Este trabalho foi apoiado pela concessão do National Science Foundation IOS-0817658.

Os procedimentos de peixe-zebra neste protocolo ter sido aprovado pelo Comitê Institucional de Animal Care and Use (IACUC) da Virginia Commonwealth University. 1. Preparação dos Reagentes <ol><li> 4% PFA / PBS. Pesar 8 g de paraformaldeído (PFA), na hotte. Enquanto ainda no exaustor de fumos, dissolve-se o PFA seco em ~ 80 mL de H2O destilada com agitação e aquecimento para 50 ° C. Enquanto se agita, adicionar 3 - 10 gotas de NaOH 1N fresco até que esteja completamente dissolvido PFA e solução fique límpida. Retire do fogo e adicione 100 ml 2x tampão fosfato salino (PBS). Levar o volume a 200 ml com H2O destilada Arrefecer até à TA e confirmar pH de 7,4 antes da utilização. Guardar no escuro a 4 ° C, mas usar dentro de uma semana. </li><li> Usar 100% de metanol sem quaisquer suplementos. Use etanol a 95%, sem quaisquer suplementos. </li><li> Prepare com fosfato Tween (PBT). Suplemento de solução salina tamponada com fosfato (PBS) com 0,1% de Tween-20. Adicionar 0.5 ml de uma solução de Tween-20 da 20% a 100 ml de 1x PBS. Armazenar à temperatura ambiente. </li><li> Prepara-fosfato tamponada de Triton-X (PbTx). Suplemento de PBS com 0,1% de Triton X-100. Adicionar 0,5 ml de Triton um estoque 20% X-100 a 100 ml de PBS. Armazenar à temperatura ambiente. </li><li> Preparar 10% NGS / PbTx. blocos normais de soro de cabra (NGS) Os locais de ligação não específicos. NGS armazenar em alíquotas a -20 ° C. Para usar, adicionar 1 ml de 9 ml PbTx. </li><li> Preparar 50% de glicerol / PBS. Misturar completamente partes iguais de 2x PBS e 100% de glicerol. Armazenar à temperatura ambiente. </li></ol> Fixação 2. Embrião <ol><li> . (. Por exemplo, β-actina: CAAX-GFP) Breeding Obter tipo selvagem (AB e WIK) ou transgênico embriões através de acasalamentos naturais. Se desejado, injectar embriões com construções ou morfolinos, como descrito 15,16. </li><li> Elevar embriões. Recolhe embriões e incuba-se a 28,5 ° C, na presença de 0,003% de 1-fenil-2-tioureia (PTU) para bloquear a pigmentação, como descrito. 17 </su<html> p&gt; </li><li> Fix. Quando os embriões atingiram o estágio de desenvolvimento desejado, 18, &#160; anestesiar os embriões utilizando MESAB, remover tanta água quanto possível do sistema e adicionar fresco PFA a 4% / PBS durante 3-4 horas à temperatura ambiente. NOTA: Às vezes com menos de 20 horas após a fertilização (hpf), corrigir antes dechorionation. Às vezes após 20 hpf, dechorionate antes de fixação com dois pares de pinças de ponta fina sob um microscópio de dissecação. </li><li> Mudando soluções. Transferência de embriões utilizando uma ampla pipeta furo, como descrito. 17 soluções para a mudança em 1,5 ml tapados microtubos, simplesmente permitindo embriões para resolver pela força da gravidade, sem centrifugação. </li><li> Pós-fixador Após 3 -. 4 horas, remova PFA / PBS, substitua com 100% de metanol e armazenar a -20 ° C durante pelo menos 48 horas. NOTA: Para máxima imunorreactividade de P-CaMK-II, limitar o tempo de armazenamento total de metanol a uma semana. </li></ol>&quot;&gt; 3. Imunomarcação Whole Embriões <ol><li> Coloque um mínimo de 10 embriões para cada condição experimental em tubo de microcentrífuga de 1,5 ml tampado, e rotulá-los. </li><li> Permitir que os embriões para resolver. Remover e descartar o metanol e re-hidratados com lavagens progressivas contendo 0,5 ml de concentrações decrescentes de etanol, tal como indicado no passo seguinte. Cada etapa é uma lavagem de 5 minutos com balanço. </li><li> Progressivamente hidratar com estes 5 Soluções: 66% de etanol / 33% PbTx, 33% de etanol / 66% PbTx, 100% PbTx, 100% PbTx (esta é a primeira repetição de PbTx), 100% PbTx (esta é a segunda repetição PbTx). </li><li> Remover última lavagem PbTx. Adicionar 0,5 ml de 10% NGS / PbTx. Incuba-se durante pelo menos 1 h à temperatura ambiente com agitação suave e em seguida, remover e descartar solução. </li><li> Preparar a solução de anticorpo primário por diluição em 10% NGS / PbTx. Se co-imunocoloração, utilize o anticorpo maior afinidade em primeiro lugar. Para este estudo, incubar com o anticorpo monoclonal de ratinho anti tubulina-acetilado diluído a 1: 500. para example, se existem 10 amostras, combinam 6 ul de solução de anticorpo com 3 ml de estoque de 10% NGS / PbTx e, em seguida, distribuem 0,3 mL desta a cada tubo. </li><li> Adicionar 0,2 - 0,5 ml do anticorpo primário diluído a cada tubo de mergulhar todos os embriões. Incube com agitação suave O / N à temperatura ambiente. </li><li> Na parte da manhã, remova e elimine a solução de anticorpo. Adicionar 0,5 ml de 2% NGS / PbTx para lavar anticorpo primário em excesso. Agite delicadamente por 5 min. Remover e solução descartar. Repita duas vezes. </li><li> Dim luzes do tecto e dilui-se o anticorpo secundário fluorescente apropriado conjugado em 10% NGS / PbTx de modo que cada condição contém, pelo menos, 0,5 ml. Com o anticorpo primário tubulina de ratinho anti-acetilado, utilizar a IgG anti-ratinho de cabra corante fluorescente verde a uma diluição de 1: 500. </li><li> Incubar anticorpo secundário durante 4 h, com agitação suave à TA no escuro. A partir de agora, o processo as amostras sob luzes se apagaram e incubar em um recipiente escuro ou por envolvimento em folha de alumínio. </li><li>No final da incubação, remover e descartar a solução de anticorpo secundário. Adicionar 0,5 ml de 2% NGS / PbTx para lavar. Agite delicadamente por 5 min. Remover e solução descartar. Repita duas vezes. </li><li> Se for co-imunocoloração, obter o segundo anticorpo primário a partir de uma espécie diferente do que o primeiro anticorpo primário. Nestes estudos, a seguir o anticorpo de coelho anti-fosfo-CaMK II pelo anticorpo secundário IgG conjugado com corante de cabra anti-coelho-vermelho fluorescente. </li><li> Dilui-se anticorpo de coelho anti-fosfo-II CaMK anticorpo 01:20. Para cada tubo de embriões, suspensão em 0,3 ml de 10% NGS / PbTx, em seguida, adicionar 15 ul de solução de anticorpo estoque. </li><li> Certifique-se de que os embriões são imersos e luzes se apagarem. Incube com agitação suave O / N à temperatura ambiente no escuro. </li><li> Na parte da manhã sob as luzes ofuscantes, remova e elimine a solução de anticorpo. Adicionar 0,5 ml de 2% NGS / PbTx. Agite delicadamente por 5 min. Remover e solução descartar. Repita duas vezes. </li><li> Mantenha luzes do teto dim e diluir o suficiente da fluorescência-conju apropriadaanticorpo secundário fechado de modo a que cada condição contém, pelo menos, 0,5 ml. Neste estudo, diluir o corante de cabra conjugado com anticorpo anti-coelho-vermelho fluorescente IgG secundário (canal vermelho) 1: 500 em 10% NGS / PbTx. </li><li> Incubar segundo anticorpo secundário durante 4 horas com agitação suave à TA no escuro. </li><li> No final desta incubação e sob luz fraca, remover e descartar a solução de