Estamos muito gratos a Anders Bovin que forneceu a música para o nosso filme e Stocks Michael Elliot para o voluntariado para ser a voz do altifalante. Apoio foi fornecido por Carl Trygger Foundation, o programa estratégico de pesquisa agrícola Genômica Funcional (AgriFunGen) da Universidade Sueca de Ciências Agrícolas, o sueco Research Council (VR), eo Conselho de Pesquisa sueco do Ambiente, Ciências Agrárias e do Ordenamento do Território (FORMAS ).

<ol>
	<li>Carr, S. M., Irish, V. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Floral+homeotic+gene+expression+defines+developmental+arrest+stages+in+Brassica+oleracea+L.+vars.+botrytis+and+italica.">Floral homeotic gene expression defines developmental arrest stages in Brassica oleracea L. vars. botrytis and italica.</a> Planta. 201 (2), 179-188 (1997).</li><li>Coen, E. S., Meyerowitz, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+war+of+the+whorls:+genetic+interactions+controlling+flower+development.">The war of the whorls: genetic interactions controlling flower development.</a> Nature. 353 (6339), 31-37 (1991).</li><li>Jackson, D., Gurr, S. J., McPherson, M. J., Bowles, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+hybridization+in+plants.">In situ hybridization in plants.</a> Molecular Plant Patology: a practical approach. , (1991).</li><li>Leitch, A. R., Schwarzacher, T., Jackson, D., Leitch, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> In situ hybridization: a practical guide. , (1994).</li><li>Smyth, D. R., Bowman, J. L., Meyerowitz, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+flower+development+in+Arabidopsis.">Early flower development in Arabidopsis.</a> Plant Cell. 2 (8), 755-767 (1990).</li><li>Azevedo, H., Lino-Neto, T., Tavares, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+method+for+high-quality+RNA+isolation+from+needles+of+adult+maritime+pine+trees.">An improved method for high-quality RNA isolation from needles of adult maritime pine trees.</a> Plant Molecular Biology Reporter. , 333-338 (2003).</li><li>Sundstrom, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MADS-box+genes+active+in+developing+pollen+cones+of+Norway+spruce+(Picea+abies)+are+homologous+to+the+B-class+floral+homeotic+genes+in+angiosperms.">MADS-box genes active in developing pollen cones of Norway spruce (Picea abies) are homologous to the B-class floral homeotic genes in angiosperms.</a> Dev Genet. 25 (3), 253-266 (1999).</li><li>Tandre, K., Svenson, M., Svensson, M. E., Engstrom, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conservation+of+gene+structure+and+activity+in+the+regulation+of+reproductive+organ+development+of+conifers+and+angiosperms.">Conservation of gene structure and activity in the regulation of reproductive organ development of conifers and angiosperms.</a> Plant J. 15 (5), 615-623 (1998).</li><li>Winter, K. U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MADS-box+genes+reveal+that+gnetophytes+are+more+closely+related+to+conifers+than+to+flowering+plants.">MADS-box genes reveal that gnetophytes are more closely related to conifers than to flowering plants.</a> Proc Natl Acad Sci U S A. 96 (13), 7342-7347 (1999).</li><li>Savard, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chloroplast+and+nuclear+gene+sequences+indicate+late+Pennsylvanian+time+for+the+last+common+ancestor+of+extant+seed+plants.">Chloroplast and nuclear gene sequences indicate late Pennsylvanian time for the last common ancestor of extant seed plants.</a> Proc Natl Acad Sci U S A. 91 (11), 5163-5167 (1994).</li><li>Jack, T., Brockman, L. L., Meyerowitz, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+homeotic+gene+APETALA3+of+Arabidopsis+thaliana+encodes+a+MADS+box+and+is+expressed+in+petals+and+stamens.">The homeotic gene APETALA3 of Arabidopsis thaliana encodes a MADS box and is expressed in petals and stamens.</a> Cell. 68 (4), 683-697 (1992).</li><li>Bowman, J. L., Smyth, D. R., Meyerowitz, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genes+directing+flower+development+in+Arabidopsis.">Genes directing flower development in Arabidopsis.</a> Plant Cell. 1 (1), 37-52 (1989).</li><li>Kramer, E. M., Irish, V. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+genetic+mechanisms+controlling+petal+development.">Evolution of genetic mechanisms controlling petal development.</a> Nature. 399 (6732), 144-148 (1999).</li><li>Sundstrom, J., Engstrom, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conifer+reproductive+development+involves+B-type+MADS-box+genes+with+distinct+and+different+activities+in+male+organ+primordia.">Conifer reproductive development involves B-type MADS-box genes with distinct and different activities in male organ primordia.</a> Plant J. 31 (2), 161-169 (2002).</li><li>Carlsson, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarray+analysis+reveals+altered+expression+of+a+large+number+of+nuclear+genes+in+developing+cytoplasmic+male+sterile+Brassica+napus+flowers.">Microarray analysis reveals altered expression of a large number of nuclear genes in developing cytoplasmic male sterile Brassica napus flowers.</a> Plant J. 49 (3), 452-462 (2007).</li><li>Teixeira, R. T., Farbos, I., Glimelius, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+levels+of+meristem+identity+and+homeotic+genes+are+modified+by+nuclear-mitochondrial+interactions+in+alloplasmic+male-sterile+lines+of+Brassica+napus.">Expression levels of meristem identity and homeotic genes are modified by nuclear-mitochondrial interactions in alloplasmic male-sterile lines of Brassica napus.</a> Plant J. 42 (5), 731-742 (2005).</li><li>Ying, S., Kay, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+technique+of+in+situ+hybridization.">The technique of in situ hybridization.</a> Methods in Molecular Medicine. 56, 263-283 (2001).</li></ol>

