We thank Ueli Grossniklaus (University of Z&#252;rich) for technical and financial support. We are thankful to Valeria Gagliardini, Christof Eichenberger, Arturo Bolanos and Peter Kopf for general lab support. This research was funded by the University of Z&#252;rich, grants from the Swiss National Foundation to CB (31003A_130722) and Ueli Grossniklaus (31003A_141245 and 31003AB-126006), and the Agence Nationale de la Recherche to DG (Programme ANR-BLANC-2012).

O procedimento é descrito no fluxo de trabalho na Figura 1, e a configuração para a dissecção e a incorporação de tecidos são apresentados na Figura 2. 1. Fixação do tecido <ol><li> Recolher 20-30 carpelos em um tubo de microcentrífuga contendo feita recentemente tampão fixador BVO em gelo. </li><li> Fixar o tecido 30 min com agitação suave à temperatura ambiente. </li><li> Girar os tubos contendo as carpelos em fixador em uma microcentrífuga de bancada 1 min a 400 x g. </li><li> Retire cuidadosamente o tampão fixador e adicionar 1 ml de PBT, colocar os tubos no gelo. </li></ol> 2. Dissection and Embedding <ol><li> Preparar cinco tubos de Eppendorf com 200 ul de cada uma feita recentemente, 5% de mistura de acrilamida. </li><li> Prepare cinco lâminas Superfrost pré-limpos com etanol 70% e marcadas com um lápis. </li><li> Descongelar uma alíquota de 20% de EPA e 20% NaPS cada, em gelo. </li><li> Toma 4-5 carpelos com uma ponta de corte-end, lugarlos numa lâmina de limpeza, remover o excesso de líquido. </li><li> Faça cortes longitudinais com uma agulha fina e retire as paredes carpelo para liberar fileiras de óvulos como mostra a Figura 2, evitar a secagem cobrindo com PBS (não mais de 10 mL). </li><li> Adicione rapidamente e misturar 12 ml, 12 NaPS APS ul com uma alíquota de 200 mL da mistura de acrilamida. </li><li> Adicionar 30 ul da acrilamida activado para os óvulos dissecados. </li><li> Cubra com uma lamela mm 20 x 20, vamos polimerização à temperatura ambiente, 45-60 min. </li><li> Retirar as lamelas usando uma lâmina de barbear. Nesta fase, as amostras podem ser mantidas durante a noite a 4 ° C num frasco Coplin contendo PBS. </li></ol> 3. Processamento de Tecidos NOTA: Todas as etapas, exceto 3.2.1, 3.2.3, 3.3.2 e 3.4.3 são realizadas em frascos Coplin com 80 ml da solução sob o capô química à temperatura ambiente. Slides são transferidos com um de ponta plana fórceps. <ol><li> Clarificação tecido efixação: <ol><li> Incubar 5 min em metanol. </li><li> Incubar 5 min em etanol. </li><li> Incubar 30 minutos em etanol: xileno (1:1). </li><li> Incubar 5 min em etanol. </li><li> Incubar 5 min em metanol. </li><li> Incubar 15 minutos em metanol e PBT (1:1), complementado com 2,5% de formaldeído. </li><li> Lavar 2 x 10 minutos em PBT. Neste estágio, os slides podem ser mantidas durante a noite a 4 ° C. </li></ol></li><li> Digestão da parede celular: <ol><li> Descongelar uma aliquota da mistura de digestão da parede celular em gelo. </li><li> Tome um slide da jarra de Coplin, drenar o excesso de líquido, colocando-o verticalmente sobre uma toalha de papel. </li><li> Adicione 100 ml de mistura de digestão da parede celular sobre a almofada de acrilamida e cobrir com uma lamela mm 23 x 46. Repita o procedimento para os demais