anticorpo secundário. Adicionar 0,5 ml de PbTx. Agite delicadamente por 5 min. Remover e solução descartar. Repita duas vezes. Armazenar em qualquer PbTx ou 50% de glicerol / PBS, dependendo do procedimento de imagiologia. </li></ol> 4. confocal Imaging and Processing <ol><li> Mount embriões para a imagem latente. Coloque 1 - 5 embriões em uma lâmina de vidro. Criar uma câmara entre a lamela principal e o slide usando fragmentos lamela em cada lado da câmara. Normalmente, quatro # 1 lamelas são usados ​​para fazer estas pilhas espaçadores sem vedação. NOTA: No meio de montagem é necessária. </li><li> Use uma imersão em óleo de 100Xobjetivo trazer único embrião em foco usando luz transmitida. Apague a luz. Ligue microscópio confocal. Assegurar que os lasers apropriados (verde e / ou vermelho) estão ligados e accessary foco remoto está envolvida. </li><li> Ligue o programa confocal. No &quot;bar Acquire&quot;, selecione o próprio objecto. No bar &quot;XY Basic&quot;, definir o tamanho da imagem para 1024, clicando no botão 1.024. </li><li> No &quot;Laser e Detector&quot; bar, clique na caixa de 488 vermelho e 568 a caixa verde para ligar a laser e detector para cada canal. Definir pinhole para médio e ajustar o ganho no bar &quot;Gain&quot; de cada canal de visualizar. </li><li> No bar &quot;Configurações de Obtenção de&quot;, clique em &quot;Live&quot; para começar a aquisição de imagens. No bar &quot;Ver Definições&quot;, desmarque a caixa &quot;Forçar Integral Zoom&quot; para o centro na janela &quot;Live&quot;. </li><li> No bar &quot;Adquirir Configurações&quot;, na guia &quot;Z&quot;, percorrer as camadas da imagem. Depois de seleccionar o Number de secções ópticas de incorporar a imagem, mover-se para algum ponto no &quot;centro&quot; do plano z. </li><li> Sob a guia &quot;Z&quot;, clique na pequena caixa vermelha &quot;Referência&quot;. Isto irá zerar a RFA no ponto escolhido como o &quot;centro&quot;. Na caixa &quot;Tamanho do Passo&quot;, selecione a espessura das camadas (entre 0,25 mm e 2,0 mm é ideal). Preste atenção para a caixa &quot;Tamanho&quot; e tentar limitar a 1GB. NOTA: Normalmente, até 40 secções ópticas de 0,5-1,0 uM são obtidos, mas o número de secções podem ser determinadas empiricamente. </li><li> Para encontrar o extremo superior da imagem, clique no círculo ao lado de &quot;Top&quot; e mover-se através das camadas z. Agora clique no círculo ao lado de &quot;Bottom&quot;, o computador terá a imagem de volta para a camada como o centro. Mais uma vez se mover através das camadas para encontrar o extremo inferior da imagem. </li><li> Clique em &quot;Live&quot; novamente em &quot;Configurações de Obtenção de&quot; caixa para parar o laseraquisição. Neste painel, clique nas caixas vermelhas marcadas média e Z-stack. Estas caixas ficará verde. </li><li> Na caixa &quot;Configurações de Obtenção de&quot;, clique em &quot;Single&quot; para adquirir toda a série de imagens. Você pode monitorar o progresso como ele verifica em ambos os canais e através de todo o z-stack. </li><li> Para salvar, clique na janela de volume e clique em &quot;salvar como&quot; e nomeie o arquivo. Salvar como um arquivo &quot;.IDS&quot;. Ao volume de processar o arquivo enquanto o z-stack ainda está em aberto, selecione os dados no menu suspenso e clique em &quot;volume de render&quot;. Salve o arquivo processado como um arquivo TIFF. Esta é a imagem projectada que são mostrados nesta publicação. </li></ol>

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Os autores declaram que não têm interesses financeiros concorrentes.

O método / metanol PFA foi desenvolvido neste laboratório com o objetivo principal de otimizar a imunolocalização do fosfo-T 287 -CaMK-II epitopo durante o desenvolvimento do peixe-zebra. Este método localizada com êxito P-CaMK-II durante a formação de vários órgãos, incluindo o ciliadas KV peixe-zebra, 12 ouvido interno 14 e do rim. 13 em particular na fase KV, esta técnica foi necessário. O sucesso deste método é provavelmente devido a uma combinação de a) m...

The techniques described here are the outcome of studies that have sought to define downstream targets of Ca2+ signals during events that occur during early development, including fertilization, gastrulation, somitogenesis and trunk, eye, brain and organ formation.1-3 The original discoveries of embryonic Ca2+ signaling were dependent on the use of natural and engineered Ca2+ indicators, such as aequorin4 and fura-2.5 Even with current technology, the detection of transient elevations of Ca2+ requires cumbersome analytical tools and does not reveal the targets of such Ca2+ signals. 
This laboratory investigates Ca2+ signals that act through the Ca2+/calmodulin-dependent (multifunctional) protein kinase known as CaMK-II, an enzyme that is enriched in the central nervous system and originally identified as a regulator of long-term potentiation.6 CaMK-II is not brain-specific, is widely expressed and highly conserved throughout the entire lifespan and bodies of species throughout the animal kingdom, including invertebrates.7,8 CaMK-II has the unique capability of sustaining its own activity even after Ca2+ levels have diminished due to its ability to autophosphorylate at Thr287. In this autophosphorylated state, CaMK-II remains active in a Ca2+/CaM-independent manner, until dephosphorylated.6 Thus, the localization of phosphorylated CaMK-II (Thr287) can identify cells in which natural, relevant Ca2+ elevations have occurred. 
An antibody against autophosphorylated (P-Thr287) mammalian CaMK-II has been well-characterized and was initially used to localize activated CaMK-II in brain tissue.9 Zebrafish (Danio rerio) have seven CaMK-II genes10,11 whose protein products contain a sequence of MHRQE[pT287]VECLK in this region.10,11 This sequence is very similar to the phosphopeptide antigen used to create this rabbit polyclonal antibody (MHRQE[pT]VDCLK; Upstate/Millipore) and therefore it was not a complete surprise that this antibody cross-reacted with zebrafish CaMK-II. This laboratory showed that this antibody reacts with zebrafish CaMK-II in proportion to autophosphorylation and Ca2+/CaM-independent activity.12 Additional pan-specific CaMK-II antibodies have also been shown to cross-react with zebrafish CaMK-II.13
This antibody has been used to demonstrate that zebrafish CaMK-II is preferentially activated in cells on one side of the zebrafish Kupffer's Vesicle (KV), the ciliated organ necessary for establishment of left/right asymmetry.12 This antibody was used to demonstrate that CaMK-II is transiently activated in four adjacent cells on the left side of the KV during the exact same developmental phase that organ positioning is determined.12 In addition to the Kupffer's Vesicle (KV), autophosphorylated (P-T287) was also located in specific intracellular sites in other ciliated tissues including the kidney, neuromasts, and inner ear.12,13 In the zebrafish kidney, P-T287-CaMK-II is enriched along the apical border of ciliated ductal cells and within cloacal cilia where it influences their assembly.13 Finally, in the developing inner ear, P-T287-CaMK-II is intensely concentrated at the base of cilia and influences cell differentiation through the Delta-Notch signal pathway.14 In summary, the detection of activated CaMK-II has pinpointed sites of intracellular Ca2+ release and illuminated potential new signaling pathways.
These discoveries were completely dependent on developing a sensitive and accurate method to localize activated (P-T287-autophosphorylated) CaMK-II. The methods to fix and immunostain the zebrafish KV, kidney and inner ear are described. The limitations of this technique are also described. These techniques should be useful to any investigator who seeks to obtain high-resolution images in two fluorescent channels of not just phospho-epitopes, but any epitope, during early vertebrate development.

<table><tbody><tr><td>1-phenyl-2-thiourea (PTU)</td><td>Sigma</td><td>P-7629</td><td>0.12% Stock solution. Dilute 1:40 in system water</td></tr><tr><td>Alexa488 anti-mouse IgG</td><td>Life Technologies</td><td>A11001</td><td>Goat polyclonal, use at 1:500</td></tr><tr><td>Alexa488 anti-rabbit IgG</td><td>Life Technologies</td><td>A11008</td><td>Goat polyclonal, use at 1:500</td></tr><tr><td>Alexa488 phalloidin</td><td>Life Technologies</td><td>A12379</td><td>Preferentially binds to F-actin</td></tr><tr><td>Alexa568 anti-mouse IgG</td><td>Life Technologies</td><td>A11004</td><td>Goat polyclonal, use at 1:500</td></tr><tr><td>Alexa568 anti-rabbit IgG</td><td>Life Technologies</td><td>A11011</td><td>Goat polyclonal, use at 1:500</td></tr><tr><td>anti-acetylated &amp;#945;-tubulin</td><td>Sigma</td><td>T7451</td><td>Mouse monoclonal, use at 1:500</td></tr><tr><td>anti-phospho-T&lt;sup&gt;287&lt;/sup&gt; CaMK-II</td><td>EMD Millipore</td><td>06-881</td><td>Rabbit polyclonal, use at 1:20</td></tr><tr><td>anti-total CaMK-II</td><td>BD Biosciences</td><td>611292</td><td>Mouse monoclonal, use at 1:20</td></tr><tr><td>Ethanol</td><td>Fisher</td><td>S96857</td><td>Lab grade, 95% denatured</td></tr><tr><td>Forceps</td><td>Fine Science Tools</td><td>11252-20</td><td>Dumont #5</td></tr><tr><td>Glass coverslips</td><td>VWR</td><td>16004-330</td><td>#1&amp;nbsp; thickness</td></tr><tr><td>Glass microscope slides</td><td>Fisher</td><td>12-550-15</td><td>Standard glass slides</td></tr><tr><td>Methanol</td><td>Fisher</td><td>A411</td><td>Store in freezer</td></tr><tr><td>Microcentrifuge tubes</td><td>VWR</td><td>20170-038</td><td>capped tubes, not sterile</td></tr><tr><td>Normal goat serum</td><td>Life Technologies</td><td>16210-064</td><td>Aliquot 1 ml tubes, store in freezer</td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>P-6148</td><td>Reagent grade, crystalline</td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Quality Biological</td><td>119-069-131</td><td>10x stock solution or made in lab</td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>BP-151</td><td>10% solution in water, store at RT</td></tr><tr><td>Tween-20</td><td>Life Technologies</td><td>85113</td><td>10% solution in water, store at RT</td></tr><tr><td>Compound microscope</td><td>Nikon</td><td>E-600</td><td>Mount on vibration-free table</td></tr><tr><td>C1 Plus two-laser scanning confocal</td><td>Nikon</td><td>C1 Plus</td><td>Run by EZ-C1 program, but upgrades use &quot;Elements&quot;</td></tr></tbody></table>

immunostaining phospho-epitopes in ciliated organs of whole mount zebrafish embryos

A rápida proliferação das células, a expressão específica de tecido dos genes e o surgimento de redes de sinalização caracterizar o desenvolvimento embrionário no início de todos os vertebrados. A cinética e localização de sinais - mesmo dentro de uma única célula - no embrião em desenvolvimento complementa a identificação de genes de desenvolvimento importantes. técnicas de imunocoloração são descritas que têm sido mostrados para definir a cinética de sinais intracelulares e animais inteiros em estruturas tão pequenas quanto cílios. As técnicas para imagens de fixação, de imagem e de processamento usando um laser de varredura composto microscópio confocal pode ser concluído em apenas 36 horas. Peixe-zebra (Danio rerio) é um organismo desejável para os investigadores que buscam realizar estudos com uma espécie de vertebrados que é acessível e relevante para a doença humana. nocautes genéticos ou knockdowns deve ser confirmada pela perda do produto de proteína real. Essa confirmação da perda de proteínapode ser conseguido usando as técnicas descritas aqui. Pistas sobre as vias de sinalização também pode ser decifrada por utilização de anticorpos que são reactivos com as proteínas que foram modificadas pós-translacionalmente pela fosforilação. Preservar e otimizar o estado fosforilada de um epitopo é, portanto, fundamental para esta determinação e é realizado por este protocolo. Este estudo descreve técnicas para fixar o primeiro embriões durante 72 h de desenvolvimento e de co-localizar uma variedade de epitopos relevantes com ciliares de vesículas de Kupffer (KV), o rim e o ouvido interno. Estas técnicas são simples, não exigem dissecção e pode ser completada num período relativamente curto de tempo. Projectar pilhas de imagens confocal em uma única imagem é um meio útil de apresentar estes dados.

Condições ideais para a visualização de fosfo-epítopos Métodos que descrevem a imunolocalização de epitopos da proteína em embriões de peixe-zebra foram relativamente escassas em comparação com aqueles para a localização de ARNm através de hibridação in situ. Os fixadores usados ​​na localização de epítopos de proteínas em células ciliadas de embriões de peixe-zebra incluír...

Watch this Scientific Journal Video about Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos at JoVE.com

Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos

Técnicas são descritas a imunocoloração fosfo-epitopos em embriões de peixe-zebra inteiros e em seguida, realizar duas cores localização confocal fluorescente em estruturas celulares tão pequenos quanto cílios. As técnicas de fixação e de imagem pode definir a localização e cinética da aparência ou a activação de proteínas específicas.

Imunocoloração fosfo-epitopos em ciliadas Órgãos da Whole Mount embriões Zebrafish

The rapid proliferation of cells, the tissue-specific expression of genes and the emergence of signaling networks characterize early embryonic development ...

immunostaining-phospho-epitopes-ciliated-organs-whole-mount-zebrafish

Research

JoVE Journal

Chemistry

8.0K visualizações.  Virginia Commonwealth University.  Técnicas são descritas a imunocoloração fosfo-epitopos em embriões de peixe-zebra inteiros e em seguida, realizar duas cores localização confocal fluorescente em estruturas celulares tão pequenos quanto cílios. As técnicas de fixação e de imagem pode definir a localização e cinética da aparência ou a activação de proteínas específicas. 

Vídeo: Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos

High Precision Zinc Isotopic Measurements Applied to Mouse Organs

We present a procedure to measure with high precision zinc isotope ratios in mouse organs. Zinc is composed of 5 stable isotopes (64Zn, 66Zn, 67Zn, 68Zn and 70Zn) which are naturally fractionated between mouse organs. We first show how to dissolve the different organs in order to free the Zn atoms; this step is realized by a mixture of HNO3 and H2O2. We then purify the zinc atoms from all the other elements, in particular from isobaric interferences (e.g., Ni), by anion-exchange chromatography in a dilute HBr/HNO3 medium. These first two steps are performed in a clean laboratory using high purity chemicals. Finally, the isotope ratios are measured by using a multi-collector inductively-coupled-plasma mass-spectrometer, in low resolution. The samples are injected using a spray chamber and the isotopic fractionation induced by the mass-spectrometer is corrected by comparing the ratio of the samples to the ratio of a standard (standard bracketing technique).&#160;This full typical&#160;procedure produces an isotope ratio with a 50 ppm (2 s.d.) reproducibility.

We present the technique to measure with high precision zinc isotope ratios in mouse organs.

Medições de alta precisão de zinco isotópicas Aplicadas à mouse Órgãos

We present a procedure to measure with high precision zinc isotope ratios in mouse organs. Zinc is composed of 5 stable isotopes (64Zn, 66Zn, 67Zn, 68Zn ...

Química

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and optical transparency. In particular, the zebrafish embryo has become an important organism to study vertebrate kidney organogenesis as well as multiciliated cell (MCC) development. To visualize MCCs in the embryonic zebrafish kidney, we have developed a combined protocol of whole-mount fluorescent in situ hybridization (FISH) and whole mount immunofluorescence (IF) that enables high resolution imaging. This manuscript describes our technique for co-localizing RNA transcripts and protein as a tool to better understand the regulation of developmental programs through the expression of various lineage factors.

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

Cilia development is vital to proper organogenesis. This protocol describes an optimized method to label and visualize ciliated cells of the zebrafish.

Visualização de células Multiciliated no Zebrafish através de um protocolo combinado de toda montagem fluorescente In Situ da hibridação e imunofluorescência

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and ...

Biologia do Desenvolvimento

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 ...

Biologia

Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. Although many immunohistochemistry protocols have been optimized for mammalian tissue and tissue sections, these protocols often require modification and optimization for non-mammalian model organisms. Zebrafish are increasingly used as a model system in basic, biomedical, and translational research to investigate the molecular, genetic, and cell biological mechanisms of developmental processes. Zebrafish offer many advantages as a model system but also require modified techniques for optimal protein detection. Here, we provide our protocol for whole-mount fluorescence immunohistochemistry in zebrafish embryos and larvae. This protocol additionally describes several different mounting strategies that can be employed and an overview of the advantages and disadvantages each strategy provides. We also describe modifications to this protocol to allow detection of chromogenic substrates in whole mount tissue and fluorescence detection in sectioned larval tissue. This protocol is broadly applicable to the study of many developmental stages and embryonic structures.

Here, we present a protocol for fluorescent antibody-mediated detection of proteins in whole preparations of zebrafish embryos and larvae.

Imunohistoquímica do Monte Inteiro em Embriões e Larvas de Zebrafish

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. ...

Single Cell Fate Mapping in Zebrafish

The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, lymphatic, and blood vessel lineages arise in developing embryos requires fate mapping and lineage tracing of undifferentiated precursor cells. Recently, photoactivatable proteins which include: Eos1, 2, PAmCherry3, Kaede4-7, pKindling8, and KikGR9, 10 have received wide interest as cell tracing probes. The fluorescence spectrum of these photosensitive proteins can be easily converted with UV excitation, allowing a population of cells to be distinguished from adjacent ones. However, the photoefficiency of the activated protein may limit long-term cell tracking11. As an alternative to photoactivatable proteins, caged fluorescein-dextran has been widely used in embryo model systems7, 12-14. Traditionally, to uncage fluorescein-dextran, UV excitation from a fluorescence lamp house or a single photon UV laser has been used; however, such sources limit the spatial resolution of photoactivation. Here we report a protocol to fate map, lineage trace, and detect single labeled cells. Single cells in embryos injected with caged fluorescein-dextran are photoactivated with near-infrared laser pulses produced from a titanium sapphire femtosecond laser. This laser is customary in all two-photon confocal microscopes such as the LSM 510 META NLO microscope used in this paper. Since biological tissue is transparent to near-infrared irradiation15, the laser pulses can be focused deep within the embryo without uncaging cells above or below the selected focal plane. Therefore, non-linear two-photon absorption is induced only at the geometric focus to uncage fluorescein-dextran in a single cell. To detect the cell containing uncaged fluorescein-dextran, we describe a simple immunohistochemistry protocol16 to rapidly visualize the activated cell. The activation and detection protocol presented in this paper is versatile and can be applied to any model system. 
Note: The reagents used in this protocol can be found in the table appended at the end of the article.

A method is described to photoactivate single cells containing a caged fluorescent protein using two-photon absorption from a Ti:Sapphire femtosecond laser oscillator. To fate map the photoactivated cell, immunohistochemistry is used. This technique can be applied to any cell type.

Mapeamento Destino única célula em Zebrafish

The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, ...

An Efficient Protocol to Assess ERK Activity Modulation in Early Zebrafish Noonan Syndrome Models via Live FRET Microscopy and Immunofluorescence

RASopathies are genetic syndromes caused by ERK hyperactivation and resulting in multisystemic diseases that can also lead to cancer predisposition. Despite a broad genetic heterogeneity, germline gain-of-function mutations in key regulators of the RAS-MAPK pathway underlie the majority of the cases, and, thanks to advanced sequencing techniques, potentially pathogenic variants affecting the RAS-MAPK pathway continue to be identified. Functional validation of the pathogenicity of these variants, essential for accurate diagnosis, requires fast and reliable protocols, preferably in vivo. Given the scarcity of effective treatments in early childhood, such protocols, especially if scalable in cost-effective animal models, can be instrumental in offering a preclinical ground for drug repositioning/repurposing. 
Here we describe step-by-step the protocol for rapid generation of transient RASopathy models in zebrafish embryos and direct inspection of live disease-associated ERK activity changes occurring already during gastrulation through real-time multispectral F&#246;rster resonance energy transfer (FRET) imaging. The protocol uses a transgenic ERK reporter recently established and integrated with the hardware of commercial microscopes. We provide an example application for Noonan syndrome (NS) zebrafish models obtained by expression of the Shp2D61G. We describe a straightforward method that enables registration of ERK signal change in the NS fish model before and after pharmacological signal modulation by available low-dose MEK inhibitors. We detail how to generate, retrieve, and assess ratiometric FRET signals from multispectral acquisitions before and after treatment and how to cross-validate the results via classical immunofluorescence on whole embryos at early stages. We then describe how, via examining standard morphometric parameters, to query late changes in embryo shape, indicative of a resulting impairment of gastrulation, in the same embryos whose ERK activity is assessed by live FRET at 6 h post fertilization.

RASopathies are multisystem genetic syndromes caused by RAS-MAPK pathway hyperactivation. Potentially pathogenic variants awaiting validation emerge continuously while poor preclinical evidence limits therapy. Here, we describe our in vivo protocol to test and cross-validate RASopathy-associated ERK activation levels and its pharmacological modulation during embryogenesis by live FRET imaging in Teen-reporter zebrafish.

Um protocolo eficiente para avaliar a modulação da atividade de ERK em modelos iniciais da síndrome de Noonan do peixe-zebra por meio de microscopia FRET ao vivo e imunofluorescência

RASopathies are genetic syndromes caused by ERK hyperactivation and resulting in multisystemic diseases that can also lead to cancer predisposition. ...

Bioquímica

Immunostaining of Dissected Zebrafish Embryonic Heart 

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology and genetics 1,2. About 100-200 embryos are readily available every week from a single pair of adult fish. The transparent embryos that develop ex utero make them ideal for assessing cardiac defects 3. The expression of any gene can be manipulated via morpholino technology or RNA injection 4. Moreover, forward genetic screens have already generated a list of mutants that affect different perspectives of cardiogenesis 5. 
Whole mount immunostaining is an important technique in this animal model to reveal the expression pattern of the targeted protein to a particular tissue 6. However, high resolution images that can reveal cellular or subcellular structures have been difficult, mainly due to the physical location of the heart and the poor penetration of the antibodies. 
 Here, we present a method to address these bottlenecks by dissecting heart first and then conducting the staining process on the surface of a microscope slide. To prevent the loss of small heart samples and to facilitate solution handling, we restricted the heart samples within a circle on the surface of the microscope slides drawn by an immEdge pen. After the staining, the fluorescence signals can be directly observed by a compound microscope. 
Our new method significantly improves the penetration for antibodies, since a heart from an embryonic fish only consists of few cell layers. High quality images from intact hearts can be obtained within a much reduced procession time for zebrafish embryos aged from day 2 to day 6. Our method can be potentially extended to stain other organs dissected from either zebrafish or other small animals.

Immunostaining of Dissected Zebrafish Embryonic Heart

A rapid way to conduct immunostaining of zebrafish embryonic heart is described. Compared to the whole mount immunostaining approach, this method dramatically increases the penetration of the antibodies, which allows obtaining high resolution images that reveal cellular/subcellular structures in the heart within a much reduced processing time.

Imunomarcação de Dissecada Coração Zebrafish embrionárias

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology ...

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression both in terms of tissue distribution and developmental stage. Protocols are described for the use of WISH of zebrafish embryos using antisense RNA probes labeled with digoxigenin. Probes are generated by incorporating digoxigenin-linked nucleotides through in vitro transcription of gene templates that have been cloned and linearized. The chorions of embryos harvested at defined developmental stages are removed before incubation with specific probes. Following a washing procedure to remove excess probe, embryos are incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase. By employing a chromogenic substrate for alkaline phosphatase, specific gene expression can be assessed. Depending on the level of gene expression the entire procedure can be completed within 2-3 days.

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

The zebrafish, a small tropical fish, has become a popular model for studying gene function during vertebrate development and disease. The temporal and spatial expression of target genes can be determined by in situ hybridization. Our improved protocol allows for the detection of low abundant transcripts with low non-specific background signal.

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

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

In Situ Hybridization Combined with Immunohistochemistry in Cryosectioned Zebrafish Embryos

As a vertebrate, the zebrafish has been widely used in biological studies. Zebrafish and humans share high genetic homology, which allows its use as a model for human diseases. Gene function study is based on the detection of gene expression patterns. Although immunohistochemistry offers a powerful way to assay protein expression, the limited number of commercially available antibodies in zebrafish restricts the application of costaining. In situ hybridization is widely used in zebrafish embryos to detect mRNA expression. This protocol describes how to obtain images by combining in situ hybridization and immunohistochemistry for zebrafish embryo sections. In situ hybridization was performed prior to cryosectioning, followed by antibody staining. Immunohistochemistry and the imaging of a single cryosection were performed after in situ hybridization. The protocol is helpful to unravel the expression pattern of two genes, first by in situ transcript detection and then by immunohistochemistry against a protein in the same section.

In Situ Hybridization Combined with Immunohistochemistry in Cryosectioned Zebrafish Embryos

This protocol describes how to obtain images by combining in situ hybridization and immunohistochemistry of zebrafish embryonic sections. In situ hybridization was performed prior to cryosectioning, followed by antibody staining. It is useful to detect the expression patterns of two genes in zebrafish if there is a paucity of antibodies.

In situ Hibridização Combinada com Imunohistoquímica em Embriões de Zebrafish Crioseccionados

As a vertebrate, the zebrafish has been widely used in biological studies. Zebrafish and humans share high genetic homology, which allows its use as a ...

Neurociência

Sequential Immunofluorescence and Immunohistochemistry on Cryosectioned Zebrafish Embryos

Investigation of intercellular interactions often requires discrete labeling of specific cell populations and precise protein localization. The zebrafish embryo is an excellent tool for examining such interactions with an in vivo model. Whole-mount immunohistochemical and immunofluorescence assays are frequently applied in zebrafish embryos to assess protein expression. However, it can be difficult to achieve accurate mapping of co-localized proteins in three-dimensional space. In addition, some studies may require the use of two antibodies that are not compatible with the same technique (e.g., antibody 1 is only suitable for immunohistochemistry and antibody 2 is only suitable for immunofluorescence). The purpose of the method described herein is to perform sequential immunofluorescence and/or immunohistochemistry on individual cryosections derived from early-stage zebrafish embryos. Here we describe the use of sequential rounds of immunofluorescence, imaging, immunohistochemistry, imaging for a single cryosection in order to achieve precise identification of protein expression at the single-cell level. This methodology is suitable for any study in early-stage zebrafish embryos that requires accurate identification of multiple protein targets in individual cells.

This protocol demonstrates sequential immunofluorescence and immunohistochemistry on cryosections from early-stage zebrafish embryos enabling precise colocalization analyses in specific cell populations.

Imunofluorescência sequencial e imuno-histoquímica em embriões de zebrafish Crioseccionados

Investigation of intercellular interactions often requires discrete labeling of specific cell populations and precise protein localization. The zebrafish ...