Dois aspectos do P. abies protocolo foram otimizadas em relação ao A. thaliana / B. napus protocolo, RNase tratamento e proteinase K concentração. A RNase remove sinais de fundo e assim aumentar a especificidade do sinal 17. O tratamento proteinase K é necessária para permeabilizar o tecido para que a sonda pode entrar e hibridizar para a piscina RNA de interesse. A sonda antisense DAL13 deu um maior sinal sem RNase-tratamento (Fig. 3C, E, G) em comparação com seções tratadas com RNase por 30 min (Figura 2D, F e H). Desde a RNase-tratamento não melhorou o sinal que foi retirado do protocolo. O tratamento proteinase K foram testadas em três concentrações diferentes; 1, 3 e 5 mg / ml. No caso de P. abies um sinal de baixa ou nenhuma foi observada usando 1 mg / ml proteinase K (Fig. 3C-D). Um sinal forte foi obtida com ambos mg 3 / ml (Fig. 3E-F) e 5 mg / ml (Fig. 3G-H) proteinase K. Uma vez que não aparente diferença na força do sinal quando foi encontrado usando 5 mg / ml proteinase K em comparação com 3 mg / ml, optamos por utilizar 3 mg / ml em nosso protocolo. É provável que a necessidade de uma maior concentração proteinase K na P. abies cones masculinos em relação ao A. thaliana e B. botões florais napus é devido a mais de tecido compacto, com níveis mais elevados de compostos fenólicos e polissacarídeos. Durante a fixação e incorporação de algumas diferenças entre o A. thaliana / B. napus protocolo eo P. abies protocolo são evidentes. O tecido de juro é fixa para fornecer retenção de RNA e este é um passo crítico, pois materiais mal fixa pode dar um sinal de baixa ou indetectável mesmo que o mRNA é altamente abundante. O tecido é desidratado, limpo e embebido em cera em mudanças graduais para evitar danos aos tecidos e, em alguns protocolos de NaCl 0,85% é adicionado ao etanol na desidratação para continuar a evitar o encolhimento e inchaço da 3 células. A etapa de desidratação durante a incorporação pode ser realizada no gelo para manter a atividade RNase no mínimo. No P. abies protocolo de corrigir a solução de 4% paraformaldeído contém glutaraldeído 0,25%, o que é suposto dar uma melhor retenção de RNA 17, apesar de que alguns argumentam que ele provavelmente não fazem diferença 3. Depois de cortar o material é fixado em lâminas e pré-tratados (isto é, desparafinados, reidratados, permeabilizadas, tratado para reduzir a ligação não específica da sonda e, finalmente, desidratadas) antes da hibridização. No protocolo aqui apresentado o procedimento de detecção de todo pode ser realizado em laboratórios comuns, o sinal se desenvolve normalmente dentro de 1-3 dias ea relação entre o sinal sobre o fundo pode ser continuamente monitorado. A DIG sondas marcadas costumam dar um sinal mais distinta em comparação com sondas marcadas radioativos, são mais estáveis ​​e podem ser reutilizados em experimentos consecutivos. Por outro lado, a sensibilidade é reduzida em comparação com sondas radioativas 17. A hibridização in situ detecta expressão usando uma sonda marcada específico para um determinado mRNA. A marcação é um método de etiquetagem robusta e sensível, mas requer a manipulação da radioatividade e leva várias semanas antes que os resultados podem ser detectados. Antígeno marcado com sondas, por outro lado, são mais estáveis, menos perigosos e exigem uma exposição para o meio de detecção por alguns dias apenas 17 anos. Assim, o antígeno-rotulagem métodos são atualmente dominante. O passo mais crítico independentemente do protocolo é o projeto da sonda. Cuidados devem ser tomados para garantir que a sonda tem uma alta especificidade e é rotulado com UTPs de alta qualidade. Às vezes, a temperatura de hibridação e / ou comprimento de reação tem que ser ajustado para sondas específicas. Nosso protocolo modificado com base na DIG sondas marcadas irá facilitar a localização de expressão de mRNA, simultaneamente, em espécies que variam de A. thaliana a P. abies, e deve, com pequenos ajustes ser usado em outras espécies vegetais. O protocolo tem, ao lado de cones masculinos, também foi testado em diferentes estágios de brotos vegetativos e em embriões de ambos os zigóticos e somáticos de P. abies com bons resultados (resultados não publicados;. Karlgren et al).

