Os autores agradecem SI Bernstein (San Diego State University) para a leitura crítica e sugestões úteis sobre a preparação deste manuscrito. Este trabalho foi financiado pelo NIH concede a SI Bernstein, SDSU, e de R. Bodmer, BIMR, e por uma bolsa de pós-doutorado da filial dos Estados do Oeste da Associação Americana do Coração para G. Vogler e A. Cammarato.

Antes de começar a <ol><li> Prepare as seguintes soluções: <ol class="lalpha"><li> buffer de relaxamento (artificial Drosophila hemolinfa (ADH) (ver &quot;Visualizando o coração batendo em Drosophila&quot;) que contém 10 mM EGTA) </li><li> fixador (formaldeído 4% em PBS 1x) </li><li> PBSTx (PBS contendo 0,1% Triton-X-100) </li><li> Apropriadamente diluída primário e espécie-específicos fluorescente etiquetado anticorpos secundários em PBSTx </li></ol></li><li> Dissecar Drosophila para expor os tubos cardíacos (de moscas e / ou larvas), após <ol class="lalpha"><li> o semi-intactas Drosophila protocolo de preparação do coração em &quot;Visualizar o coração batendo em Drosophila&quot;, ou </li><li> Drosophila larval Dissection MNJ um protocolo com as seguintes modificações: Use oxigenado ADH durante a dissecção larval e deslocar o pino posterior ligeiramente da linha média ventral. As larvas são cortados ao longo da linha média ventral. Não remova qualquer tecido antes da fixação. </li></ol></li></ol> A coloração fluorescente <ol><li> Verifique se todos os corações estão batendo ritmicamente na ADH oxigenado. Substituir rapidamente ADH relaxante com buffer. Examine cada tubo cardíaca para assegurar contrações são inibidas. </li><li> Corrigir os corações, substituindo o buffer relaxante com fixador. Incubar à temperatura ambiente por 20 minutos, agitando suavemente. (Para agitar a preparação larval não é necessário em qualquer etapa, e pode ser considerada prejudicial à integridade do tecido cardíaco). </li><li> Lavar as amostras três vezes por dez minutos com PBSTx em temperatura ambiente com agitação contínua. </li><li> Para os adultos, apare cuidadosamente de volta as bordas ventral da cutícula abdominal tal que o que resta é mais elíptico e menos arredondadas. Além disso, com um único corte entre o abdômen e tórax, com cuidado e limpa separar segmentos de corpo e garantir o mínimo dano à região anterior do coração. Para corações larval, remova cuidadosamente a gordura corporal usando uma pinça fina. A remoção de gordura deve ser executada com extrema cautela, pois o coração larval é muito frágil e tem muito pouco apoio de outros músculos ou tecido conjuntivo. Não remova os ramos traqueais, pois isso pode danificar o coração. </li><li> Transferência da região dorsal aparados da cutícula abdominal pelas bordas, evitando contato com o tubo de uma localização central cardíaca, em uma placa de 96 poços de bem com que contêm 5-10 mL de anticorpo primário diluído em PBSTx. Coloque no máximo 12 amostras por poço. Incubar à temperatura ambiente por 2 horas com agitação contínua. Incubação pode ser feito também durante a noite a 4 ° C. </li><li> Remoção da solução de anticorpo primário. Lavar os corações de três vezes por dez minutos com 100 mL PBSTx em temperatura ambiente com agitação contínua. </li><li> Após a remoção do tampão de lavagem final, adicionar 100 ul do anticorpo secundário em PBSTx, suplementado com Alexa594-phalloidin (1:1000). Incubar durante uma hora com agitação contínua à temperatura ambiente. Manter as amostras coberta para evitar fluoróforo branqueamento. </li><li> Após a incubação secundário, lave os corações de três vezes por dez minutos com 100 ul de PBSTx em temperatura ambiente com agitação contínua. Mantenha as amostras cobertas durante todas as etapas de lavagem. </li><li> Para a remoção de 100-Triton-X, lavar os corações pela última vez em 100 ul de PBS por 10 minutos. As amostras podem ser armazenadas no escuro a 4 ° C por vários dias antes da montagem. </li></ol> Corações de montagem para Adultos <ol><li> Aderir dois 18 x 18 mm a lamínulas uma lâmina de microscópio com 10 ml de Vectashield meio de montagem. As lamelas devem ser espaçadas ~ 10-15 mm. Coloque uma pequena gota terço do meio de montagem em entre as duas lamelas. </li><li> Coloque 20 l de Vectashield no centro de uma lamela terceiros. </li><li> Remova cuidadosamente cada um dos corações da solução de lavagem PBS pelas bordas extremas da cutícula, e colocá-los suavemente coração voltada para baixo sobre a queda de meio de montagem da lamínula terceiros. Lugar não mais de cinco corações por gota na lamela. </li><li> Verificar sob um microscópio para garantir que todos os corações estão voltados para baixo. </li><li> Cuidadosamente inverter a lamela contendo os corações e