Gostaríamos de agradecer as contribuições de Dorthe Jülich, Round Jennifer e Mara Andrew no desenvolvimento deste protocolo. Apoio à pesquisa fornecido pelo NICHD, a Sociedade Americana do Câncer ea March of Dimes.

1. FIXAÇÃO <ol><li> Fixar os embriões durante a noite a 4 ° C com paraformaldeído a 4% (PFA) em PBS. </li><li> Remover corrigir e lave 2 x PBS, 5 minutos cada em temperatura ambiente (RT). </li><li> Embriões manualmente dechorionate na PBS em uma placa de vidro com a pinça depressão relojoeiro. Depois dechorionation, os embriões são transferidos usando um fogo-de vidro polido pipeta Pasteur, pois podem ficar com pipetas de polipropileno. </li><li> Transferência de embriões através de uma série de 25%, 50% e metanol 75% em PBS por 5 minutos cada. </li><li> Substituir o líquido com metanol 100%, incubar 5 minutos e em seguida, substitua com metanol fresco. </li><li> Lugar embriões a -20 ° C por um mínimo de uma hora. (Normalmente, incubar os embriões durante a noite. Padrão de hibridização in situ funcionará em embriões armazenados há mais de um ano, mas não temos examinado se a alta resolução da hibridização fluorescente in situ é perdido por armazenamento prolongado.) </li><li> Lavar embriões por 5 minutos cada em 75%, 50%, metanol 25% em PBST na RT. Lavar duas vezes por 5 minutos cada, em PBST na RT. </li><li> Fix novamente por 20 minutos em PFA 4% em PBS a RT. </li><li> Lavar duas vezes por 5 minutos cada, em PBST na RT. </li></ol> NOTA SOBRE PFA: Nós armazenamos PFA 4% em alíquotas a -20 º C (ver tabela de reagentes). Para a fixação inicial, só usamos PFA que nunca tenha sido previamente descongeladas. Para fixações posteriores, muitas vezes usamos PFA que tenha sido previamente descongeladas. A fixação inicial parece ser crítico para a coloração de sucesso com este protocolo. 2. Proteinase e POSTFIXATION <ol><li> Digest com proteinase K (5μg/ml em PBST) a RT por 3 a 12 minutos para permeabilizar os embriões. (O tempo de incubação depende da idade dos embriões como estágios mais jovens são mais sensíveis. Também depende do lote de enzima.) Para embriões em estágio somitogenesis, nós tipicamente permeabilizar por 3-4 minutos. Durante esta incubação, nós colocamos o tubo de microcentrífuga de lado. </li><li> Lave brevemente em PBST e lave uma vez por 5 minutos em PBST. </li><li> Fix novamente por 20 minutos em PFA 4% em PBS a RT. </li><li> Lavar duas vezes, por 5 minutos cada, em PBST na RT. </li></ol> 3. PREHYBRIDIZATION <ol><li> Incubar os embriões durante 5 minutos a 65 º C em HYB. </li><li> Prehybridize a 65 º C durante pelo menos 1 hora em HYB +. </li></ol> 4. HIBRIDIZAÇÃO <ol><li> Remover todos, mas 50 ul do preHYB, mas certifique-se de manter os embriões completamente submerso. </li><li> Adicione 1-2 mL de cada riboprobe (digoxigenina e fluoresceína marcado riboprobes) para os embriões e misture delicadamente flicking do tubo. A quantidade de sonda é tipicamente 1-2μl de uma reação 20μl da sonda de síntese. </li><li> Dado que a fluoresceína é sensível à luz, os tubos devem ser embrulhados em papel alumínio ou expostos à luz mínima a partir deste ponto em diante. </li><li> Incubar os embriões durante a noite a 65 º C. </li></ol> 5. REMOÇÃO PROBE Note-se que as soluções