slides. Incubar durante 2 horas a 37 ° C numa câmara húmida (descrito em Materiais). </li><li> Lavar as lâminas 2 x 5 minutos em PBT. </li></ol></li><li> RNase A de tratamento: <ol><li> Tome um slide da jarra de Coplin, escorra ªe o excesso de líquido, como antes. </li><li> Incubar cada lâmina com 100 ul de ARNase A a 100 ng / mL em PBS com 1% de Tween-20 durante 1 hora a 37 ° C numa câmara húmida. </li><li> Lavar as lâminas durante 2 x 5 minutos em PBT. </li></ol></li><li> Pós-fixação e permeabilização: <ol><li> Post-correção para 20 min no recentemente feito PBT-F. </li><li> Lavar as lâminas por 10 minutos em PBT. </li><li> Permeabilizar durante 2 horas em PBS com 2% de Tween-20 a 4 ° C. </li><li> Lavar as lâminas por 2 x 5 minutos em PBT. </li></ol></li></ol> 4. Imunocoloração NOTA: Para este passo, a concentração óptima do anticorpo primário tem de ser testada por meio de diferentes diluições (1:200, 1:500, 1:1000) dos anticorpos. <ol><li> Incubar cada lâmina com 100 uL de anticorpo primário diluído em PBS com 0,2% de Tween-20 durante 12-24 horas a 4 ° C. </li><li> Lavar as lâminas em PBT durante 2-4 horas à temperatura ambiente, sob agitação suave. </li><li> Aplique o1:200 de anticorpo secundário em PBS + 0,2% de Tween-20 durante 24 horas a 4 ° C. </li><li> Lavar as lâminas em PBT durante uma hora à temperatura ambiente, sob agitação suave. </li><li> Contracoloração com 10 ug / ml de iodeto de propidio em PBS durante 15 minutos, depois enxaguar 15 min em PBS, sob agitação suave, à temperatura ambiente. </li><li> Monte em anti-desbotamento montagem líquido suplementado com 10 mg / ml de iodeto de propídio. Deixe o meio de montagem endurecer por 1 hora antes de adquirir imagens por CLSM. </li></ol> 5. Imagens quantitativas <ol><li> A aquisição de imagens: <ol><li> Adquirir imagens de alta resolução usando CLSM, o ideal é usar um modo de digitalização de ressonância, o que permite uma melhor preservação dos sinais fluorescentes sobre imagem prolongada 23, e uma lente de imersão 63X de glicerol. </li><li> Teste os parâmetros de aquisição, como a intensidade do laser, o ganho, pinhole, tamanho voxel e fator de zoom no início do experimento para definir um procedimento de aquisição padrão a seguir rigorosamente todotodos os slides para medições quantitativas consistentes. </li><li> Determinar a ausência de cross-talk entre fluorocromos. Se estiver presente, configurar uma verificação seqüencial. Adquirir imagens de transmissão separadamente e não simultaneamente. </li><li> Realize serial, aquisição de imagem tridimensional com resolução mais alta possível nos dimensões X e Y e com oversampling 2x na dimensão z (regra de Nyquist). </li></ol></li><li> Processamento de imagem: <ol><li> Reconstruir imagens de série em três dimensões usando o software de fonte comercial ou aberto. </li><li> Definir superfícies de contorno ao redor de cada núcleo (ou célula) de interesse em 3D. </li><li> Quantificar a fluorescência em cada canal, como a soma da intensidade dos pixels de cada objecto. </li><li> Exportar os dados para o Excel para análises estatísticas. Normalizar sinais de anticorpos contra os sinais de coloração, por exemplo DNA. </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