rapidamente colocá-lo no slide que contém o par de lamelas de tal forma que a gota com fusíveis cardíaca tubos com a gota Vectashield entre o par lamela. A &quot;ponte&quot; deve ser formado por as lamelas. Os corações serão suspensas entre uma lamela ea lâmina de microscópio. </li><li> Verificar sob um microscópio para assegurar que os corações estão agora viradas para cima. </li><li> Fix lamínulas em suas bordas com unha polonês. </li><li> O coração agora está pronto para ser trabalhada por meio de microscopia fluorescente ou confocal. </li></ol><ol><li> Colocar uma gota (~ mL 20) Vectashield numa lâmina de microscópio. </li><li> Cuidado de transferência de até dois espécimes larval na Vectashield e orientar lado deles dorsal para baixo, usando agulhas de tungstênio. </li><li> Individualmente arrastar os espécimes para fora do meio de montagem e alinhar cada em paralelo. </li><li> Coloque uma lamela em lados opostos da amostra. Utilizando uma pinça, coloque uma lamela terceiro em cima, colocando primeiro um lado sobre a lamela posterior e baixando-o para a lamela anterior formando uma ponte. Forças capilares fará com que um fluxo de Vectashield de posterior para anterior, que ajuda o alinhamento adequado do coração larval. </li><li> Corrigir todas as lamínulas no lugar, em suas bordas, com unha polonês e cuidadosamente preencher o espaço entre as lamelas com 20-30 mL Vectashield. </li><li> Seal com unha polonês e armazenar a 4 ° C, ou para armazenamento de longo prazo a -20 ° C. </li></ol> Resultados representante Quando executado corretamente, todos os componentes e tecidos associados do vaso dorsal deve permanecer intacta e ser facilmente visualizado. A fluorescência de fundo deve ser mínima. Para os adultos, as manchas ventrais camada muscular longitudinal muito bem e produz um sinal significativo (Figura 1 e Figura 2). Os cardiomiócitos subjacente circular no entanto, tendem a não produzir como um sinal intenso que o da camada sobrejacente ventral. Os miócitos da &quot;câmara cônica&quot; anterior do coração adulto contém uma quantidade substancial de material contrátil e, conseqüentemente, a região aparece como o parente mais robusta para o restante do tubo cardíaco. O coração larval mostra um arranjo miofibrilar espiral semelhante à dos cardiomiócitos adultos contráteis (Figura 3). <img src="/files/ftp_upload/1423/1423fig1atmb.jpg" alt="Figura 1a" border="0" /> <img src="/files/ftp_upload/1423/1423fig1btmb.jpg" border="0" alt="Figura 1b" /> Figura 1: Top. A micrografia fluorescentes mostrando todo o tubo cardíaco de um adulto Drosophila melanogaster que expressa miosina-GFP. A imagem foi obtida com um microscópio Zeiss Z1 Imager fluorescentes equipado com um módulo ApoTome deslizante. Miosina-GFP é mostrado em verde, actina está manchado com AlexaFluor ® 594 phalloidin (vermelho) e α-actinina é rotulado com anti-α-actinina anticorpos (azul). Observe o procedimento preparativa permita a cobrança de amostras de coração com estruturas bem preservadas. CC = cônica câmara; AM = muscular Alary; V = interna da válvula; VLL = camada muscular longitudinal ventral. Inferior. A região do tubo cardíaco de adultos do CC através do segmento 3 abdominal do coração logo abaixo da camada ventral longitudinal mostrando o arranjo espiral miofibrila contrátil dos cardiomiócitos. Por favor, <a href="https://www.jove.com/files/ftp_upload/1423/1423fig1a.jpg">clique aqui</a> para uma versão maior da Figura 1a, e <a href="https://www.jove.com/files/ftp_upload/1423/1423fig1b.jpg">aqui</a> para uma versão ampliada da figura 1b <img border="0" src="/files/ftp_upload/1423/1423fig2tmb.jpg" alt="Figura 2" /> Figura 2: Representante pilhas confocal de uma porção anterior do coração adulto corados com AlexaFluor ® 594 phalloidin (vermelho) e um anti-Pericardin (colágeno tipo IV) (verde) do anticorpo. Pericardin está associada com o coração ao longo da superfície ventral, provavelmente provenientes do miofibrilas sentido longitudinal da camada muscular ventral. Por favor, <a href="https://www.jove.com/files/ftp_upload/1423/1423fig2.jpg">clique aqui</a> para uma versão ampliada da figura 2. <img border="0" src="/files/ftp_upload/1423/1423fig3tmb.jpg" alt="Figura 3" /> Figura 3: A micrografia fluorescentes do segmento A7 do coração próprio de um estágio larva Drosophila três. A imagem foi obtida com um microscópio Zeiss Z1 Imager fluorescentes equipado com um módulo ApoTome deslizante. Actina é rotulado com AlexaFluor ® 594 phalloidin (verde) e α-actinina está manchado com anti-α-actinina anticorpos (vermelho). Por favor, <a href="https://www.jove.com/files/ftp_upload/1423/1423fig3.jpg">clique aqui</a> para uma versão ampliada da figura 3.