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<tr>
<td>Acetic anhydride</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>A6404-200ml</td>
<td>minimum 98%</td>
</tr>
<tr>
<td>Acrylamide, linear</td>
<td>&nbsp;</td>
<td>Ambion</td>
<td>9520</td>
<td>&nbsp;</td>
<tr>
<td>Anti-DIG-antibodies</td>
<td>&nbsp;</td>
<td>Roche Applied Science</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Blocking reagent</td>
<td>&nbsp;</td>
<td>Roche Applied Science</td>
<td>11 175 041 910 or 11 096 176 001</td>
<td>&nbsp;</td>
<tr>
<td>Bovine serum albumin</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>A7030-50g</td>
<td>minimum 98%</td>
</tr>
<tr>
<td>Deionized formamide</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Denhardts solution</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>D2532-5ml</td>
<td>&nbsp;</td>
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<td>DEPC, diethylpyrocarbonate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Dextran sulfate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>DIG RNA Labeling Kit (SP6/T7)</td>
<td>&nbsp;</td>
<td>Roche Applied Science</td>
<td>11175025910</td>
<td>&nbsp;</td>
<tr>
<td>Glutaraldehyde (25%)</td>
<td>&nbsp;</td>
<td>Histolab products AB</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Glycogen</td>
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<td>Ambion</td>
<td>9510G</td>
<td>&nbsp;</td>
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<td>Histoclear II</td>
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<td>Histowax</td>
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<td>Histolab products AB</td>
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<td>Paraformaldehyde</td>
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<td>P6148-500g</td>
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<td>Paraplast plus</td>
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<td>Sigma-Aldrich</td>
<td>P3683-1kg</td>
<td>Histowax or Paraplast can be used for embedding.</td>
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<td>Phusion&#x2122; High-Fidility DNA Polymerase</td>
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<td>Finnzymes</td>
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<td>Probe-on Plus slides</td>
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<td>Fischer Scientific</td>
<td>22-230-900</td>
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<td>Proteinase K</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P2308-5mg</td>
<td>&nbsp;</td>
<tr>
<td>RNase A</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>R5503-100mg</td>
<td>&nbsp;</td>
<tr>
<td>Tissue-clear</td>
<td>&nbsp;</td>
<td>Sakura Finetek Europ&#233; BV</td>
<td>&nbsp;</td>
<td>You may use Tissue-clear, Histoclear II, xylen or something similar when embedding your tissue.</td>
</tr>
<tr>
<td>Triethanolamine</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>T1377-100ml</td>
<td>minimum 98%</td>
</tr>
<tr>
<td>Triton X-100</td>
<td>&nbsp;</td>
<td>use your standard product in the lab</td>
<td>&nbsp;</td>
<td>Use one or both of Triton X-100 and Tween-20.</td>
</tr>
<tr>
<td>tRNA</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>83853-100mg</td>
<td>&nbsp;</td>
<tr>
<td>Tween-20</td>
<td>&nbsp;</td>
<td>use your standard product in the lab</td>
<td>&nbsp;</td>
<td>Use one or both of Triton X-100 and Tween-20.</td>
</tr>
<tr>
<td>Western Blue</td>
<td>&nbsp;</td>
<td>Promega Corp.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
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non-radioactive in situ hybridization protocol applicable for norway spruce and a range of plant species

A tecnologias de alta capacidade de análise de expressão disponíveis hoje dar aos cientistas um estouro de perfis de expressão, mas a sua resolução em termos de expressão tecido específico é limitado devido a problemas em dissecar tecidos individual. Dados de expressão deve ser confirmado e complementado com padrões de expressão usando, por exemplo hibridização in situ, uma técnica utilizada para localizar a expressão de RNAm de células específicas. O método de hibridização in situ é trabalhoso, demorado e muitas vezes exige otimização extensa dependendo da espécie e do tecido. Em experimentos in situ são relativamente mais difíceis de executar em espécies lenhosas, como a Noruega coníferas spruce (Picea abies). Aqui apresentamos uma DIG modificado no protocolo de hibridização in situ, que é rápido e aplicável em uma ampla gama de espécies de plantas, incluindo P. abies. Com apenas alguns ajustes, incluindo alteradas RNase tratamento e concentração proteinase K, poderíamos usar o protocolo para estudar a expressão tecido específico de genes homólogos nos órgãos reprodutivos masculinos de uma gimnosperma e duas espécies de angiospermas; P. abies, Arabidopsis thaliana e Brassica napus. O protocolo funcionou tão bem para as espécies e genes estudados. AtAP3 e BnAP3 foram observados no segundo e terceiro whorl órgãos florais em A. thaliana e B. napus e DAL13 em microsporophylls de cones masculinos de P. abies. Para P. abies a concentração de K proteinase, usado para permeablize os tecidos, teve que ser aumentada para 3 g / ml em vez de 1 g / ml, possivelmente devido aos tecidos mais compactos e níveis mais elevados de compostos fenólicos e polissacarídeos. Para todas as espécies o tratamento RNase foi removido devido a intensidade do sinal reduzida sem um aumento correspondente na especificidade. Ao comparar os padrões de expressão tecido específico de genes homólogos de ambas as plantas floridas e uma árvore conífera demonstramos que a DIG situ no protocolo aqui apresentado, com apenas pequenos ajustes, pode ser aplicado a uma grande variedade de espécies de plantas. Assim, o protocolo evita tanto a otimização espécies extensa específicas ea utilização de sondas trabalhoso marcado radioativamente, em favor de sondas DIG rotulados. Nós escolhemos para ilustrar os passos tecnicamente exigente do protocolo em nosso filme. Anna Karlgren e Jenny Carlsson contribuíram igualmente para este estudo. Autores correspondentes: Anna Karlgren em Anna.Karlgren @ ebc.uu.se e Sundström Jens F. em Jens.Sundstrom @ vbsg.slu.se

Watch this Scientific Journal Video about Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species at JoVE.com

Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species

Nós descrevemos um DIG modificado<em&gt; In situ</em&gt; Protocolo de hibridização, que é rápido e aplicável em uma ampla gama de espécies de plantas, incluindo a Noruega abeto. Com apenas alguns ajustes, incluindo alteradas RNase tratamento e concentração proteinase K, o protocolo pode ser utilizado em estudos de diferentes tecidos e espécies.