a partir deste ponto em diante a falta de detergente. Eliminação de detergente aparece para ajudar as reações de coloração, mas faz com que os embriões para se tornar um pouco pegajosa. <ol><li> Remover o riboprobe. </li><li> Lave 2 x 30 minutos a 65 º C em formamide/2xSSC 50%. </li><li> Lavar por 15 minutos a 65 º C em 2 x SSC. </li><li> Lavagem para 30 minutos 65 º C em 0,2 x SSC. </li></ol> 6. ANTI-Fluoresceína incubação com anticorpo <ol><li> Bloco para pelo menos 1 hora em temperatura ambiente em 500μl de uma solução de 1x tampão de ácido maleico, mais 2% de reagente de bloqueio (ver tabela de reagentes). </li><li> Adicione o anticorpo anti-fluoresceína-POD, como fornecido pela Roche, na diluição 1:500 em solução de bloqueio. </li><li> Incubar overnight a 4 º C. Durante esta incubação, nós colocamos o tubo de microcentrífuga de lado. </li><li> Lavar 4 x 20 minutos em tampão ácido maleico 1x. Lavar duas vezes por 5 minutos cada em PBS. </li></ol> 7. DETECÇÃO DE fluoresceína DOS RÓTULOS PROBE <ol><li> Incubar 30-60 minutos em solução de fluoresceína TSA Plus. (Spin para baixo substrato TSA antes de fazer solução de coloração. Para a reação, diluir o reagente tiramida 1:50 em tampão Perkin Elmer diluente de amplificação.) Durante esta incubação, nós colocamos o tubo de microcentrífuga de lado. Tempo de reação devem ser determinadas empiricamente para cada sonda. Infelizmente, a reação de coloração não pode ser visualmente monitorado como o substrato é fluorescente, e vai ver uma fluorescência verde onipresentes em toda a reação de coloração. </li><li> Lavagem para 10 minutos cada em 30%, 50%, 75% e 100% de metanol em PBS. </li><li> Incubar em uma solução de 1% H 2 0 2 em metanol por 30 minutos para inativar a peroxidase em primeiro lugar. </li><li> Lavar 10 minutos cada em 75%, 50% e metanol 30% em PBS. Em seguida, lave duas vezes para 10 minutos cada em PBS. É importante que todos os conhecihanol ser removido. </li></ol> NOTAS SOBRE A amplificação de sinal tiramida: Temos vindo a utilizar a Perkin Elmer Kits TSA. Nós achamos que os substratos Alexa-tiramida mancha bem usando a Perkin Elmer tampão diluente amplificação, mas não tiveram sucesso usando o buffer fornecido com a coloração Kits Invitrogen / Molecular Probes. Por fim, descobrimos que fluorescence Cy5 é eliminada por subseqüentes 2 O metanol / H 2 tratamento, enquanto fluoresceína e Alexa-647 não são afetados. Cy3 também pode ser adversamente afetado pela 2 O metanol / H 2 tratamento, uma vez que está estruturalmente relacionado com Cy5. Por esta razão, Cy3 e Cy5 reações TSA só deve ser utilizado para a reação de coloração segundo de uma fluorescente duplo em protocolo situ. 8. ANTI-digoxigenina incubação com anticorpo <ol><li> Bloco os embriões novamente por pelo menos uma hora em temperatura ambiente em uma solução de 1x tampão de ácido maleico mais 2% de reagente de bloqueio. </li><li> Adicione o anticorpo anti-DIG POD como fornecidos pela Roche na diluição de 1:1000 em solução de bloqueio acima </li><li> Incubar overnight a 4 º C. Durante esta incubação, nós colocamos o tubo de microcentrífuga de lado. </li><li> Lavar 4 x 20 minutos em tampão ácido maleico 1x. Lavar duas vezes por 5 minutos cada em PBS. </li></ol> 9. DETECÇÃO DE digoxigenina-DOS RÓTULOS PROBE <ol><li> Incubar 30-60 minutos em TSA Além disso Cy5 Solution (Spin para baixo substrato TSA antes de fazer solução de coloração. Para a reação, diluir o reagente tiramida 1:50 em tampão diluente amplificação) Durante esta incubação, nós colocamos o tubo de microcentrífuga de lado. Tempo de reação devem ser determinadas empiricamente para cada sonda. </li><li> Lavar três vezes por 10 minutos cada, em PBST. </li></ol> 10. Propídio COLORAÇÃO iodeto <ol><li> Lavar duas vezes por 5 minutos cada, em 2 x SSC. </li><li> Incubar os embriões durante 30 minutos a 37 ° C em 50 mL 2 x SSCT com 10 mL de RNAse (para uma concentração final de 100μg/ml). </li><li> Lavar seis vezes por 3 minutos cada, em 2 x SSC a RT. </li><li> Mancha embriões por 8 minutos em uma solução de iodeto de propídio 330μg/ml em 2 x SSC. </li><li> Lavar seis vezes por 3 minutos cada, em 2 x SSC a RT. </li><li> Correção para 20 minutos em PFA 4% em PBS a RT. </li><li> Lavar duas vezes, por 5 minutos cada, em PBST na RT. </li></ol> 11. MONTAGEM <ol><li> Incubar os embriões para 10 minutos cada em 25% e glicerol a 50% em PBST. Claro durante a noite em glicerol 75% a 4 º C. </li><li> Dissecamos e deyolk os embriões e plana montá-los em uma lâmina de microscópio. A gema pode produzir fluorescência de fundo significativo. Dissecção é muito mais fácil depois de os embriões terem desaparecido ovenight em gylcerol. </li></ol> SOLUÇÕES: <table border="1"><tbody><tr><td> PBST </td><td> PBS Tween mais 0,1% </td><td></td></tr><tr><td> SSCT </td><td> SSC Tween mais 0,1% </td><td></td></tr><tr><td> HYB- </td><td> Formamida 50% 5xSSC 0,1% Tween-20 </td><td></td></tr><tr><td> HYB + </td><td> HYB- 5mg/ml torula (levedura) RNA 50μg/ml de heparina </td><td style="width: 300px;"> O RNA torula é preparado por proteinase K digestão de RNA com fenol subseqüentes, do fenol-clorofórmio, clorofórmio e-extraction.The RNA é precipitado e dissolvido em água tratada DEPC-. </td></tr><tr><td> 1 x tampão ácido maleico </td><td> 150mm de ácido maleico, NaCl 100mM (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. Os resultados representativos do monte todo dupla hibridização fluorescente in situ. (AC). Imagens mostram uma única seção confocal com a região posterior de um embrião de peixe-zebra na fase de dez somito. A vista é dorsal anterior com o topo. Coradas com iodeto de propídio núcleos são de cor azul. (A) deltaC mRNA foi detectada usando um riboprobe digoxigenina-rotulados e TSA-Cy5 reagente. (B) mRNA transcrito do gene HER1 foi detectado com um riboprobe fluoresceína rotulados e TSA-fluoresceína reagente. (C) A fusão dos canais verde e vermelho identifica inequivocamente regiões distintas da expressão do gene ou sobreposição dos dois genes. (D, E) Detalhe da HER1 expressão demonstrando a alta resolução deste procedimento. Localização subcelular de mRNA pode ser claramente discernida. As setas indicam uma célula exibindo transcrição ativa, revelado por pontos de coloração no núcleo. Setas indicam uma célula mostrando a localização citoplasmática de mRNA. (F, G) coloração duplo HER1 e deltaC revela células que são a transcrição de ambos os genes (seta branca), ou seja gene separadamente (setas vermelhas e verdes).

<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>

O protocolo apresentado aqui funciona bem com sondas que dar um sinal forte limpa após coloração por 30-45 minutos em uma fosfatase alcalina mediada típico de reação. Antes de realizar a fluorescente no protocolo de hibridização in situ, sempre testamos nossas sondas utilizando o protocolo não fluorescente padrão (desde que no material suplementar juntamente com o protocolo de síntese de sonda). Temos tido menos sucesso com as fluorescentes em protocolo de hibridização in situ utilizando sondas quando mais ...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<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>
		<tbody>
		
<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

Montar toda<em&gt; In situ</emHibridização&gt; é uma das técnicas mais utilizadas em biologia do desenvolvimento. Aqui, apresentamos uma alta resolução de dupla fluorescentes<em&gt; In situ</em&gt; Protocolo de hibridização para analisar o padrão de expressão precisa de um único gene e para determinar a sobreposição dos domínios de expressão dos dois genes. O protocolo é uma versão modificada do padrão<em&gt; In situ</em&gt; Hibridização com fosfatase alcalina e substratos tais como NBT / BCIP e Red Rápido<sup&gt; 1,2</sup&gt;. Este protocolo utiliza sondas padrão digoxigenina e fluoresceína rotulados junto com amplificação de sinal tiramida (TSA)<sup&gt; 3</sup&gt;. Os kits disponíveis comercialmente TSA permitem delineamento experimental flexível como emissão de fluorescência do verde ao vermelho-extremo pode ser usado em combinação com várias manchas nuclear, como o iodeto de propídio, ou imunohistoquímica de fluorescência para as proteínas. TSA produz um substrato reativo fluorescente que rapidamente covalentemente liga-se a metades, tipicamente resíduos de tirosina, nas imediações do riboprobe antisense rotulados. Os padrões de coloração resultantes são de alta resolução em que a localização subcelular do mRNA pode ser observado através de varredura a laser microscopia confocal<sup&gt; 3,4</sup&gt;. Pode-se observar transcrições nascente no loci cromossômicos, distinguir coloração nuclear e citoplasmática e visualizar outros padrões, como localização cortical de mRNA. Estudos em<em&gt; Drosophila</em&gt; Indicam que aproximadamente 70% dos mRNAs exibem padrões específicos de localização subcelular que freqüentemente se correlacionam com a função da proteína codificada<sup&gt; 5</sup&gt;. Quando combinado com computer-aided reconstrução de conjuntos de dados 3D confocal, o nosso protocolo permite a análise detalhada da distribuição de mRNA com sub-celular resolução em embriões vertebrados todo.

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

Montar toda a hibridização in situ é uma das técnicas mais utilizadas em biologia do desenvolvimento. Aqui, apresentamos uma alta resolução de dupla fluorescentes em protocolo de hibridização in situ para analisar o padrão de expressão precisa de um único gene e para determinar a sobreposição dos domínios de expressão dos dois genes. Incluímos uma propidium iodeto nuclear contra-coloração para destacar a organização do tecido.

Zebrafish Whole Mount Alta Resolução Duplo Fluorescente In Situ Hibridização

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 visualizações.  Yale University. Montar toda a hibridização in situ é uma das técnicas mais utilizadas em biologia do desenvolvimento. Aqui, apresentamos uma alta resolução de dupla fluorescentes em protocolo de hibridização in situ para analisar o padrão de expressão precisa de um único gene e para determinar a sobreposição dos domínios de expressão dos dois genes. Incluímos uma propidium iodeto nuclear contra-coloração para destacar a organização do tecido.

Vídeo: 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.

Monte Whole dupla hibridização in situ de embriões de galinha precoce

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

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.

Método para coloração Whole Mount anticorpos em Pintainho

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.

Usando Monte Whole In situ Hibridação a ligação de Biologia Molecular e Organísmicos

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.

Monte todo In Situ Hibridação de E8.5 de Embriões E11.5 rato

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.

Análise de uma única célula de transcrição do gene pela RNA Fluorescente<em&gt; In Situ</em&gt; A hibridização (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.

Tempo cromossomo Replicando Combinado com Fluorescente<em&gt; Em situ</em&gt; Hibridização

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.

Todo o Monte RNA Fluorescente<em&gt; In situ</em&gt; A hibridação de<em&gt; Drosophila</em&gt; Embriões

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; Em situ</em&gt; Hibridação em embriões de Mount inteiros e histologia de células Adaptado para Elasmobrânquios Marinhos

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 resolução Monte Whole<em&gt; In Situ</em&gt; Hibridização dentro embriões Zebrafish para estudar a expressão gênica e função

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.

Análise de apoptose em embriões Zebrafish por Whole-mount imunofluorescência para detectar Caspase 3 ativado

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