<ol>
	<li>Brent, J. R., Werner, K. M., McCabe, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+larval+NMJ+dissection.">Drosophila larval NMJ dissection.</a> J Vis Exp. , (2009).</li><li>Molina, M. R., Cripps, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ostia,+the+inflow+tracts+of+the+Drosophila+heart,+develop+from+a+genetically+distinct+subset+of+cardial+cells.">Ostia, the inflow tracts of the Drosophila heart, develop from a genetically distinct subset of cardial cells.</a> Mech Dev. 1, 51-59 (2001).</li><li>Dulcis, D., Levine, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamatergic+innervation+of+the+heart+initiates+retrograde+contractions+in+adult+Drosophila+melanogaster.">Glutamatergic innervation of the heart initiates retrograde contractions in adult Drosophila melanogaster.</a> J Neurosci. 2, 271-280 (2005).</li><li>Zeitouni, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signalling+pathways+involved+in+adult+heart+formation+revealed+by+gene+expression+profiling+in+Drosophila.">Signalling pathways involved in adult heart formation revealed by gene expression profiling in Drosophila.</a> PLoS Genet. 10, 1907-1921 (2007).</li><li>Robertson, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+metamorphosis+of+Drosophila+melanogaster,+including+an+accurately+timed+account+of+the+principal+morphological+change.">The metamorphosis of Drosophila melanogaster, including an accurately timed account of the principal morphological change.</a> J. Morphol. 2, 351-399 (1936).</li><li>Miller, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The internal anatomy and histology of the imago of Drosophila melanogaster. , (1950).</li><li>Rizki, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+circulatory+system+and+associated+cells+and+tissues.">The circulatory system and associated cells and tissues.</a> , (1978).</li><li>Cammarato, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myosin+transducer+mutations+differentially+affect+motor+function,+myofibril+structure,+and+the+performance+of+skeletal+and+cardiac+muscles.">Myosin transducer mutations differentially affect motor function, myofibril structure, and the performance of skeletal and cardiac muscles.</a> Mol Biol Cell. 2, 553-562 (2008).</li><li>Taghli-Lamallem, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dystrophin+deficiency+in+Drosophila+reduces+lifespan+and+causes+a+dilated+cardiomyopathy+phenotype.">Dystrophin deficiency in Drosophila reduces lifespan and causes a dilated cardiomyopathy phenotype.</a> Aging Cell. 2, 237-249 (2008).</li><li>Kelso, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=a+database+documenting+a+GFP+protein-trap+insertion+screen+in+Drosophila+melanogaster.">a database documenting a GFP protein-trap insertion screen in Drosophila melanogaster.</a> Nucleic Acids Res. 32, D418-D420 (2004).</li><li>Dietzl, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genome-wide+transgenic+RNAi+library+for+conditional+gene+inactivation+in+Drosophila.">A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila.</a> Nature. 7150, 151-156 (2007).</li><li>Mery, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+muscle+LIM+protein,+Mlp84B,+is+essential+for+cardiac+function.">The Drosophila muscle LIM protein, Mlp84B, is essential for cardiac function.</a> J Exp Biol. 211, 15-23 (2008).</li></ol>

