Los autores agradecen SI Bernstein (San Diego State University) para la lectura crítica y sugerencias relativas a la preparación de este manuscrito. Este trabajo fue apoyado por subvenciones del NIH para SI Bernstein, SDSU, y R. Bodmer, BIMR, y por una beca post-doctoral de la Filial Oeste de los Estados de la Asociación Americana del Corazón para G. Vogler y A. Cammarato.

Antes de empezar <ol><li> Prepare las siguientes soluciones: <ol class="lalpha"><li> buffer relajante (artificial hemolinfa Drosophila (ADH) (véase &quot;Visualización de los latidos del corazón en Drosophila&quot;) que contiene 10 mM EGTA) </li><li> fijador (formaldehído al 4% en PBS 1x) </li><li> PBSTx (PBS con 0,1% Triton-X-100) </li><li> Convenientemente diluida primaria y especies específicas de la etiqueta fluorescente secundaria de anticuerpos en PBSTx </li></ol></li><li> Diseccionar Drosophila para exponer los tubos cardíacos (de moscas adultas y / o larvas), tras <ol class="lalpha"><li> la semi-intacta corazón Drosophila preparación del protocolo de &quot;visualizar el corazón latiendo en Drosophila&quot;, o </li><li> Drosophila larval NMJ disección 1 del protocolo con las siguientes modificaciones: Use oxigenada ADH durante la disección de larvas y desplazar el pasador posterior ligeramente de la línea media ventral. Las larvas se cortan a lo largo de la línea media ventral. No remover el tejido antes de la fijación. </li></ol></li></ol> La tinción fluorescente <ol><li> Compruebe que todos los corazones están latiendo en rítmicamente oxigenada ADH. Colocar inmediatamente ADH con relajantes buffer. Examine cada tubo cardíaco para asegurar las contracciones se inhiben. </li><li> Fijar los corazones mediante la sustitución de la memoria intermedia de relax con fijador. Incube a temperatura ambiente durante 20 minutos con agitación suave. (Para agitar la preparación de las larvas no es necesario en cualquier etapa, y puede ser considerado perjudicial para la integridad del tejido cardíaco). </li><li> Lávese las muestras tres veces durante diez minutos con PBSTx a temperatura ambiente con agitación continua. </li><li> Para los adultos, cuidadosamente Recorte los bordes de la cutícula ventral abdominal, que lo que queda es más elíptica y menos redondeada. Además, con un solo corte entre el abdomen y el tórax, cuidadosa y limpiamente separar ambos segmentos del cuerpo y asegurar el mínimo daño a la región anterior del corazón. Por los corazones de larvas, retire con cuidado la grasa corporal utilizando unas pinzas finas. La eliminación de grasa deben ser ejecutados con extrema precaución, ya que el corazón de larvas es muy frágil y tiene muy poco apoyo de otros músculos o del tejido conectivo. No quite las ramas traqueal, ya que puede dañar el corazón. </li><li> Transferencia de la región dorsal recortada de la cutícula abdominal por los bordes, evitando el contacto con el tubo situado en el centro cardíaco, en una placa de 96 pocillos con pozos que contienen desde 50 hasta 100 l de anticuerpo primario diluido en PBSTx. Por no más de 12 muestras por pozo. Incube a temperatura ambiente durante 2 horas con agitación continua. Incubación también se puede hacer durante la noche a 4 ° C. </li><li> Eliminar la solución de anticuerpo primario. Lavar el corazón tres veces durante diez minutos con 100 PBSTx l a temperatura ambiente con agitación continua. </li><li> Tras la retirada del tampón de lavado final, añadir 100 ml de anticuerpo secundario en PBSTx, complementado con Alexa594-phalloidin (1:1000). Incubar durante una hora con agitación continua a temperatura ambiente. Mantener las muestras cubiertas para evitar fluoróforo blanqueo. </li><li> Después de la incubación secundaria, lavar el corazón tres veces durante diez minutos con 100 l de PBSTx a temperatura ambiente con agitación continua. Mantener las muestras cubiertas a lo largo de las etapas de lavado. </li><li> Para la eliminación de Triton-X-100, aclare el corazón por última vez en 100 l de PBS durante 10 minutos. Las muestras se pueden almacenar en la oscuridad a 4 ° C durante varios días antes del montaje. </li></ol> Montaje de los corazones de adultos <ol><li> Se adhieren dos de 18 x 18 mm cubreobjetos a un portaobjetos de microscopio con 10 l de medio Vectashield montaje. Los cubreobjetos se deben espaciar ~ 10-15 mm. Coloque una pequeña gota de tercero medio de montaje en entre los dos cubreobjetos. </li><li> Ponga 20 l de Vectashield en el centro de un cubre terceros. </li><li> Retire con cuidado cada uno de los corazones de la solución de lavado PBS por los bordes extremos de la cutícula, y suavemente coloque su corazón hacia abajo sobre la gota de medio de montaje sobre el cubreobjetos terceros. Por no más de cinco corazones por gota sobre el cubreobjetos. </li><li> Compruebe bajo un microscopio para asegurar que todos los corazones están