Não-radioativo In situ Protocolo de hibridação Aplicável para a Noruega Spruce e uma variedade de espécies de plantas

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

non-radioactive-situ-hybridization-protocol-applicable-for-norway

Research

JoVE Journal

Biology

Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species

20.9K visualizações.  Uppsala University. Nós descrevemos um DIG modificado In situ Protocolo de hibridização, que é rápido e aplicável em uma ampla gama de espécies de plantas, incluindo a Noruega abeto. Com apenas alguns ajustes, incluindo alteradas RNase tratamento e concentração proteinase K, o protocolo pode ser utilizado em estudos de diferentes tecidos e espécies.

Vídeo: Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species

In situ Protocol for Butterfly Pupal Wings Using Riboprobes

Here we present, in video format, a protocol for in situ hybridizations in pupal wings of the butterfly Bicyclus anynana using riboprobes.  In situ hybridizations, a mainstay of developmental biology, are useful to study the spatial and temporal patterns of gene expression in developing tissues at the level of transcription. If antibodies that target the protein products of gene transcription have not yet been developed, and/or there are multiple gene copies of a particular protein in the genome that cannot be differentiated using available antibodies, in situs can be used instead. While an in situ technique for larval wing discs has been available to the butterfly community for several years, the current protocol has been optimized for the larger and more fragile pupal wings.

In order to examine gene expression in the pupal wing tissue of Bicyclus anynana, we present an optimized protocol for in situ hybridizations using riboprobes. We also provide guidelines for the further optimization of this protocol for use in pupal wings of other Lepidopteran species.

No Protocolo situ de asas da borboleta Pupal Usando Riboprobes

Here we present, in video format, a protocol for in situ hybridizations in pupal wings of the butterfly Bicyclus anynana using riboprobes.  In situ ...

Biologia

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 ...

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

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

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

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

Zebrafish 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 ...

Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic process in the parents. In human males, these errors can lead to the production of spermatozoa with numerical chromosome abnormalities which represent an increased risk of transmitting these anomalies to the offspring.
For this reason, the technique of fluorescence in situ hybridization (FISH) on sperm nuclei has become a protocol widely incorporated in the context of clinical diagnosis. This practice provides an estimate of the frequencies of numerical chromosome abnormalities in the gametes of the patients that seek for genetic reproductive advice.
To date, the chromosomes most frequently included in sperm FISH analysis are chromosomes X, Y, 13, 18 and 21.
This video-article describes, step by step, how to process and fix a human semen sample, how to decondense and denature the sperm chromatin, how to proceed to obtain sperm FISH preparations, and how to visualize the results at the microscope. Special remarks of the most relevant steps are given to achieve the best results.

Fluorescence in situ hybridization FISH Protocol in Human Sperm

This video-article describes, step by step, how to process a semen sample to achieve good-quality fluorescence in situ hybridization on human spermatozoa. Preparations obtained can be used for aneuploidy screening in the context of clinical diagnosis.

Fluorescência In situ Hybridization (FISH) Protocolo no esperma humano

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic ...

A Noninvasive Hair Sampling Technique to Obtain High Quality DNA from Elusive Small Mammals

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using noninvasive sampling techniques to investigate population genetics and demography of wild populations1. This approach has proven to be especially useful when dealing with rare or elusive species2. While a number of these methods have been developed to sample hair, feces and other biological material from carnivores and medium-sized mammals, they have largely remained untested in elusive small mammals. In this video, we present a novel, inexpensive and noninvasive hair snare targeted at an elusive small mammal, the American pika (Ochotona princeps). We describe the general set-up of the hair snare, which consists of strips of packing tape arranged in a web-like fashion and placed along travelling routes in the pikas&#x2019; habitat. We illustrate the efficiency of the snare at collecting a large quantity of hair that can then be collected and brought back to the lab. We then demonstrate the use of the DNA IQ system (Promega) to isolate DNA and showcase the utility of this method to amplify commonly used molecular markers including nuclear microsatellites, amplified fragment length polymorphisms (AFLPs), mitochondrial sequences (800bp) as well as a molecular sexing marker. Overall, we demonstrate the utility of this novel noninvasive hair snare as a sampling technique for wildlife population biologists. We anticipate that this approach will be applicable to a variety of small mammals, opening up areas of investigation within natural populations, while minimizing impact to study organisms.

