamen sular altında embriyolar tutmak için emin olun. </li><li> Tüp hafifçe Flicking embriyo ve karıştırmak için her Spastine Özgü Riboprop 1-2 ul (digoxygenin ve floresein etiketli riboprobes) ekleyin. Prob miktarı genellikle 1-2μl 20μl prob sentez reaksiyonu. </li><li> Floresein ışığa duyarlı olduğu göz önüne alındığında, tüpler alüminyum folyo ile sarılmış veya başka bir şekilde bu noktadan itibaren en az ışığa maruz olmalıdır. </li><li> Embriyoların gece 65 º C&#39;de inkübe </li></ol> 5. PROBE GİDERİLMESİ Bu noktadan ileriye eksikliği deterjan çözümler unutmayın. Deterjan giderilmesi boyama reaksiyonları yardımcı olmak için görünür ama embriyoların oldukça yapışkan hale neden. <ol><li> Spastine Özgü Riboprop çıkarın. </li><li> 65 º C&#39;de% 50 formamide/2xSSC içinde 2 x 30 dakika yıkayın. </li><li> 2 x SSC 65 º C&#39;de 15 dakika süreyle yıkayın. </li><li> 0.2 x SSC - 65 º C &#39;de 30 dakika süreyle yıkayın. </li></ol> 6. ANTİ-floresein ANTİKOR KULUÇKA <ol><li> 1x maleik asit tampon artı% 2 engelleme reaktifi (reaktiflerin tabloya bakınız) bir çözüm 500μl oda sıcaklığında en az 1 saat Blok. </li><li> Roche tarafından verilen anti-Floresein-POD antikor, 1:500 dilüsyon engelleme çözüm ekleyin. </li><li> 4 º C&#39;de bir gece inkübe Bu İnkübasyon sırasında, mikrosantrifüj tüp yan yattı. </li><li> 1x maleik asit tampon 4 x 20 dakika yıkayın. PBS içinde her 5 dakika için iki kez yıkayın. </li></ol> 7. Floresein Etiketli SONDA VE ALGILAMA <ol><li> TSA Plus Floresein Çözüm 30-60 dakika inkübe edin. (Reaksiyon için boyama çözüm yapmadan önce TSA substrat aşağı Spin tyramide reaktif Perkin Elmer amplifikasyon seyreltici tampon 01:50 sulandırmak.) Bu kuluçka sırasında, yan mikrosantrifüj tüp yatıyordu. Reaksiyon zamanı, her sonda için ampirik olarak tespit edilmelidir. Ne yazık ki, boyama reaksiyon yüzey floresan gibi görsel olarak izlenebilir edemez ve bir boyama reaksiyon boyunca her yerde yeşil floresan göreceksiniz. </li><li> % 30,% 50,% 75 ve% 100 PBS içinde metanol her biri için 10 dakika yıkayın. </li><li> Ilk peroksidaz inaktive etmek için 30 dakika boyunca metanol,% 1 H 2 0 2 bir çözüm inkübe edin . </li><li> % 75,% 50 ve% 30 metanol PBS içinde her biri 10 dakika yıkayın. Sonra her biri 10 dakika PBS içinde iki kez yıkayın. Bu önemli olduğunu görüştü, tümhanol kaldırıldı. </li></ol> TYRAMIDE SİNYAL YÜKSELTME İLE İLGİLİ NOTLAR: Biz Perkin Elmer TSA Kitleri kullanıyoruz. Alexa-Tyramide yüzeyler Perkin Elmer amplifikasyon seyreltici tampon kullanarak leke olduğunu bulmak, ama biz Invitrogen / Moleküler Problar Kitleri ile sağlanan boyama tampon kullanarak başarılı olmadı. Son olarak, floresein ve Alexa-647 etkilenmez Cy5 floresan sonraki Metanol / H 2 O 2 tedavisi ile ortadan olduğunu bulduk. Cy3 yapısal Cy5 ile ilgili olarak Metanol / H 2 O 2 tedavisi de olumsuz etkilenebilir. Bu nedenle, Cy3 ve Cy5 TSA reaksiyonlar sadece bir çift floresan in situ protokolde ikinci boyama reaksiyon için kullanılan olmalıdır. 