We thank Hongjae Sunwoo for helpful advice for oligonucleotide design in RNA FISH. We also thank Serenity Curtis for editing the manuscript. N.Y. was supported by a Postdoctoral Fellowship for Research Abroad of the Japan Society for the Promotion of Science (JSPS). This work was supported by the NIH (RO1-GM102184), the March of Dimes Research Foundation (#6-FY12-337), and the Developmental Fund and Trustee Grant at Cincinnati Children's Hospital Medical Center to Y.O.

1. Preparação Probe <ol><li> Obter vários oligonucleotídeos únicas em Xist (oligonucleotídeos 20-30 nucleotídeos de comprimento, 63-65 ºC de temperatura de fusão, a Tabela 1) com uma modificação 5&#39;-amino e suspender em água. </li><li> Piscina quantidades equimolares de oligonucleotídeos com 5&#39;-amino modificação (total 4,5 g) e etiqueta com corante fluorescente amino-reactivo seguindo as instruções do fabricante. <ol><li> Dissolve-se os oligonucleótidos reunidos no final de 5 ul de água isenta de nuclease e adicionar 3 mL de 1 M de bicarbonato de sódio à solução de oligonucleótido. </li><li> Adicionar 2 mL de DMSO para o frasco de corante e vortex brevemente reactivo com amina. </li><li> Misturar a solução de oligonucleótido com o corante reactivo em DMSO e incubar a mistura à temperatura ambiente durante 1 h no escuro. </li></ol></li><li> Purifica-se as sondas oligonucleotídicas marcadas com fluorescência por coluna G-25 seguido de precipitação com etanol. <ol><li&gt; Centrífuga coluna G-25 a 750 xg durante 1 min para remover o excesso de tampão de armazenamento. </li><li> Adicionar 40 ul livre de nuclease de água à mistura de rotulagem produzido no Passo 1.2.3 e aplicar à coluna de G-25. Centrifugar a 750 xg por 2 min. </li><li> Adicionar 50 ul de água livre de nuclease, 10 ul de acetato de sódio 3M (pH 5,2) e 250 ul de etanol a sonda purificada a partir do passo 1.3.2. Armazenar a -80 ° C durante 30 min e centrifuga-se a 21.000 xg durante 15 min a 4 ° C. </li><li> Lavar os oligonucleótidos precipitados uma vez com 1 ml de etanol a 70%. </li><li> Re-suspender em 50 ul de água isenta de nuclease ou TE (10 mM Tris-HCl, pH 7,5; EDTA 1 mM) para uma concentração final de aproximadamente 10 pM. </li></ol></li><li> Dilui-se as sondas de oligonucleótidos marcados com fluorescência para uma concentração final de 250 nM com tampão de hibridação (formamida a 10%; 2 x SSC [NaCl 300 mM; citrato de sódio 30 mM], 2 mg / ml de BSA, 10% de sulfato de dextrano). NOTA: Com este protocolo FISH rápida, oligonucleotídeo pmantos de Xist foram concebidos e utilizados com uma concentração mais elevada do que o protocolo normal de FISH utilizando sondas oligonucleotídicas, a fim de encurtar o tempo de incubação para a hibridação. </li></ol> 2. Deslize Preparação NOTA: As lâminas podem ser preparados para imuno-FISH utilizando o rato-tronco embrionárias (ES) ou corpo embryoid (EB) diferenciação de células 20, 21 ou por cultivá-las diretamente em um slide tratados com poli-lisina ou usando cytospin com suspensão de células. Aqui, preparação da lâmina por cytospin é mostrado. <ol><li> Aspirar o meio de cultura em um prato de 6 poços. Lavar com PBS (temperatura ambiente), e aspirado cuidadosamente. </li><li> Adicionar 0,5 ml de tripsina para o prato de 6 poços, incubar durante 5-10 min a 37 ° C. </li><li> Adicionar 5 ml de meio e dispersar as células por suavemente pipetando para cima e para baixo várias vezes. </li><li> Transferir as células para um tubo de 15 ml, centrifugar durante 5 minutos a 200 xg à temperatura ambiente. </li><li> Descartar o meio de umd suavemente suspender as células em 2 ml de PBS. </li><li> Contagem de células utilizando um hemocitómetro, e dilui-se a suspensão celular a 4 x 10 5 células / ml por PBS. </li><li> Montar uma corrediça, um cartão de filtro e uma câmara com um grampo e colocá-los dentro das ranhuras adequadas no rotor do cytospin. Adicionar 250 mL de cada amostra preparada no passo 2.6 em cada câmara das unidades montadas na cytospin. Centrifuga-se a 1.500 rpm durante 10 minutos à temperatura ambiente. </li><li> Encher frascos de 3 coplin sobre gelo com 40 mL de cada um de