We present a noninvasive sampling approach to efficiently collect hair samples from elusive small mammals, as shown for the American pika. We demonstrate the utility of this method by extracting DNA from sampled hair and amplifying several types of molecular markers commonly used in studies of wildlife ecology and conservation.

A técnica de amostragem não invasiva cabelo obter DNA de alta qualidade a partir de Elusive Pequenos Mamíferos

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using ...

In Situ Hybridization for the Precise Localization of Transcripts in Plants

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene expression. Microarray and next generation sequencing approaches are being used to investigate developmental, physiological and stress response processes, dissect epigenetic and small RNA pathways, and build large gene regulatory networks1-3. While these techniques facilitate the simultaneous analysis of large gene sets, they typically provide a very limited spatiotemporal resolution of gene expression changes. This limitation can be partially overcome by using either profiling method in conjunction with lasermicrodissection or fluorescence-activated cell sorting4-7. However, to fully understand the biological role of a gene, knowledge of its spatiotemporal pattern of expression at a cellular resolution is essential. Particularly, when studying development or the effects of environmental stimuli and mutants can the detailed analysis of a gene's expression pattern become essential. For instance, subtle quantitative differences in the expression levels of key regulatory genes can lead to dramatic phenotypes when associated with the loss or gain of expression in specific cell types. 
Several methods are routinely used for the detailed examination of gene expression patterns. One is through analysis of transgenic reporter lines. Such analysis can, however, become time-consuming when analyzing multiple genes or working in plants recalcitrant to transformation. Moreover, an independent validation to ensure that the transgene expression pattern mimics that of the endogenous gene is typically required. Immunohistochemical protein localization or mRNA in situ hybridization present relatively fast alternatives for the direct visualization of gene expression within cells and tissues. The latter has the distinct advantage that it can be readily used on any gene of interest. In situ hybridization allows detection of target mRNAs in cells by hybridization with a labeled anti-sense RNA probe obtained by in vitro transcription of the gene of interest. 
Here we outline a protocol for the in situ localization of gene expression in plants that is highly sensitivity and specific. It is optimized for use with paraformaldehyde fixed, paraffin-embedded sections, which give excellent preservation of histology, and DIG-labeled probes that are visualized by immuno-detection and alkaline-phosphatase colorimetric reaction. This protocol has been successfully applied to a number of tissues from a wide range of plant species, and can be used to analyze expression of mRNAs as well as small RNAs8-14.

In Situ Hybridization for the Precise Localization of Transcripts in Plants

The in situ hybridization protocol described here allows a direct localization of mRNA and small RNA expression at the cellular level with high sensitivity and specificity. The procedure is optimized for paraffin-embedded plant tissue sections, is applicable to a wide range of plants and tissues, and can be completed within ten days.

Hibridização In Situ para a localização precisa de transcrições em Plantas

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

Detecção de RNA viral por fluorescência<em&gt; In situ</em&gt; Hybridization (FISH)

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

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 ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

Visualização e análise de moléculas de mRNA Usando Fluorescência<em&gt; In Situ</em&gt; A hibridação em<em&gt; Saccharomyces cerevisiae</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA probes. This protocol, originally developed by Kloosterman and colleagues for broad use with Exiqon miRNA probes1, has been modified to overcome challenges inherent in miRNA analysis in kidney tissues. These include issues such as structure identification and hard to remove residual probe and antibody. Use of relatively thin, 5 mm thick, tissue sections allowed for clear visualization of kidney structures, while a strong probe signal was retained in cells. Additionally, probe concentration and incubation conditions were optimized to facilitate visualization of microRNA expression with low background and nonspecific signal. Here, the optimized protocol is described, covering the initial tissue collection and preparation through the mounting of slides at the end of the procedure. The basic components of this protocol can be altered for application to other tissues and cell culture models.

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

This article describes an in situ hybridization protocol optimized for colormetric detection of microRNA expression in formalin fixed kidney sections.

MicroRNA<em&gt; Em situ</em&gt; Hibridização de tecidos de rim fixadas em formalina

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA ...