Les auteurs remercient SI Bernstein (San Diego State University) pour une lecture critique et des suggestions utiles concernant la préparation de ce manuscrit. Ce travail a été soutenu par des subventions du NIH pour SI Bernstein, SDSU, et à R. Bodmer, BIMR, et par une bourse post-doctorale de l&#39;affiliation Ouest des États de l&#39;American Heart Association à G. Vogler et à A. Cammarato.

Avant de commencer <ol><li> Préparer les solutions suivantes: <ol class="lalpha"><li> mémoire tampon de détente (artificielle drosophile hémolymphe (ADH) (voir &quot;Visualisation du cœur battant de la drosophile&quot;) qui contient 10 mM EGTA) </li><li> fixateur (formaldéhyde à 4% dans du PBS 1X) </li><li> PBSTx (PBS contenant 0,1% de Triton-X-100) </li><li> Convenablement diluée primaire et spécifique à l&#39;espèce marqué par fluorescence des anticorps secondaire dans PBSTx </li></ol></li><li> Disséquer la drosophile pour exposer les tubes cardiaques (des mouches adultes et / ou larves) suite <ol class="lalpha"><li> le protocole de semi-intactes drosophile préparation cardiaque dans «Visualiser le cœur battant de la drosophile», ou </li><li> Drosophile larvaire Dissection JNM 1 du protocole avec les modifications suivantes: Utilisez oxygénée ADH pendant la dissection des larves et de déplacer la broche postérieure légèrement de la ligne médiane ventrale. Les larves sont coupés le long de la ligne médiane ventrale. Ne pas enlever tout tissu avant la fixation. </li></ol></li></ol> Révélation <ol><li> Vérifier que tous les cœurs sont frappant en cadence dans oxygénée ADH. Remplacer rapidement l&#39;ADH à la détente de la mémoire tampon. Examinez chaque tube cardiaque à assurer les contractions sont inhibées. </li><li> Fixer les cœurs en remplaçant le tampon de détente avec fixateur. Incuber à température ambiante pendant 20 minutes en remuant doucement. (Pour secouer la préparation des larves n&#39;est pas nécessaire à n&#39;importe quelle étape, et peut être considéré comme préjudiciable à l&#39;intégrité du tissu cardiaque). </li><li> Lavez échantillons à trois reprises pendant dix minutes avec PBSTx à température ambiante avec agitation continue. </li><li> Pour les adultes, soigneusement tailler les bords ventraux de la cuticule abdominale telle que ce qui reste est plus elliptique et moins arrondis. En outre, avec une seule coupure entre l&#39;abdomen et du thorax, avec soin et proprement séparées deux segments du corps et assurer un minimum de dommages à la région antérieure du cœur. Pour les cœurs des larves, retirez soigneusement le corps gras à l&#39;aide de pinces fines. L&#39;élimination de la graisse doit être exécuté avec une extrême prudence, puisque le cœur des larves est particulièrement fragile et a très peu de soutien d&#39;autres muscles ou les tissus conjonctifs. Ne pas enlever les branches trachéale car cela peut endommager le coeur. </li><li> Transfert de la région dorsale parés de la cuticule abdominale par les bords, en évitant tout contact avec le tube situé cardiaque, dans une plaque de 96 puits avec des puits qui contiennent de 50 à 100 pi d&#39;anticorps primaire dilué dans PBSTx. Ne placez pas plus de 12 spécimens par puits. Incuber à température ambiante pendant 2 heures avec une agitation continuelle. L&#39;incubation peut aussi être fait durant la nuit à 4 ° C. </li><li> Retirer solution d&#39;anticorps primaire. Lavez le cœur à trois reprises pendant dix minutes avec 100 ul PBSTx à température ambiante avec une agitation continuelle. </li><li> Après élimination du tampon de lavage final, ajouter 100 pi d&#39;anticorps secondaire dans PBSTx, complétée par Alexa594-phalloïdine (1:1000). Incuber pendant une heure avec agitation constante à température ambiante. Conserver les échantillons couverts pour éviter fluorophore blanchiment. </li><li> Après incubation du secondaire, se laver le cœur à trois reprises pendant dix minutes avec 100 pi de PBSTx à température ambiante avec une agitation continuelle. Conserver les échantillons couvertes au long des étapes de lavage. </li><li> Pour le retrait de Triton-X-100, rincer les cœurs une dernière fois dans 100 ul de PBS pendant 10 minutes. Les échantillons peuvent être stockés dans l&#39;obscurité à 4 ° C pendant plusieurs jours avant le montage. </li></ol> Montage pour Hearts adultes <ol><li> Adhérer deux 18 x 18 mm à lamelles une lame de microscope avec 10 ul de milieu Vectashield montage. Les lamelles doivent être espacés de ~ 10-15 mm. Placer une petite goutte tiers de milieu de montage en entre les deux lamelles. </li><li> Placer 20 ul de Vectashield dans le centre d&#39;une lamelle tiers. </li><li> Retirez soigneusement chacun des coeurs de la solution de lavage PBS par les bords extrêmes de la cuticule, et placez doucement les coeur vers le bas sur la goutte de milieu de montage sur la lamelle tiers. Ne placez pas plus de cinq coeurs par goutte sur la lamelle. </li><li> Vérifiez sous un microscope pour s&#39;assurer que tous les cœurs sont vers le bas. </li><li> Retourner avec précaution la lamelle contenant le cœur et rapidement le placer sur la diapositive contenant la paire de lamelles de telle sorte que la goutte avec fusibles tubes cardiaque avec la gouttelette Vectashield entre la paire de lamelle. Un «pont» doit être formé par les lamelles. Les coeurs seront suspendus entre une lamelle et la lame de microscope. </li><li> Vérifiez sous un microscope pour s&#39;assurer que les cœurs sont maintenant confrontés à la hausse. </li><li> Fixer des lamelles à leurs bords avec des vernis à ongles. </li><li> Les cœurs sont maintenant prêts à être imagée par microscopie fluorescente ou confocale. </li></ol><ol><li> Déposer une goutte (~ 20 pl) Vectashield sur une lame de microscope. </li><li> Soigneusement de transfert jusqu&#39;à deux spécimens de larves dans le Vectashield et les orienter face dorsale vers le bas en utilisant des aiguilles de tungstène. </li><li> Individuellement glisser les spécimens hors du milieu de montage et d&#39;aligner chacune en parallèle. </li><li> Placer une lamelle sur des côtés opposés de l&#39;échantillon. En utilisant des pinces, placer une lamelle troisième sommet, d&#39;abord par la pose d&#39;un côté sur la lamelle postérieure et l&#39;abaisser jusqu&#39;à la lamelle antérieure formant ainsi un pont. Forces capillaires provoquera un flux de Vectashield d&#39;arrière en avant qui permet un bon alignement du cœur larvaire. </li><li> Fixer tous les lamelles en place, à leurs bords, avec du vernis à ongles et remplissez soigneusement l&#39;espace entre les lamelles avec 20-30 Vectashield ul. </li><li> Sceller avec du vernis à ongles et stocker à 4 ° C, ou pour stockage à long terme à -20 ° C. </li></ol> Les résultats représentatifs Lorsqu&#39;il est exécuté correctement, tous les composants et les tissus associés du vaisseau dorsal doit rester intact et être facilement visualisés. La fluorescence de fond devrait être minime. Pour les adultes, la ventrale longitudinale taches couche musculaire très bien et produit un signal important (Figure 1 et Figure 2). Les cardiomyocytes sous-jacente circulaires ont cependant tendance à ne pas produire un signal plus intense que celle de la couche recouvrant ventrale. Les myocytes de l&#39;antérieur &quot;chambre conique» du cœur adulte contient une quantité importante de matériel contractile et, par conséquent, cette région apparaît comme le parent le plus robuste pour le reste du tube cardiaque. Le cœur des larves montre un agencement en spirale myofibrillaire similaire à celle de l&#39;cardiomyocytes adultes contractiles (Figure 3). <img src="/files/ftp_upload/1423/1423fig1atmb.jpg" alt="Figure 1a" border="0" /> <img src="/files/ftp_upload/1423/1423fig1btmb.jpg" border="0" alt="Figure 1b" /> Figure 1: Haut. Une micrographie montrant le tube fluorescent toute cardiaque d&#39;un adulte de Drosophila melanogaster qui exprime la myosine-GFP. L&#39;image a été prise avec un microscope Zeiss Z1 Imager fluorescents équipés d&#39;un module Apotome coulissantes. Myosine-GFP est représenté en vert, l&#39;actine est coloré avec AlexaFluor phalloïdine ® 594 (rouge) et α-actinine est étiqueté avec des anti-α-actinine anticorps (bleu). Notez la procédure de préparation permet la récupération des échantillons coeur avec des structures bien conservées. CC = coniques chambre; AM = muscle Alary; V = interne de soupape; VLL = couche musculaire ventrale longitudinale. Bottom. Une région du tube cardiaques adultes de la CC à travers le 3ème segment abdominal de coeur juste sous la couche ventrale longitudinale montrant l&#39;agencement en spirale myofibrille des cardiomyocytes contractiles. S&#39;il vous plaît <a href="https://www.jove.com/files/ftp_upload/1423/1423fig1a.jpg">cliquez ici</a> pour une version agrandie de la figure 1a, et <a href="https://www.jove.com/files/ftp_upload/1423/1423fig1b.jpg">ici</a> pour une version agrandie de la figure 1b <img border="0" src="/files/ftp_upload/1423/1423fig2tmb.jpg" alt="Figure 2" /> Figure 2: Représentant des piles confocale d&#39;une partie antérieure du cœur adulte colorés avec AlexaFluor phalloïdine ® 594 (rouge) et un anti-Pericardin (collagène de type IV) (vert) d&#39;anticorps. Pericardin est associé avec le cœur le long de la surface ventrale, vraisemblablement originaires du myofibrilles orientées longitudinalement de la couche musculaire ventrale. S&#39;il vous plaît <a href="https://www.jove.com/files/ftp_upload/1423/1423fig2.jpg">cliquez ici</a> pour une version agrandie de la figure 2. <img border="0" src="/files/ftp_upload/1423/1423fig3tmb.jpg" alt="Figure 3" /> Figure 3: Une micrographie fluorescence du segment A7 du cœur d&#39;un bon trois étapes larve de drosophile. L&#39;image a été prise avec un microscope Zeiss Z1 Imager fluorescents équipés d&#39;un module Apotome coulissantes. L&#39;actine est étiqueté avec AlexaFluor phalloïdine ® 594 (vert) et α-actinine est taché avec des anti-α-actinine anticorps (rouge). S&#39;il vous plaît <a href="https://www.jove.com/files/ftp_upload/1423/1423fig3.jpg">cliquez ici</a> pour une version agrandie de la figure 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>

Nous présentons ici un protocole utile pour la préparation et la coloration du navire Drosophila melanogaster dorsale et des tissus attenants à l&#39;imagerie par microscopie fluorescente ou confocale. Nous fournissons un compte rendu concis des étapes affiné par et couramment employées dans notre laboratoire pour la coloration efficace qui permet de bien résolu à l&#39;imagerie in situ de la larve et adulte tubes cardiaques chez la drosophile. D&#39;autres ont décrit des méthodes si...

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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>Polysciences Inc.</td>
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<td>Triton-X-100</td>
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<td>Sigma-Aldrich&reg;</td>
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<td>Alexa Fluor&reg; 594 phalloidin</td>
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fluorescent labeling of drosophila heart structures

L&#39;<em&gt; Drosophila melanogaster</em&gt; Vaisseau dorsal, ou du coeur, est une structure tubulaire composé d&#39;une seule couche de cardiomyocytes contractiles, cellules péricardiques qui s&#39;alignent le long de chaque côté de la paroi cardiaque, les muscles de soutien et Alary, chez les adultes, une couche de cellules musculaires longitudinales ventrales. La maison contractiles des fibres conservé constituants de la cytoarchitecture musculaires y compris les faisceaux denses et complexes des myofibrilles protéine du cytosquelette / submembranous, qui interagissent avec les composantes homologues de la matrice extracellulaire. Nous décrivons ici un protocole pour la fixation et le marquage fluorescent des éléments particuliers du myocarde du cœur des larves disséquées et semi-intactes pour adultes<em&gt; Drosophile</em&gt;. Plus précisément, nous démontrons l&#39;étiquetage des sarcomériques F-actine et d&#39;α-actinine dans les coeurs des larves. De plus, nous effectuons l&#39;étiquetage des F-actine et α-actinine dans myosine-GFP exprimer mouches adultes et d&#39;α-actinine et pericardin, un collagène de type IV de la matrice extracellulaire, dans les cœurs sauvages adulte type. Une attention particulière est accordée à une stratégie de fixation pour les adultes semi-coeurs intacts qui minimise la manipulation et optimise la possibilité de maintenir l&#39;intégrité des tubes cardiaques et les tissus associés. Ces préparations sont adaptés à l&#39;imagerie par microscopie fluorescente et confocale. Globalement, cette procédure permet une analyse attentive et détaillée des caractéristiques structurelles du coeur d&#39;un puissant système de modèle génétiquement maniable.

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

Fluorescent Labeling of Drosophila Heart Structures

Nous décrivons ici un protocole de base pour le marquage fluorescent des différents éléments de tubes coeur de la larve et l&#39;adulte<em&gt; Drosophila melanogaster</em&gt;. Ces spécimens sont bien adaptés pour l&#39;imagerie par microscopie fluorescente ou confocale. Cette technique permet une analyse détaillée des caractéristiques structurelles des cœurs d&#39;un organisme modèle puissant.

Marquage fluorescent Drosophile</emOuvrages d&#39;art> Coeur

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 vues.  San Diego State University. Nous décrivons ici un protocole de base pour le marquage fluorescent des différents éléments de tubes coeur de la larve et l'adulte Drosophila melanogaster. Ces spécimens sont bien adaptés pour l'imagerie par microscopie fluorescente ou confocale. Cette technique permet une analyse détaillée des caractéristiques structurelles des cœurs d'un organisme modèle puissant.

Vidéo: 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.

Étiquetage des cellules souches avec des colorants fluorescents pour les non-invasive de détection avec l&#39;imagerie optique

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 ...

Recherche

Biologie

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.

Visualiser le Live Drosophile Gliales-jonction neuromusculaire avec des colorants fluorescents

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.

Visualiser le cœur battant Drosophile</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.

Surveillance de la fonction cardiaque chez les larves Drosophila melanogaster Pour les études physiologiques

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.

Préparation d&#39;embryons pour la microscopie électronique des Drosophile Tube cardiaque embryonnaire

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.

Préparation des Courges drosophile chromosomes polytènes pour marquage de l&#39;anticorps

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.

Marquage fluorescent du COS-7 exprimant des protéines de fusion SNAP-tag pour imagerie de cellules vivantes

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.

Toute Mont ARN fluorescent<em&gt; In situ</emHybridation&gt; d&#39;<em&gt; Drosophila</em&gt; Embryons

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.

Microscopie intravitale pour l&#39;imagerie Subcellular ouvrages chez des souris vivantes exprimant des protéines 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 Préparation et imagerie longitudinale de la fonction cardiaque in vivo en utilisant cohérence optique Microscopie (OCM)

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

Marquage fluorescent Drosophile Coeur