tampão PBS, CSK gelado (10 mM PIPES, pH 6,8; NaCl 100 mM; sacarose 300 mM; MgCl2 3 mM) e tampão CSKT (CSK com tampão de 0,5% de Triton X-100), respectivamente. </li><li> Após a citocentrifugação está terminado, rapidamente transferir a corrediça em PBS gelado e incubar durante 5 min. </li><li> Transferir as lâminas para tampão CSK gelada e incubar durante 1 min. </li><li> Transferir as lâminas para tampão CSKT gelada e incubar durante 5 min. </li><li> Transferir as lâminas de volta para gelada CSK buffer e incubar durante 1 min. </li><li> Transferir as lâminas em 4% de paraformaldeído em PBS e incubar durante 10 min à temperatura ambiente. </li></ol> 3. Imunofluorescência <ol><li> Colocar as lâminas em um frasco coplin com PBST (1x PBS com 0,1% de Tween-20) durante vários segundos. </li><li> Colocar as lâminas em uma inclinação permitindo tampão restante flua no escorregador. Limpe cuidadosamente o excesso de líquido para fora do slide e colocá-lo em uma caixa de pipeta-tip vazio com água. Sobrepor a lâmina com 40 ul de solução de bloqueio (PBS 1x, 1% BSA, 0,1% de Tween-20), colocar suavemente uma lamela, e incubar durante 10 min à temperatura ambiente. </li><li> Retirar as lamelas, remover a solução de bloqueamento e adicionar 40 ul de anticorpo de histona H3K27me3 primário diluído para uma concentração de 1: 500 em solução de bloqueio. Coloque a lamela de volta sobre a lâmina e incubar durante 30 min à temperatura ambiente. NOTA: Um inibidor RNase na solução de bloqueio pode ser útil para preservar RNA neste RNA FISProtocolo de H quando são utilizados anticorpos policlonais. </li><li> Wash deslizar 2 vezes em frascos coplin cheios de PBST, durante 5 minutos cada, agitando suavemente durante a lavagem. </li><li> Colocar as lâminas em uma inclinação permitindo tampão restante flua no escorregador. Limpe cuidadosamente o excesso de líquido e coloque a lâmina de volta na caixa de ponta. Sobreposição com 40 ul do anticorpo secundário (1: 500) diluídos em solução de bloqueio, colocar uma lamela, e incubar durante 15 min no escuro. Para os passos seguintes, manter o slide no escuro em todos os momentos. </li><li> Lavar duas vezes em frascos Coplin com PBST como no passo 3.4. </li></ol> 4. FISH RNA com Oligonucleotide Sondas <ol><li> Pré-aquecer uma caixa de pipeta-tip vazio com água na parte inferior para evitar slides de secagem a 37 ° C por RNA FISH no passo 4.3. </li><li> Colocar as lâminas em uma inclinação permitindo tampão restante flua no escorregador. Limpe cuidadosamente o excesso de líquido para fora do slide a partir do passo 3.6 e coloque a lâmina na caixa de pipeta-ponta com water na parte inferior. Adicionar 500 ul de tampão de lavagem de FISH (10% de formamida em 2 x SSC) sobre a lâmina e incubar durante 5 min à temperatura ambiente. </li><li> Remover solução de lavagem FISH, adicione 8 mL de sondas de oligonucleotídeos para o slide, coloque uma lamela, e transferir o slide para uma caixa de pipeta-ponta pré-aquecido com água na parte inferior; incubar a 37 ° C durante 1-2 horas. </li><li> Coloque 3 frascos coplin pré-aquecido em banho-maria a 37 ° C com tampão de lavagem FISH, tampão de lavagem FISH com DAPI, e 2x SSC. </li><li> Depois de 1-2 incubação hr para RNA FISH, coloque o slide no tampão de lavagem FISH pré-aquecido. </li><li> Incubar a lâmina durante alguns minutos até que a lamela cai fora da lâmina. </li><li> Colocar a lâmina de volta para o tampão de lavagem, e continuar a incubação durante 5 min. </li><li> Transferir as lâminas para outro frasco coplin com tampão de lavagem pré-aquecido FISH com 0,5 ng / ml DAPI e incubar por 5 min. </li><li> Transferir as lâminas para outro frasco coplin com pré-aquecido 2x SSC e incubar por 5 min. </li><li> Colocar as lâminas em uma inclinação permitindo tampão restante flua no escorregador. Limpe cuidadosamente o excesso de líquido e coloque a lâmina de volta em uma caixa de ponta seca. Adicione 4 l de antifade com DAPI para o slide e colocar uma lamela. </li><li> Selar a lamela com unha polonês e observar ao microscópio. A corrediça pode ser armazenado a 4 ° C no escuro, por alguns meses. </li></ol>

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No conflicts of interest declared.

Neste artigo, apresentamos um protocolo de imuno-FISH rápida, que leva menos de 5 horas para completar a preparação slide, Xist RNA FISH, e imunofluorescência para H3K27me3. Em comparação com a abordagem geral imuno-FISH para Xist RNA de detecção, que normalmente precisa de incubação durante a noite para RNA FISH, este protocolo não só reduz significativamente o tempo gasto, mas também melhora a sensibilidade imunológica-FISH usando sondas de oligonucleotídeos. Uma vez que é ...

Mammalian inactivação do cromossoma X (XCI) é uma estratégia para compensar o desequilíbrio de dosagem do gene ligado ao X e XY entre XX, em que um dos dois cromossomas X em fêmeas é transcricionalmente inactiva 1. Inativação do cromossomo X é um grande sistema modelo para RNA longo não-codificante (lncRNA) pesquisa. Inativação do cromossomo X é regulada por vários lncRNAs, e tem sido amplamente estudado ao longo das últimas décadas para descobrir os mecanismos de crosstalk entre lncRNAs, transcrição, estrutura da cromatina e organização nuclear 2, 3. O centro X inactivação (XIC) localizado no cromossoma X é um locus genético complexo composto de um número de genes que produzem não codificantes RNAs 4. X-inativo transcrição específico (Xist) lncRNA em mamíferos eutherian é um desses lncRNA que desempenha um papel crucial na inativação do cromossomo X 5,6. Transcrições XIST rodeiam o local do future Xi para iniciar inativação do cromossomo X, e aparecem como uma nuvem quando visualizada usando RNA FISH; esta formação é referido como o &quot;Xist Nuvem&quot; 7. Desde Xist ARN interage com vários enzimas de modificação de cromatina, a co-localização das nuvens XIST com diferentes modificações epigenética para cromatina silenciosa e repressor de transcrição é observado durante a inactivação do cromossoma X 8. Por exemplo, Xist ARN interage com Polycomb complexo repressor 2 (PRC2) que é responsável pela H3K27me3 e induz um estado repressivo 9 cromatina. A ocorrência da nuvem Xist no Xi e sua co-localização com a modificação intensiva H3K27me3 representa uma paisagem heterocromatina facultativa do Xi 10,11. Técnicas de citogenética, como o DNA / RNA FISH têm percorreu um longo caminho desde o método tradicional utilizando sondas radiomarcados 12 às técnicas recentes e avançados, com maior sensibilidade eimagens fluorescentes usando várias sondas de oligonucleotídeos 13,14. PEIXES ADN / ARN acoplado com a imunofluorescência foi rotineiramente usado como uma ferramenta para compreender citológico organização espaço-temporal nuclear, ARN de localização, a estrutura da cromatina e modificações. A preparação mais padrão sonda para RNA FISH envolve o uso de plasmídeo ou cromossoma artificial (BAC) clones bacterianas e sua rotulagem subsequente quer com tradução nick ou iniciação ao acaso 15. No entanto, cerca de 70% dos genes em ratos e 40% de genes em humanos mostram uma sobreposição de transcritos de sentido e anti-16, exigindo assim um método FISH específico de cadeia, a fim de distinguir o sentido e anti-transcrições. Em sondas de ARN transcritos in vitro (ribossonda ) são frequentemente utilizados para específicos de cadeia de ARN FISH 17,18; No entanto, isto envolve a preparação de um clone de plasmídeo ou com o produto de PCR de T7, SP6 ou T3 promotor e ribossondas sintetizar. Além disso, ribossondas derivado genomic DNA ou cDNA geralmente contêm regiões não-específicas e elementos repetitivos, que resultam em alto ruído de fundo. Outra questão é que ribossondas, que são algumas centenas de nucleótidos de comprimento e contêm vários fluoróforos, não pode penetrar de forma eficiente para o núcleo. Para contornar este, várias sondas de oligonucleótidos mais curtos marcados com um fluoróforo único no final foram desenvolvidos que possuem uma boa sensibilidade, a intensidade do sinal uniforme, e facilidade de purificação e de manuseamento 14. Além disso, os oligonucleótidos de ADN são geralmente mais estáveis ​​do que o RNA. Nós aplicamos uma estratégia similar de usar oligonucleotídeos