mirando hacia abajo. </li><li> Con cuidado, invertir el cubreobjetos que contiene el corazón y la rapidez con que el lugar de la diapositiva que contiene el par de cubreobjetos de tal manera que la gota con fusibles tubos cardíaco con la gota Vectashield entre los dos cubreobjetos. Un &quot;puente&quot; debe ser formado por el cubreobjetos. Los corazones serán suspendidos entre un cubreobjetos y portaobjetos de microscopio. </li><li> Compruebe bajo un microscopio para asegurarse de que los corazones están mirando hacia arriba. </li><li> Fix cubreobjetos en sus bordes con esmalte de uñas. </li><li> El corazón está listo para ser fotografiado por fluorescentes o de cmicroscopía onfocal. </li></ol> Montaje de los corazones de larvas <ol><li> Coloque una gota (aproximadamente 20 l) Vectashield en un portaobjetos de microscopio. </li><li> Cuidado de transferencia de hasta dos muestras de larvas en el Vectashield y orientarlos lado dorsal por el uso de agujas de tungsteno. </li><li> Individualmente arrastrar las muestras de medio de montaje y de alinear cada en paralelo. </li><li> Coloque un cubreobjetos en lados opuestos de la muestra. Con unas pinzas, coloque un cubreobjetos tercera en la parte superior, por primera vez por un lado, por la que se sobre el cubreobjetos posterior y bajándola hasta el cubreobjetos anterior, formando así un puente. Las fuerzas capilares causará un flujo de Vectashield de posterior a anterior, que ayuda a la correcta alineación del corazón larval. </li><li> Arreglar todos los cubreobjetos en su lugar, en sus bordes, con esmalte de uñas y cuidado rellenar el espacio entre los cubreobjetos Vectashield con 20-30 l. </li><li> Sellar con esmalte de uñas y se almacenan a 4 ° C, o para almacenamiento a largo plazo a -20 ° C. </li></ol> Resultados representante Cuando se ejecuta correctamente, todos los componentes y los tejidos del vaso dorsal deben permanecer intactos y se visualiza fácilmente. La fluorescencia de fondo debe ser mínimo. Para los adultos, el ventral manchas longitudinales capa muscular muy bien y produce una señal importante (Figura 1 y Figura 2). Los cardiomiocitos circular subyacente sin embargo, tienden a no producir una señal tan intenso como el de la capa que recubre ventral. Los miocitos de la anterior &quot;cámara cónica&quot; del corazón adulto contienen una cantidad considerable de material contráctil y, en consecuencia, esta región aparece como la relación más sólida con el resto del tubo cardíaco. El corazón de larvas muestra una espiral arreglo miofibrilar similar a la de los cardiomiocitos adultos contráctil (Figura 3). <a href="https://www.jove.com/files/ftp_upload/1423/1423fig1a.jpg"><img src="/files/ftp_upload/1423/1423fig1atmb.jpg" alt="Figura 1a" border="0" /></a> <a href="https://www.jove.com/files/ftp_upload/1423/1423fig1b.jpg"><img src="/files/ftp_upload/1423/1423fig1btmb.jpg" border="0" alt="Figura 1b" /></a> Figura 1: Inicio. Una micrografía fluorescente que muestra todo el tubo cardíaco de un adulto Drosophila melanogaster, que expresa la miosina-GFP. La imagen fue tomada con un microscopio Zeiss Imager Z1 fluorescentes equipados con un módulo ApoTome deslizamiento. Miosina-GFP se muestra en verde, la actina se tiñe con AlexaFluor phalloidin ® 594 (roja) y α-actinina se etiqueta con anti-α-actinina anticuerpos (azul). Tenga en cuenta el procedimiento de preparación permite la recuperación de las muestras de corazón con estructuras bien conservadas. CC = cónica cámara; AM = músculo Alary, V = válvula interna; VLL = capa muscular longitudinal ventral. Parte inferior. Una región de la trompa de un adulto cardiaca de la CC a través del segmento abdominal tercero del corazón justo debajo de la capa ventral longitudinal que muestra la disposición de las miofibrillas en espiral de los cardiomiocitos contráctiles. <a href="https://www.jove.com/files/ftp_upload/1423/1423fig2.jpg"><img border="0" src="/files/ftp_upload/1423/1423fig2tmb.jpg" alt="Figura 2" /></a> Figura 2: Representante confocal pilas de una porción anterior del corazón adulto teñidas con phalloidin AlexaFluor ® 594 (rojo) y un anti-Pericardin (colágeno tipo IV) (verde) de anticuerpos. Pericardin se asocia con el corazón a lo largo de la superficie ventral, probablemente procedentes de las miofibrillas orientadas longitudinalmente de la capa muscular ventral. <a href="https://www.jove.com/files/ftp_upload/1423/1423fig3.jpg"><img border="0" src="/files/ftp_upload/1423/1423fig3tmb.jpg" alt="Figura 3" /></a> Figura 3: Una micrografía fluorescente del segmento A7 del corazón propio de una etapa de tres larvas de Drosophila. La imagen fue tomada con un microscopio Zeiss Imager Z1 fluorescentes equipados con un módulo ApoTome deslizamiento. Actina se etiqueta con AlexaFluor phalloidin ® 594 (verde) y α-actinina se tiñeron con anti-α-actinina anticuerpos (rojo).

<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>

Aquí se presenta un protocolo útil para la preparación y la tinción de la embarcación Drosophila melanogaster dorsal y los tejidos a través de imágenes de microscopía de fluorescencia confocal o. Ofrecemos un relato conciso de los pasos y refinado por empleado comúnmente en nuestro laboratorio para la tinción de efectivo que permita bien resueltos en las imágenes in situ de las larvas y adultos tubos corazón Drosophila. Otros han descrito métodos similares en forma abrevi...

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<td>Ethylene glycol-bis (2-amino-ethylether) -N,N,N&#x2019;,N&#x2019;-tetra-acetic acid (EGTA)</td>
<td>Reagent</td>
<td>Sigma-Aldrich&reg;</td>
<td>E4378</td>
<td>&nbsp;</td>
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<td>Formaldehyde, 10%, methanol free, Ultra Pure</td>
<td>Reagent</td>
<td>Polysciences Inc.</td>
<td>50-00-0</td>
<td>&nbsp;</td>
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<td>Triton-X-100</td>
<td>Reagent</td>
<td>Sigma-Aldrich&reg;</td>
<td>9002-93-1</td>
<td>&nbsp;</td>
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<td>Alexa Fluor&reg; 594 phalloidin</td>
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<td>Vectashield&reg; Mounting Medium for Fluorescence with DAPI</td>
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<td>Vector Laboratories, Inc.</td>
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fluorescent labeling of drosophila heart structures

La<em&gt; Drosophila melanogaster</em&gt; Vaso dorsal, o el corazón, es una estructura tubular que consta de una sola capa de cardiomiocitos contráctiles, células pericárdicas que se alinean a lo largo de cada lado de la pared del corazón, los músculos de apoyo Alary y, en adultos, una capa de células de músculo longitudinal ventral. La casa de las fibras contráctiles conservan los componentes de la citoarquitectura músculos incluidos los paquetes compactos de las miofibrillas y complejos de proteínas del citoesqueleto / submembranous, que interactúan con homólogos de los componentes de la matriz extracelular. Aquí se describe un protocolo para la fijación y el marcaje fluorescente de elementos particulares de miocardio del corazón de las larvas y adultos disecados semi-intacta<em&gt; Drosophila</em&gt;. En concreto, se demuestra el etiquetado de los sarcoméricas F-actina y α-actinina en el corazón de las larvas. Además, llevamos a cabo el etiquetado de los F-actina y α-actinina en la miosina-GFP expresando las moscas adultas y de la α-actinina y pericardin, un colágeno tipo IV de la matriz extracelular, en el medio silvestre corazones de tipo adulto. Se presta especial atención a una estrategia de fijación de los corazones de adultos semi-intacta que minimiza el manejo y optimiza la oportunidad de mantener la integridad de los tubos cardíacos y los tejidos asociados. Estas preparaciones son adecuadas para las imágenes a través de microscopía de fluorescencia y confocal. En general, este procedimiento permite realizar un análisis cuidadoso y detallado de las características estructurales del corazón de un potente sistema genético modelo manejable.

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

Fluorescent Labeling of Drosophila Heart Structures

Aquí se describe un protocolo básico para el marcaje fluorescente de diferentes elementos de los tubos de corazón de la larva y el adulto<em&gt; Drosophila melanogaster</em&gt;. Estas muestras son adecuadas para las imágenes a través de microscopía de fluorescencia confocal o. Esta técnica permite el análisis estructural detallado de las características de los corazones de un organismo modelo de gran alcance.

Marcaje fluorescente de Drosophila Estructuras del Corazón

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

13.5K vistas.  San Diego State University. Aquí se describe un protocolo básico para el marcaje fluorescente de diferentes elementos de los tubos de corazón de la larva y el adulto Drosophila melanogaster. Estas muestras son adecuadas para las imágenes a través de microscopía de fluorescencia confocal o. Esta técnica permite el análisis estructural detallado de las características de los corazones de un organismo modelo de gran alcance.

Video: 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.

Etiquetado de las células madre con tintes fluorescentes para la detección no invasiva con imágenes 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 ...

Investigación

Biología

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.

Visualizar el vivo Drosophila Gliales-unión neuromuscular con tintes 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 el corazón latiendo en 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.

Monitorización de la función del corazón en las larvas Drosophila melanogaster Para los estudios 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.

Preparación de embriones para Microscopía Electrónica de la Drosophila Tubo cardíaco embrionario

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.

Preparación de los cromosomas politénicos de Drosophila Calabazas para el Etiquetado de anticuerpos

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.

Marcaje fluorescente de COS-7 Expresando etiqueta SNAP-proteínas de fusión de imágenes 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 el monte ARN fluorescente<em&gt; In situ</em&gt; La hibridación de<em&gt; Drosophila</em&gt; Los embriones

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 formar imágenes de estructuras subcelulares en ratones vivos que expresan proteínas fluorescentes

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 Preparación e Imagen longitudinal de la función cardiaca in vivo utilizando microscopía de coherencia óptica (OCM)

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