Em plantas com flores, a linhagem reprodutor feminino é cercado por várias camadas de células, incluindo o nucelo e os tegumentos óvulo, tornando assim a coloração citológica em toda montagem tecnicamente desafiador. Aqui apresentamos um protocolo eficiente que permite a preparação e processamento de um grande número de óvulos adequados para a coloração citológica como imunomarcação, a coloração do DNA e hibridização fluorescente in situ no todo-mount. Foi utilizado com sucesso para...

Em plantas com flores, o estabelecimento de linhagens de reprodução inicia-se com a diferenciação de células musculares lisas, MMC feminino e célula-mãe microspore masculino. A MMC desenvolve a partir de uma célula nucelar sub-epidérmica na ponta distal do primórdio óvulo, e a célula mãe micrósporos se desenvolve a partir de tecido esporogênico na antera lóculo, que estão localizados no fundo dos órgãos florais 1. SMCs sofrer meiose para produzir esporos haplóides, que depois dão origem aos gametófitos sobre mitose. O gametófito feminino ou saco embrionário, consiste de uma célula-ovo, uma célula central, dois sinérgides e três antípodas. O gametófito masculino, ou o pólen, é composto de uma célula vegetativa e duas células espermáticas. Enquanto o gametófito masculino continua a ser um objeto de estudo-de-relativamente acessível, o gametófito feminino é incorporado dentro do óvulo, por sua vez incluído no carpelo flor, e, assim, coloca desafios específicos para análises moleculares e citológicas. Recentemente, no entanto, de laser assistidamicrodissection ofereceu uma solução elegante que permite análises transcriptomic no MMC e células gametófitos femininos 2-4. Em adição à expressão do gene candidato analisa, por exemplo, utilizando ARN de hibridação in situ ou ensaios de gene repórter, análises citológicas permite investigar a dinâmica de componentes celulares endógenas utilizando coloração celular directa específica ou imunocoloração indirecta. Particularmente, coloração citogenética utilizando FISH e coloração do DNA, juntamente com imunocoloração de modificações da cromatina ou componentes de cromatina são abordagens centrais para elucidar a dinâmica da cromatina e organização nuclear em Arabidopsis 5. Normalmente, a meiose implica dinâmica cromossômicas específicas que tem sido bem investigados em planta macho meiócitos 6,7; ainda em grande escala, específico de célula cromatina reorganização, provavelmente refletindo a reprogramação epigenética dinâmica foi descrito durante o desenvolvimento do pólen 8-10. Por outro lado, devidoà relativa inacessibilidade do meiocyte fêmea e gametófito, estas investigações continuam tecnicamente difíceis de aplicar, e exigem muitas vezes a dissecção de corte ou manual e digestão enzimática (ver abaixo). Além disso, o prevalente falta de clareza óptica em todo o monte é um obstáculo para imagens de alta resolução de células reprodutivas em óvulos intactas. Um método clássico para a análise citológica da organização cromossômica em óvulos inteiros de montagem usa coloração de Feulgen 11-13. Trata-se hidrólise ácida (utilizando um ácido hipocloroso) do ADN o que resulta em desnaturação das proteínas e, assim, causa a destruição da estrutura da cromatina. Alternativamente, organização cromossomo em meiócitos femininos e células gametófitos podem ser observados por meio da coloração DAPI e imunocoloração em seções semi-finas ou sacos de embriões dissecados e MMC (por exemplo ver 14-18). Claramente, no entanto, a dissecção manual e corte pode ser de trabalho intensivo eimpede a análise qualitativa e quantitativa de um grande número de epítopos de cromatina. Aqui nós fornecemos um protocolo eficiente para preparar um grande número de óvulos Arabidopsis adequados para uma variedade de coloração citológica jusante em todo-mount. Em breve, botões de flores são incubadas numa solução fixadora, linhas de óvulos são dissecados do carpelo e incorporado em acrilamida na corrediça, como feito para meiócitos pólen 19,20. Os óvulos são incorporados mais afastada e fixadas em metanol, etanol, e xileno, antes da digestão da parede celular e permeabilização. São discutidas possíveis variações destes passos. As amostras podem em seguida ser utilizado para a coloração de ADN, a imunocoloração, e FISH. O modo de preparação é eficiente e permite a paralela conjunto experimental (até 16 lâminas pode ser preparado de um dia para análise a jusante diferente). Os tratamentos descritos permitem sinais homogêneas em todo-mount e bem preservado histológico, celular,e organização nuclear em células reprodutivas e células nucelares vizinhas que beneficiam comparações qualitativas e quantitativas entre os tipos de células. Calibrado, imagens de alta resolução baseado em CLSM seguido de reconstrução em 3 dimensões permite medições quantitativas significativas de sinais fluorescentes. Temos utilizado com sucesso este procedimento para analisar a dinâmica da cromatina no MMC diferenciando 21 e desenvolvendo gametófito feminino 22; Apresentamos aqui resultados representativos de análise heterochromatin, immunostaining cromatina, GFP imunocoloração e peixes em todo-montagem óvulos. Além disso, acreditamos que o nosso protocolo irá ser adequado para outros tecidos e espécies vegetais.

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			<td style="width:481px;">2mM EGTA, pH7.5, 1% (v/v) Formaldehyde,10% DMSO, 1&#215; PBS, 0.1% Tween-20</td>
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			<td height="42" style="height:42px;">30% acrylamide:bisacrylamide&#160;</td>
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			<td style="width:481px;">3g acrylamide, 0.33g bisacrylamide, 1&#215; PBS (prepare 10ml, store at 4&#176;C)</td>
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			<td height="42" style="height:42px;">200mL&#160; 5% acrylamide mix in PBS&#160;</td>
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			<td style="width:119px;"></td>
			<td style="width:481px;">34mL 30% acrylamide:bisacrylamide, 166mL 1&#215; PBS (make fresh from 30% stock)</td>
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			<td height="42" style="height:42px;">20% ammoniumpersulfte&#160;</td>
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			<td style="width:481px;">0.2g ammoniumpersulfte, 1mL sterile water (prepare aliquots with 1mL and store at -20&#176;C)</td>
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			<td height="42" style="height:42px;">20% sodium sulfite&#160;</td>
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			<td style="width:119px;"></td>
			<td style="width:481px;">0.2g sodium sulfite, 1mL sterile water (prepare aliquots with 1mL and store at -20&#176;C)</td>
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			<td style="width:135px;"></td>
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			<td style="width:481px;">0.5% (w/v) cellulase, 1% (w/v) driselase, 0.5% (w/v) pectolyase</td>
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			<td>F1635</td>
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			<td>D5879</td>
			<td style="width:481px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Tris</td>
			<td>Amaresco</td>
			<td>0497</td>
			<td style="width:481px;"></td>
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			<td>Schaurlau</td>
			<td>ET00102500</td>
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			<td>1794</td>
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			<td>D8037</td>
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			<td>Sigma</td>
			<td>E-4378</td>
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			<td height="21" style="height:21px;">acrylamide</td>
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			<td style="width:481px;"></td>
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			<td height="21" style="height:21px;">bisacrylamide</td>
			<td>Sigma</td>
			<td>M2022</td>
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			<td>Sigma</td>
			<td>A9164</td>
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			<td>71988</td>
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			<td height="21" style="height:21px;">Anti-trimethyl-Histone H3 (Lys4)</td>
			<td>Upstate</td>
			<td>07-473</td>
			<td style="width:481px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Anti- monomethyl-Histone H3 (Lys27)</td>
			<td>Upstate</td>
			<td>07-448</td>
			<td style="width:481px;"></td>
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		<tr height="21">
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			<td>A11008</td>
			<td style="width:481px;"></td>
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			<td>P36934</td>
			<td style="width:481px;"></td>
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			<td height="21" style="height:21px;">Propidium iodide</td>
			<td>Sigma</td>
			<td>P4170</td>
			<td style="width:481px;">toxic</td>
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			<td>Sigma</td>
			<td>D9542</td>
			<td style="width:481px;">toxic</td>
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			<td height="21" style="height:21px;">RNAse A</td>
			<td>Roche</td>
			<td>10109169001</td>
			<td style="width:481px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Coplin jar</td>
			<td>Huber &#38; CO</td>
			<td>10.055</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Forceps</td>
			<td>DUMONT BIOLOGY</td>
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			<td style="width:481px;"></td>
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			<td height="21" style="height:21px;">Shaker</td>
			<td>Heidolph</td>
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			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td style="width:481px;">A plastic box with damp paper towel inside, a plastic support is put into the box for supporting the slides and keep slides from the water.</td>
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			<td>Thermo Fisher</td>
			<td>J1800AMNZ</td>
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			<td height="21" style="height:21px;">FISH Tag DNA KIt</td>
			<td>Invitrogen</td>
			<td>F32947</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:259px;">GFP booster</td>
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			<td style="width:119px;"></td>
			<td style="width:481px;"></td>
		</tr>
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</table>

an efficient method for quantitative, single-cell analysis of chromatin modification and nuclear architecture in whole-mount ovules in arabidopsis