em Xist RNA FISH 19 com imunofluorescência para entender a dinâmica epigenéticas do Xi induzidas por Xist lncRNA durante o processo de inativação do cromossomo X. Este protocolo descreve a criação de sondas de oligonucleótidos e preparação de células apropriada, bem como a utilização de imunofluorescência e ARN FISH. Xist RNA FISH usando oligonucle múltiplaotides é o custo abordagem eficaz a longo prazo, se Xist RNA FISH é realizada rotineiramente em um laboratório. Esta técnica pode ser usada para identificar lncRNAs em células, ao mesmo tempo mapeando sua co-localização com modificações epigenética ou factores. Uma das principais vantagens do protocolo é a capacidade de modificá-lo facilmente para atender um de interesses de pesquisa.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">oligonucleotides with 5&#8217;-amine modification</td>
			<td style="width:187px;">IDT</td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Alexa Fluor 488 Reactive Dye</td>
			<td style="width:187px;">Life Technologies</td>
			<td style="width:119px;">A32750</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">MicroSpin G-25 columns</td>
			<td style="width:187px;">GE Healthcare</td>
			<td style="width:119px;">27-5325-01</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Cytospin 2</td>
			<td style="width:187px;">Shandon</td>
			<td align="right" style="width:119px;">59900102</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Bovine Serum Albumin, Molecular Biology Grade (for hybridization buffer)</td>
			<td style="width:187px;">Roche</td>
			<td style="width:119px;">10715859103</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Histone H3K27me3 antibody</td>
			<td style="width:187px;">Active Motif</td>
			<td style="width:119px;">61017</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Accutase</td>
			<td style="width:187px;">Innovative Cell Technologies</td>
			<td style="width:119px;">AT104</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Alexa Fluor 555 Goat Anti-Mouse, highly cross-adsorbed</td>
			<td style="width:187px;">Life Technologies</td>
			<td style="width:119px;">A21424</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">ProLong Gold antifade reagent</td>
			<td style="width:187px;">Life Technologies</td>
			<td style="width:119px;">P36931</td>
		</tr>
	</tbody>
</table>

quick fluorescent in situ hybridization protocol for xist rna combined with immunofluorescence of histone modification in x-chromosome inactivation

Combinando ARN hibridização in situ fluorescente (FISH) com a imunofluorescência (imuno-FISH) cria uma técnica que pode ser empregue no nível da célula individual para detectar a dinâmica espacial de localização de ARN com visão simultânea para a localização de proteínas, modificações epigenética e outros detalhes que pode ser destacado por imunofluorescência. Inativação do cromossomo X é um paradigma para longo RNA não-codificante (lncRNA) silenciamento gênico mediado. Transcrição específico (Xist) acumulação lncRNA X-inactiva (chamado uma nuvem Xist) em um dos dois X-cromossomas em fêmeas de mamíferos é um passo crítico para iniciar a inactivação do cromossoma X. RNA Xist direta ou indiretamente interage com várias enzimas modificadoras da cromatina e introduz paisagens epigenéticas distintas para os inactivos cromossomo X (Xi). Uma característica conhecida da epigenética Xi representa a histona H3 trimetil-lisina 27 modificação (H3K27me3). Aqui, descrevemos um imuno-FISH simples e rápidaprotocolo para a detecção Xist RNA usando RNA FISH com várias sondas de oligonucleotídeos juntamente com imunofluorescência de H3K27me3 para examinar a localização dos Xist RNA e modificações epigenéticas associadas. Usando sondas oligonucleotídicas resulta em um tempo de incubação mais curto e de detecção mais sensível do Xist ARN em comparação com in vitro transcritos de sondas de ARN (ribossondas). Este protocolo fornece uma poderosa ferramenta para a compreensão da dinâmica de lncRNAs e sua modificação epigenética associados, estrutura da cromatina, organização nuclear e de regulação da transcrição.