8. ANTİ-Digoxygenin ANTİKOR KULUÇKA <ol><li> 1x maleik asit tampon artı% 2 engelleme reaktif bir çözüm oda sıcaklığında en az 1 saat embriyoların tekrar engelleyin. </li><li> Yukarıdaki çözüm engelleme 1:1000 dilüsyon Roche tarafından verilen anti-DIG POD antikor ekle </li><li> 4 º C&#39;de bir gece inkübe Bu İnkübasyon sırasında, mikrosantrifüj tüp yan yattı. </li><li> 1x maleik asit tampon 4 x 20 dakika yıkayın. PBS içinde her 5 dakika için iki kez yıkayın. </li></ol> 9 - Digoxygenin Etiketli SONDA VE ALGILAMA <ol><li> (TSA substrat aşağı boyama çözüm yapmadan önce Spin reaksiyonu, amplifikasyon seyreltici tampon tyramide reaktif 01:50 sulandırmak) TSA Plus Cy5 Çözüm 30-60 dakika inkübe bu kuluçka sırasında, yan mikrosantrifüj tüp yatıyordu. Reaksiyon zamanı, her sonda için ampirik olarak tespit edilmelidir. </li><li> PBST içinde her 10 dakikada üç kez yıkayın. </li></ol> 10 Propidium iyodür BOYAMA <ol><li> 2 x SSC her 5 dakika için iki kez yıkayın. </li><li> 37 az 30 dakika boyunca inkübe embriyolar ° C, 10 ul RNAse (100μg/ml son bir konsantrasyon için) ile 50 ul 2 x SSCT. </li><li> RT az 2 x SSC her 3 dakikada altı kez yıkayın. </li><li> 8 dakika 2 x SSC 330μg/ml propidium iyodür bir çözüm için embriyolar Leke. </li><li> RT az 2 x SSC her 3 dakikada altı kez yıkayın. </li><li> 4 oda sıcaklığında PBS içinde% PFA 20 dakika saptamak için. </li><li> RT PBST, her 5 dakika için, iki kez yıkayın. </li></ol> 11. MONTAJ <ol><li> Embriyoların her biri% 25 ve PBST% 50 gliserol 10 dakika inkübe edin. 4 º C&#39;de% 75 gliserol gecede temizleyin </li><li> Biz incelemek ve deyolk embriyoların ve düz bir mikroskop lamı üzerine monte. Yumurta sarısı önemli bir eşiğe üretebilir. Diseksiyon embriyolar gylcerol ovenight temizlenir sonra çok daha kolay. </li></ol> ÇÖZÜMLER: <table border="1"><tbody><tr><td> PBST </td><td> PBS artı% 0.1 Tween </td><td></td></tr><tr><td> SSCT </td><td> SSC artı% 0.1 Tween </td><td></td></tr><tr><td> HYB- </td><td> 50% formamid 5xSSC % 0.1 Tween-20 </td><td></td></tr><tr><td> HYB + </td><td> HYB- 5mg/ml Torula (maya) RNA 50μg/ml heparin </td><td style="width: 300px;"> Torula RNA RNA proteinaz K sindirimi tarafından hazırlanan sonraki fenol, fenol-kloroform ve kloroform-extraction.The RNA çöktürülmüş ve DEPC arıtılmış su içinde çözünmüş. </td></tr><tr><td> 1 x maleik asit tampon </td><td> 150mm maleik asit, 100 mM NaCl (pH 7.5) </td><td></td></tr></tbody></table><img src="/files/ftp_upload/1229/Brend_Holley_Fig1.png" alt="Şekil 1" width="600" height="688" /> Şekil 1 tüm montaj Temsilcisi sonuçları çift floresan in situ hibridizasyon. (AC). Görüntüler zebrafish on hücre gruplarının aşamada embriyo posterior bölge üzerinden tek bir konfokal bölümünde gösteriyor. Görünümü, üst anterior ile birlikte dorsal. Propidium iyodür ile Çekirdekler boyanmış mavi renkli. (A) deltaC mRNA bir digoxygenin etiketli Spastine Özgü Riboprop ve TSA-Cy5 reaktif kullanılarak tespit edildi. (B) her1 gen transkripsiyonu mRNA bir floresein etiketli Spastine Özgü Riboprop ve TSA-floresein reaktifi ile tespit edildi. (C) net bir şekilde yeşil ve kırmızı kanalları birleştirme iki gen veya örtüşen farklı gen ifade bölgeleri tanımlar. (D, E) Bu prosedürün yüksek çözünürlük göstermektedir. Her1 ifade Detay MRNA hücre içi lokalizasyonu açıkça ayırt edilebilir. Arrowheads çekirdeğinde boyama noktalar ortaya koyduğu aktif transkripsiyon sergileyen bir hücre göstermektedir. Oklar mRNA sitoplazmik lokalizasyon gösteren bir hücre göstermektedir. Her1 ve deltaC için (F, G) Çift boyama, hem genler (beyaz ok ucu), ya da ayrı ayrı gen (kırmızı ve yeşil ok başları) transkripsiyonu hücreleri ortaya koymaktadır .

<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>

Burada sunulan protokol tipik bir alkalin fosfataz aracılı reaksiyon 30-45 dakika boyandıktan sonra temiz ve güçlü bir sinyal vermek problar ile iyi çalışır. Floresan in situ hibridizasyon protokolü performans öncesinde, her zaman bizim probları standart floresan olmayan protokolü (prob sentezi protokolü ile birlikte tamamlayıcı malzeme) kullanarak test edin. Zayıf problar kullanarak, floresan in situ hibridizasyon protokol az başarı oldu, ama bazı durumlarda mümkün olabilir. Β-katenin 3</su...

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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>
		</thead>
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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

Tüm montaj<em&gt; In situ</em&gt; Hibridizasyon gelişim biyolojisi en yaygın olarak kullanılan teknikleri biridir. Burada, yüksek çözünürlüklü bir çift floresan mevcut<em&gt; In situ</em&gt; Hibridizasyon, tek bir genin kesin ifadelerde desen analiz ve iki gen ifadesi etki çakışma belirlenmesi için protokol. Protokol, standart modifiye edilmiş bir versiyonu<em&gt; In situ</em&gt; Hibridizasyon kullanarak alkalin fosfataz ve NBT / BCIP ve Hızlı Kırmızı gibi yüzeylerde<sup&gt; 1,2</sup&gt;. Bu protokol tyramide sinyal amplifikasyon (TSA) ile birlikte standart digoxygenin ve flöresein etiketli probları kullanır<sup&gt; 3</sup&gt;. Piyasada bulunan TSA kitleri uzak kırmızı yeşil floresan emisyon olarak esnek bir deneysel tasarım propidium iyodür veya proteinler için floresan immünohistokimya gibi çeşitli nükleer lekeleri ile birlikte kullanılabilir izin verir. TSA hızlı kovalent etiketli antisens Spastine Özgü Riboprop hemen yakınında, moieties genellikle tirozin kalıntılarının bağlanır reaktif bir floresan substrat üretir. Konfokal mikroskobi lazer tarama kullanarak boyanma mRNA bu hücre içi lokalizasyonu yüksek çözünürlükte görülebilir<sup&gt; 3,4</sup&gt;. Bir kromozom bölgeleri, doğmakta olan transkript gözlemlemek nükleer ve sitoplazmik boyanma ayırt ve mRNA kortikal lokalizasyon gibi diğer desen görselleştirmek. Yapılan çalışmalar<em&gt; Drosophila</em&gt; MRNA&#39;ların kabaca% 70, sık sık kodlanmış proteinin fonksiyonu ile korelasyon Subselüler localisation özel kalıplara uyduğunu gösterir<sup&gt; 5</sup&gt;. 3D konfokal veri setleri, bilgisayar destekli bir yeniden yapılanma ile birlikte kullanıldığında, bütün omurgalı embriyolarının alt hücresel çözünürlük ile mRNA dağıtım protokolü detaylı analiz sağlar.

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

In situ hibridizasyon Tüm montaj, gelişim biyolojisi en yaygın olarak kullanılan teknikleri biridir. Burada, tek bir genin kesin ifadelerde desen analiz ve iki genin ifadesi etki çakışma belirlenmesi için çift floresan in situ hibridizasyon protokol yüksek çözünürlüklü sunuyoruz. Biz bir doku organizasyon vurgulamak için propidium iyodür nükleer karşı leke içerir.

Zebra balığı Tüm Dağı Yüksek Çözünürlüklü Çift Floresan Yerinde Hibridizasyon

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

23.2K Görüntüleme.  Yale University. In situ hibridizasyon Tüm montaj, gelişim biyolojisi en yaygın olarak kullanılan teknikleri biridir. Burada, tek bir genin kesin ifadelerde desen analiz ve iki genin ifadesi etki çakışma belirlenmesi için çift floresan in situ hibridizasyon protokol yüksek çözünürlüklü sunuyoruz. Biz bir doku organizasyon vurgulamak için propidium iyodür nükleer karşı leke içerir.

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.

Erken Civciv Embriyolar in situ Hibridizasyon Çift Tüm Dağı

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

Biyoloji

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.

Chick Tüm Dağı Antikor Boyama Yöntemi

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.

Tüm Dağı In situ Moleküler Biyoloji ve organizma Biyoloji Link Hibridizasyon

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

Polarized Translocation of Fluorescent Proteins in Xenopus Ectoderm in Response to Wnt Signaling

Cell polarity is a fundamental property of eukaryotic cells that is dynamically regulated by both intrinsic and extrinsic factors during embryonic development 1, 2. One of the signaling pathways involved in this regulation is the Wnt pathway, which is used many times during embryogenesis and critical for human disease3, 4, 5. Multiple molecular components of this pathway coordinately regulate signaling in a spatially-restricted manner, but the underlying mechanisms are not fully understood. Xenopus embryonic epithelial cells is an excellent system to study subcellular localization of various signaling proteins. Fluorescent fusion proteins are expressed in Xenopus embryos by RNA microinjection, ectodermal explants are prepared and protein localization is evaluated by epifluorescence. In this experimental protocol we describe how subcellular localization of Diversin, a cytoplasmic protein that has been implicated in signaling and cell polarity determination6, 7 is visualized in Xenopus ectodermal cells to study Wnt signal transduction8. Coexpression of a Wnt ligand or a Frizzled receptor alters the distribution of Diversin fused with red fluorescent protein, RFP, and recruits it to the cell membrane in a polarized fashion 8, 9. This ex vivo protocol should be a useful addition to in vitro studies of cultured mammalian cells, in which spatial control of signaling differs from that of the intact tissue and is much more difficult to analyze.

Polarized Translocation of Fluorescent Proteins in Xenopus Ectoderm in Response to Wnt Signaling

Xenopus embryonic ectoderm has become an attractive model for studies of cell polarity. An assay is described, in which subcellular distribution of fluorescent proteins is assessed in ectoderm cells. This protocol will help address questions related to spatial control of signaling.

Floresan Proteinleri Polarize Translokasyon Xenopus Wnt Sinyalizasyonu Cevabı Ektoderm

Cell polarity is a fundamental property of eukaryotic cells that is dynamically regulated by both intrinsic and extrinsic factors during embryonic ...

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.

Tüm Dağı<em&gt; In situ</emE11.5 fare embriyolarına E8.5 ve&gt; Hibridizasyon

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.

RNA Floresan tarafından Tek hücreli Gen Transkripsiyon Analizi<em&gt; In Situ</em&gt; Hibridizasyon (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.

Floresan ile birlikte Kromozom çoğaltılıyor Zamanlama<em&gt; Yerinde</em&gt; Hibridizasyon

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.

Tüm Dağı RNA Floresan<em&gt; In situ</emVe&gt; Hibridizasyon<em&gt; Drosophila</em&gt; Embriyolar

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; Yerinde</emTüm Dağı Embriyolar ve Hücre histolojisi&gt; Hibridizasyon Deniz Elasmobranchs İçin Uyarlanmış

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.

Yüksek Çözünürlüklü Tüm Dağı<em&gt; Yerinde</emGen İfade ve Fonksiyon Eğitim için Zebra balığı Embriyolar içinde&gt; Hibridizasyon

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

Methods Article

Zebra balığı Tüm Dağı Yüksek Çözünürlüklü Çift Floresan Yerinde Hibridizasyon