Em plantas com flores, a transição destino de células somáticas-to-reprodutiva é marcada pela especificação de células-mãe de esporos (PMEs) em órgãos florais da planta adulta. O SMC feminino (célula mãe de megásporo, MMC) diferencia no primórdio óvulo e sofre meiose. O megásporo haplóide selecionado então sofre mitose para formar o gametófito multicelular feminino, o que dará origem aos gametas, o óvulo ea célula central, juntamente com células acessórias. A acessibilidade limitada do MMC, meiocyte e gametófito feminino no interior do óvulo é tecnicamente desafiador para citológico e análises citogenéticas em nível de célula única. Particularmente, imunodetecção directa ou indirecta de epítopos celulares ou nucleares é prejudicada pela falta de penetração dos reagentes no interior da célula vegetal e de uma única célula de imagem é demised pela falta de claridade óptica em todo-montagem tecidos. Assim, desenvolvemos um método eficiente para analisar os nuclorganização orelha e modificação da cromatina em alta resolução de uma única célula em Todo-mount óvulos Arabidopsis incorporados. Ele baseia-se na incorporação de dissecção e óvulos fixos em uma fina camada de gel de acrilamida a uma lâmina de microscópio. Os óvulos embarcados são submetidos a tratamentos químicos e enzimáticos com o objetivo de melhorar a clareza do tecido e permeabilidade aos reagentes de positividade. Esses tratamentos preservar organização, DNA e proteínas epítopos celulares e cromatina. As amostras podem ser utilizadas para diferentes análises citológicas a jusante, incluindo a imunocoloração da cromatina, hibridação in situ fluorescente (FISH), e a coloração de ADN para análise de heterocromatina. Microscopia confocal a laser (CLSM) de imagem, com alta resolução, seguido de reconstrução 3D permite medições quantitativas na resolução de uma única célula.

Nós fornecemos um protocolo robusto para a preparação em grande escala e de processamento de óvulos Arabidopsis adequados para exames citológicos em todo-mount. Graças à incorporação, os óvulos manter uma estrutura 3-dimensional (Figura 3). Além disso, o processamento de tecidos, incluindo esclarecimento de imagem óptico permite estruturas subcelulares em alta resolução. Figura 4 mostra a coloração de DNA em todo primórdios óvulo de montagem onde heterochromat...

Watch this Scientific Journal Video about An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis at JoVE.com

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

Nós fornecemos aqui um protocolo eficiente e confiável para a imunocoloração, hibridação in situ fluorescente, a coloração do DNA seguido de quantitativo, imagem de alta resolução em Arabidopsis thaliana óvulos inteiros de montagem. Este método foi utilizado com sucesso para analisar as modificações da cromatina e arquitectura nuclear.

Um método eficiente para Quantitativa, Análise de células Único de cromatina Modificação e Arquitetura Nuclear em Todo-mount óvulos em<em&gt; Arabidopsis</em

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the ...

an-efficient-method-for-quantitative-single-cell-analysis-chromatin

Research

JoVE Journal

Biology

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

13.1K visualizações.  University of Zürich.  Nós fornecemos aqui um protocolo eficiente e confiável para a imunocoloração, hibridação in situ fluorescente, a coloração do DNA seguido de quantitativo, imagem de alta resolução em Arabidopsis thaliana óvulos inteiros de montagem. Este método foi utilizado com sucesso para analisar as modificações da cromatina e arquitectura nuclear. 

Vídeo: An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

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

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment are open and allow cations to flow steadily inwards across the membrane, depolarizing the cell. Light exposure triggers the closure of the CNG channels, blocks the inward cation current flow, and thus results in cell hyperpolarization. Based on the polarity of photoreceptors, a suction recording method was developed in 1970s that, unlike the classic patch-clamp technique, does not require penetrating the plasma membrane 1. Drawing the outer segment into a tightly-fitting glass pipette filled with extracellular solution allows recording the current changes in individual cells upon test-flash exposure. However, this well-established "outer-segment-in (OS-in)" suction recording is not suitable for mouse cone recordings, because of the low percentage of cones in the mouse retina (3%) and the difficulties in identifying the cone outer segments. Recently, an inner-segment-in (IS-in) recording configuration was developed to draw the inner segment/nuclear region of the photoreceptor into the recording pipette 2,3. In this video, we will show how to record from individual mouse cone photoresponses using single-cell suction electrode.

We will show how to record flash responses from single mouse cones using a suction electrode.

Gravações de uma única célula de sucção de cones fotorreceptores rato

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment ...

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 ...

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ binding proteins that initiate Ca2+ signaling cascades. However, we still know little about how stimulus specific Ca2+ signals are generated. The specificity of a Ca2+ signal may be attributed to the sophisticated regulation of the activities of Ca2+ channels and/or transporters in response to a given stimulus. To identify these cellular components and understand their functions, it is crucial to use systems that allow a sensitive and robust recording of Ca2+ signals at both the tissue and cellular levels. Genetically encoded Ca2+ indicators that are targeted to different cellular compartments have provided a platform for live cell confocal imaging of cellular Ca2+ signals. Here we describe instructions for the use of two Ca2+ detection systems: aequorin based FAS (film adhesive seedlings) luminescence Ca2+ imaging and case12 based live cell confocal fluorescence Ca2+ imaging. Luminescence imaging using the FAS system provides a simple, robust and sensitive detection of spatial and temporal Ca2+ signals at the tissue level, while live cell confocal imaging using Case12 provides simultaneous detection of cytosolic and nuclear Ca2+ signals at a high resolution.

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Ca2+ signaling regulates diverse biological processes in plants. Here we present approaches for monitoring abiotic stress induced spatial and temporal Ca2+ signals in Arabidopsis cells and tissues using the genetically encoded Ca2+ indicators Aequorin or Case12.

Medindo espacial e temporal Ca<sup&gt; 2 +</sup&gt; Sinais em Arabidopsis Plantas

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ ...

A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis

Whole mount in situ hybridization (WMISH) is a technique that allows for the spatial resolution of nucleic acid molecules (often mRNAs) within a &#39;whole mount&#39; tissue preparation, or developmental stage (such as an embryo or larva) of interest. WMISH is extremely powerful because it can significantly contribute to the functional characterization of complex metazoan genomes, a challenge that is becoming more of a bottleneck with the deluge of next generation sequence data. Despite the conceptual simplicity of the technique much time is often needed to optimize the various parameters inherent to WMISH experiments for novel model systems; subtle differences in the cellular and biochemical properties between tissue types and developmental stages mean that a single WMISH method may not be appropriate for all situations. We have developed a set of WMISH methods for the re-emerging gastropod model Lymnaea stagnalis that generate consistent and clear WMISH signals for a range of genes, and across all developmental stages. These methods include the assignment of larvae of unknown chronological age to an ontogenetic window, the efficient removal of embryos and larvae from their egg capsules, the application of an appropriate Proteinase-K treatment for each ontogenetic window, and hybridization, post-hybridization and immunodetection steps. These methods provide a foundation from which the resulting signal for a given RNA transcript can be further refined with probe specific adjustments (primarily probe concentration and hybridization temperature).

A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis

The goal of this protocol is to provide users with a set of methods for the high-throughput decapsulation of Lymnaea stagnalis embryos and larvae in preparation for whole mount in situ hybridization, and for subsequent pre- and post-hybridization treatments.

A Monte Whole<em&gt; In Situ</em&gt; Método de hibridação para o molusco gastrópode<em&gt; stagnalis Lymnaea</em

Whole mount in situ hybridization (WMISH) is a technique that allows for the spatial resolution of nucleic acid molecules (often mRNAs) within a ...

CUBIC Protocol Visualizes Protein Expression at Single Cell Resolution in Whole Mount Skin Preparations

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium covering most of our body, and epidermal appendages such as the hair follicles and sweat glands. The epidermis undergoes regeneration throughout life and in response to injury. This is enabled by K14-expressing basal epidermal stem/progenitor cell populations that are tightly regulated by multiple regulatory mechanisms active within the epidermis and between epidermis and dermis. This article describes a simple method to clarify full thickness mouse skin biopsies, and visualize K14 protein expression patterns, Ki67 labeled proliferating cells, Nile Red labeled sebocytes, and DAPI nuclear labeling at single cell resolution in 3D. This method enables accurate assessment and quantification of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines. The CUBIC protocol is the best method available to date to investigate molecular and cellular interactions in full thickness skin biopsies at single cell resolution.

This report describes a CUBIC protocol to clarify full thickness mouse skin biopsies, and visualize protein expression patterns, proliferating cells, and sebocytes at the single cell resolution in 3D. This method enables accurate assessment of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines.

Protocolo CUBIC Visualiza Protein Expression na resolução única célula em preparações e peles, inteiros Mount

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium ...

Visualization of 3D White Adipose Tissue Structure Using Whole-mount Staining

Adipose tissue is an important metabolic organ with high plasticity and is responsive to environmental stimuli and nutrient status. As such, various techniques have been developed to study the morphology and biology of adipose tissue. However, conventional visualization methods are limited to studying the tissue in 2D sections, failing to capture the 3D architecture of the whole organ. Here we present whole-mount staining, an immunohistochemistry method that preserves intact adipose tissue morphology with minimal processing steps. Hence, the structures of adipocytes and other cellular components are maintained without distortion, achieving the most representative 3D visualization of the tissue. In addition, whole-mount staining can be combined with lineage tracing methods to determine cell fate decisions. However, this technique has some limitations to providing accurate information regarding deeper parts of adipose tissue. To overcome this limitation, whole-mount staining can be further combined with tissue clearing techniques to remove the opaqueness of tissue and allow for complete visualization of entire adipose tissue anatomy using light-sheet fluorescent microscopy. Therefore, a higher resolution and more accurate representation of adipose tissue structures can be captured with the combination of these techniques.

The focus of the present study is to demonstrate the whole-mount immunostaining and visualization technique as an ideal method for 3D imaging of adipose tissue architecture and cellular component.

Visualização da estrutura 3D tecido adiposo branco usando a coloração de toda a montagem

Adipose tissue is an important metabolic organ with high plasticity and is responsive to environmental stimuli and nutrient status. As such, various ...

An Easy and Flexible Inoculation Method for Accurately Assessing Powdery Mildew-Infection Phenotypes of Arabidopsis and Other Plants

Reducing crop losses due to fungal diseases requires improved understanding of the mechanisms governing plant immunity and fungal pathogenesis, which in turn requires accurate determination of disease phenotypes of plants upon infection with a particular fungal pathogen. However, accurate disease phenotyping with unculturable biotrophic fungal pathogens such as powdery mildew is not easy to achieve and can be a rate-limiting step of a research project. Here, we have developed a safe, efficient, and easy-to-operate disease phenotyping system using the Arabidopsis-powdery mildew interaction as an example. This system mainly consists of three components: (i) a wooden inoculation box fitted with a removable lid mounted with a stainless steel or nylon mesh of ~50 &#181;m pores for inoculating a flat of plants with fungal spores, (ii) a transparent plastic chamber with a small front opening for minimizing spore escape while conducting inoculation inside, and (iii) a spore-dislodging and distribution method for even and effective inoculation. The protocols described here include the steps and parameters for making the inoculation box and the plastic chamber at a low cost, and a video demonstration of how to use the system to enable even inoculation with powdery mildew spores, thereby improving accuracy and reproducibility of disease phenotyping.

We present a protocol for constructing a simple spore-distribution system consisting of an inoculation box with a ~50 &#181;m mesh and a transparent plastic chamber. This can be used to evenly inoculate plants with powdery mildew spores, thereby enabling accurate and reproducible assessment of disease phenotypes of plants under study.

Um método de inoculação fácil e flexível para avaliar com precisão os fenótipos de infecção por oídio de Arabidopsis e outras plantas

Reducing crop losses due to fungal diseases requires improved understanding of the mechanisms governing plant immunity and fungal pathogenesis, which in ...

Double-Staining Method to Detect Pectin in Plant-Fungus Interaction

Plant cells use different structural mechanisms, either constitutive or inducible, to defend themselves from fungal infection. Encapsulation is an efficient inducible mechanism to isolate the fungal haustoria from the plant cell protoplast. Conversely, pectin, one of the polymeric components of the cell wall, is a target of several pectolytic enzymes in necrotrophic interactions. Here, a protocol to detect pectin and fungal hyphae through optical microscopy is presented. The pectin-rich encapsulation in the cells of coffee leaves infected by the rust fungus Hemileia vastatrix and the mesophyll cell wall modification induced by Cercospora coffeicola are investigated. Lesioned leaf samples were fixed with the Karnovsky solution, dehydrated, and embedded in glycol methacrylate for 2-4 days. All steps were followed by vacuum-pumping to remove air in the intercellular spaces and improve the embedding process. The embedded blocks were sectioned into 5-7 &#181;m thick sections, which were deposited on a glass slide covered with water and subsequently heated at 40 &#176;C for 30 min. Next, the slides were double-stained with 5% cotton blue in lactophenol to detect the fungus and 0.05% ruthenium red in water to detect pectin (acidic groups of polyuronic acids of pectin). Fungal haustoria of Hemileia vastatrix were found to be encapsulated by pectin. In coffee cercosporiosis, mesophyll cells exhibited dissolution of cell walls, and intercellular hyphae and conidiophores were observed. The method presented here is effective to detect a pectin-associated response in the plant-fungi interaction.

This protocol describes a microscopical method to detect pectin in coffee-fungus interaction.