Aqui apresentamos um protocolo útil para preparar e coloração da Drosophila melanogaster vaso dorsal e dos tecidos adjacentes para geração de imagens através de microscopia fluorescente ou confocal. Nós fornecemos um relato conciso dos passos refinado por e comumente empregado em nosso laboratório para a coloração eficaz que permite a bem-resolvido em imagens in situ de larvas e tubos coração adulto Drosophila. Outros descreveram métodos similares de forma abreviada ...

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<td>Ethylene glycol-bis (2-amino-ethylether) -N,N,N&#x2019;,N&#x2019;-tetra-acetic acid (EGTA)</td>
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<td>E4378</td>
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<td>Formaldehyde, 10%, methanol free, Ultra Pure</td>
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<td>Triton-X-100</td>
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fluorescent labeling of drosophila heart structures

O<em&gt; Drosophila melanogaster</em&gt; Embarcação dorsal, ou coração, é uma estrutura tubular composta de uma única camada de cardiomiócitos contrátil, as células do pericárdio que se alinham ao longo de cada lado da parede do coração, músculos Alary de apoio e, em adultos, uma camada de células musculares longitudinal ventral. A casa fibras contrátil conservados constituintes da citoarquitetura muscular, incluindo feixes de miofibrilas densamente e complexos de proteínas do citoesqueleto / submembranas, que interagem com homólogos componentes da matriz extracelular. Aqui nós descrevemos um protocolo para a fixação e à rotulagem de determinados elementos fluorescentes do miocárdio do coração de larvas dissecadas e semi-intactas de adultos<em&gt; Drosophila</em&gt;. Especificamente, nós demonstramos a rotulagem de sarcômero F-actina e α-actinina nos corações larval. Além disso, realizamos rotulagem de F-actina e α-actinina em miosina-GFP expressar moscas adultas e de α-actinina e pericardin, um colágeno tipo IV matriz extracelular, no coração do adulto tipo selvagem. É dada especial atenção a uma estratégia de montagem para os corações semi-intactas de adultos que minimiza a manipulação e otimiza a oportunidade para manter a integridade dos tubos e os tecidos cardíacos associados. Estas preparações são adequadas para geração de imagens através de microscopia de fluorescência e confocal. No geral, este procedimento permite a análise cuidadosa e detalhada das características estruturais do coração de um sistema poderoso modelo geneticamente tratável.

Watch this Scientific Journal Video about Fluorescent Labeling of Drosophila Heart Structures at JoVE.com

Fluorescent Labeling of Drosophila Heart Structures

Aqui nós descrevemos um protocolo básico para a rotulagem fluorescentes de diferentes elementos de tubos de coração de larva e adulto<em&gt; Drosophila melanogaster</em&gt;. Estas amostras são bem adaptados para geração de imagens através de microscopia fluorescente ou confocal. Essa técnica permite a análise estrutural detalhada das características dos corações de um organismo modelo poderoso.