Imagens representativas de rápida imuno-FISH são apresentados na Figura 1A. A co-localização do sinal de Xist ARN nuvem e H3K27me3 no Xi foi detectado na diferenciação das células do sexo feminino. No dia 12 após a diferenciação, mais de 90% das células tinha uma nuvem EB Xist (Figura 1B). Sondas oligonucleotídicas curto eficientemente penetrou nos núcleos, levando a uma visualização de quase todos H3K27me3 sinais co-localizada com Xist ARN (Figura 1C; 97...

Watch this Scientific Journal Video about Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation at JoVE.co

Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation

We developed an easily customized strand-specific fluorescent in situ hybridization (FISH) protocol combined with immunofluorescence. This allows for a detailed examination of RNA dynamics with simultaneous insight into the chromatin structure, nuclear organization, and transcriptional regulation at the single cell level.

Fluorescent Rápida<em&gt; In Situ</em&gt; Protocolo de hibridação para Xist RNA combinado com imunofluorescência de Histone Modificação na inativação do cromossomo X

Combining RNA fluorescent in situ hybridization (FISH) with immunofluorescence (immuno-FISH) creates a technique that can be employed at the single cell ...

quick-fluorescent-situ-hybridization-protocol-for-xist-rna-combined

Research

JoVE Journal

Biology

Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation

13.8K visualizações.  Cincinnati Children's Hospital Medical Center. We developed an easily customized strand-specific fluorescent in situ hybridization (FISH) protocol combined with immunofluorescence. This allows for a detailed examination of RNA dynamics with simultaneous insight into the chromatin structure, nuclear organization, and transcriptional regulation at the single cell level. 

Vídeo: Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation

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

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

The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of tissue specific expression is limited because of problems in dissecting individual tissues. Expression data needs to be confirmed and complemented with expression patterns using e.g. in situ hybridization, a technique used to localize cell specific mRNA expression. The in situ hybridization method is laborious, time-consuming and often requires extensive optimization depending on species and tissue. In situ experiments are relatively more difficult to perform in woody species such as the conifer Norway spruce (Picea abies). Here we present a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including P. abies. With just a few adjustments, including altered RNase treatment and proteinase K concentration, we could use the protocol to study tissue specific expression of homologous genes in male reproductive organs of one gymnosperm and two angiosperm species; P. abies, Arabidopsis thaliana and Brassica napus. The protocol worked equally well for the species and genes studied. AtAP3 and BnAP3 were observed in second and third whorl floral organs in A. thaliana and B. napus and DAL13 in microsporophylls of male cones from P. abies. For P. abies the proteinase K concentration, used to permeablize the tissues, had to be increased to 3 g/ml instead of 1 g/ml, possibly due to more compact tissues and higher levels of phenolics and polysaccharides. For all species the RNase treatment was removed due to reduced signal strength without a corresponding increase in specificity. By comparing tissue specific expression patterns of homologous genes from both flowering plants and a coniferous tree we demonstrate that the DIG in situ protocol presented here, with only minute adjustments, can be applied to a wide range of plant species. Hence, the protocol avoids both extensive species specific optimization and the laborious use of radioactively labeled probes in favor of DIG labeled probes. We have chosen to illustrate the technically demanding steps of the protocol in our film.
Anna Karlgren and Jenny Carlsson contributed equally to this study.
Corresponding authors: Anna Karlgren at Anna.Karlgren@ebc.uu.se and Jens F. Sundstr&#246;m at Jens.Sundstrom@vbsg.slu.se

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

We describe a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including Norway spruce. With just a few adjustments, including altered RNase treatment and proteinase K concentration, the protocol may be used in studies of different tissues and species.

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 ...

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 ...

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 ...

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 ...