Labeling fluorescentes de Drosophila Estruturas Coração

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells ...

fluorescent-labeling-of-drosophila-heart-structures

Research

JoVE Journal

Biology

Fluorescent Labeling of Drosophila Heart Structures

13.6K visualizações.  San Diego State University. Aqui nós descrevemos um protocolo básico para a rotulagem fluorescentes de diferentes elementos de tubos de coração de larva e adulto Drosophila melanogaster. Estas amostras são bem adaptados para geração de imagens através de microscopia fluorescente ou confocal. Essa técnica permite a análise estrutural detalhada das características dos corações de um organismo modelo poderoso.

Vídeo: Fluorescent Labeling of Drosophila Heart Structures

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

Rotulagem células-tronco com corantes fluorescentes para detecção não invasiva com imagens ópticas

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

Biologia

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle synapses, we developed a larval tissue preparation that had features of live intact larvae, but also had good optical properties.  This new preparation also allowed for access of perfusates to the synapse.  We used fly larvae, immersed them in artificial hemolymph, and relaxed their normal rhythmic body contractions by chilling them. Next we dissected off the posterior segments of each animal and with a blunt insect pin pushed the mouth parts backward through the body cavity.  This everted the larval body wall, like turning a sock inside-out.  We completed the dissection with ultra-fine dissection scissors and thus exposed the visceral side of the body wall muscles. The glial structures at the NMJ expressed membrane targeted GFP under the control of glial specific promoters. The post-synaptic membrane, the SSR (Subsynaptic Reticula) in muscle expressed synaptically targeted dsRed. We needed to acutely label the motor neuron terminals, the third part of the synapse. To do this we applied primary antibodies to HRP, conjugated to a far-red emitting flurophore.  To test for dye diffusion properties into the perisynaptic space between the motor neuron terminals and the SSR, we applied a solution of large Dextran molecules conjugated to far-red emitting flurophore and collected images.

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

We described structural features of the Glia-neuromuscular synapses in a novel Inside-out tissue preparation of live fly larvae using fluorescent dyes with confocal microscopy. We labeled live neuron terminals with fluorescent primary antibodies to HRP, and also visualized the perisynaptic space with fluorescent Dextrans.

Visualizando o Live Drosophila Junção neuromuscular-Glial com corantes fluorescentes

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle ...

Visualizing the Beating Heart in Drosophila

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in myogenic hearts. A key step in examining heart function in the fly is finding a way to access the heart in a manner that preserves its myogenic function while still allowing the beating heart organ to be observed and recorded. Two different methods for observing and recording the beating heart in both larva and adult Drosophila are described here. Our semi-intact preparation using adult flies allows clear visualization of the abdominal heart without interference from the pigmented cuticle and overlying fat bodies. To record larval heart beats it is necessary to immobilize the larva, which minimizes body wall movements thereby reducing heart movements that are not associated with myocardial contractions. Our methodologies produce stable adult and larval heart preparations that can beat for hours at rates of 1-3 Hz.

Visualizing the Beating Heart in Drosophila

Technique required for visualizing the beating heart in larval and adult Drosophila are presented. Each life stage requires a different methodology.

Visualizar o coração batendo no Drosophila</em

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in ...

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained and restrained whole larvae. For direct control of the environment around the heart another approach utilizes the dissected larvae and removal of the internal organs in order to bathe the heart in desired compounds. The exposed heart also allows membrane potentials to be monitored which can give insight of the ionic currents generated by the myocytes and for electrical conduction along the heart tube. These approaches have various advantages and disadvantages for future experiments that are discussed. The larval heart preparation provides an additional model besides the Drosophila skeletal NMJ to investigate the role of intracellular calcium regulation on cellular function. Learning more about the underlying ionic currents that shape the action potentials in myocytes in various species, one can hope to get a handle on the known ionic dysfunctions associated to specific genes responsible for various diseases in mammals.

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various ways to monitor heart function in the larva of Drosophila for assessing questions dealing with the function of gap junctions, ion channel mutations, modulation of pacemaker activity and pharmacological studies.

Função de monitorização cardíaca em larval Drosophila melanogaster Para estudos fisiológicos

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained  and  ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

Preparação de embriões de Microscopia Eletrônica do Drosophila Tubo cardíaco embrionário

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

Preparation of Drosophila Polytene Chromosome Squashes for Antibody Labeling

Drosophila has long been a favorite model system for studying the relationship between chromatin structure and gene regulation due to the cytological advantages provided by the giant salivary gland polytene chromosomes of third instar larvae.  In this tissue the chromosomes undergo many rounds of replication in the absence of cell division giving rise to approximately 1000 copies.  The DNA remains aligned after each replicative cycle resulting in greatly enlarged chromosomes that provide a unique opportunity to correlate chromatin morphology with the localization of specific proteins.  Consequently, there has been a high level of interest in defining the epigenetic modifications present at different genes and at different stages of the transcription process. An important tool for such studies is the labeling of polytene chromosomes with antibodies to the enzyme, transcription factor, or histone modification of interest. This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for antibody labeling.

This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for antibody labeling.

Preparação de Abóboras Drosophila cromossomos politênicos para Rotulagem de anticorpos

Drosophila has long been a favorite model system for studying the relationship between chromatin structure and gene regulation due to the cytological ...

Fluorescent Labeling of COS-7 Expressing SNAP-tag Fusion Proteins for Live Cell Imaging

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. These systems offer a broad selection of fluorescent substrates optimized for a range of imaging instrumentation. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again. There are two steps to using this system: cloning and expression of the protein of interest as a SNAP-tag fusion, and labeling of the fusion with the SNAP-tag substrate of choice. The SNAP-tag is a small protein based on human O6-alkylguanine-DNA-alkyltransferase (hAGT), a DNA repair protein. SNAP-tag labels are dyes conjugated to guanine or chloropyrimidine leaving groups via a benzyl linker. In the labeling reaction, the substituted benzyl group of the substrate is covalently attached to the SNAP-tag. CLIP-tag is a modified version of SNAP-tag, engineered to react with benzylcytosine rather than benzylguanine derivatives. When used in conjunction with SNAP-tag, CLIP-tag enables the orthogonal and complementary labeling of two proteins simultaneously in the same cells.

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again.

Labeling fluorescentes de COS-7 expressando as proteínas SNAP tag-Fusion para imagens de células vivas

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest ...

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 ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

Microscopia intravital para Estruturas de imagem Subcelulares em ratos vivos Expressando fluorescentes Proteínas

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy (OCM)

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical coherence microscopy (OCM) has been demonstrated to be capable of imaging small animal hearts with high spatial resolution and ultrahigh imaging speed. The high image contrast and noninvasive properties make OCM ideal for performing longitudinal studies without requiring tissue dissections or staining. Drosophila has been widely used as a model organism in cardiac developmental studies due to its high number of orthologous human disease genes, its similarity of molecular mechanisms and genetic pathways with vertebrates, its short life cycle, and its low culture cost. Here, the experimental protocols are described for the preparation of Drosophila and optical imaging of the heartbeat with a custom OCM system throughout the life cycle of the specimen. By following the steps provided in this report, transverse M-mode and 3D OCM images can be acquired to conduct longitudinal studies of the Drosophila cardiac morphology and function. The en face and axial sectional OCM images and the heart rate (HR) and cardiac activity period (CAP) histograms, were also shown to analyze the heart structural changes and to quantify the heart dynamics during Drosophila metamorphosis, combined with the videos constructed with M-mode images to trace cardiac activity intuitively. Due to the genetic similarity between Drosophila and vertebrates, longitudinal study of heart morphology and dynamics in fruit flies could help reveal the origins of human heart diseases. The protocol here would provide an effective method to perform a wide range of studies to understand the mechanisms of cardiac diseases in humans.

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy OCM

Here, the experimental protocols are described for preparing Drosophila at different developmental stages and performing longitudinal optical imaging of Drosophila heartbeats using a custom optical coherence microscopy (OCM) system. The cardiac morphological and dynamical changes can be quantitatively characterized by analyzing the heart structural and functional parameters from OCM images.

Drosophila Preparação e imagem longitudinal do coração Função In Vivo Usando Optical Coherence Microscopy (OCM)

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical ...