Gli autori ringraziano SI Bernstein (San Diego State University) per la lettura critica e utili suggerimenti per quanto riguarda la preparazione di questo manoscritto. Questo lavoro è stato sostenuto dalle concessioni di NIH SI Bernstein, SDSU, e di R. Bodmer, BIMR, e da un post-dottorato dal Affiliato occidentale Stati della American Heart Association a G. Vogler e ad A. Cammarato.

Prima di iniziare <ol><li> Preparare le seguenti soluzioni: <ol class="lalpha"><li> tampone rilassante (artificiale Drosophila emolinfa (ADH) (vedere &quot;Visualizzare il cuore pulsante in Drosophila&quot;) che contiene 10 mM EGTA) </li><li> fissativo (4% di formaldeide in 1x PBS) </li><li> PBSTx (PBS contenente 0,1% Triton X-100) </li><li> Opportunamente diluita anticorpi fluorescente primaria e secondaria specie-specifiche in PBSTx </li></ol></li><li> Sezionare Drosophila per esporre i tubi cardiaci (di mosche adulte e / o larve) a seguito di <ol class="lalpha"><li> il semi-intatta Drosophila preparazione protocollo cuore &quot;Visualizzare il cuore pulsante in Drosophila&quot;, o </li><li> Drosophila larvale NMJ Dissezione protocollo 1 con le seguenti modifiche: Utilizzare ossigenata ADH durante la dissezione larvale e spostare il perno posteriore leggermente dalla linea mediana ventrale. Le larve sono tagliati lungo la linea mediana ventrale. Non rimuovere i tessuti prima di fissazione. </li></ol></li></ol> La colorazione fluorescente <ol><li> Controllare che tutti i cuori sono ritmicamente battendo in ossigenato ADH. Sostituire rapidamente ADH con rilassanti buffer. Esaminare ogni tubo cardiaco per assicurare le contrazioni sono inibiti. </li><li> Fissare il cuore, sostituendo il buffer di relax con fissativo. Incubare a temperatura ambiente per 20 minuti agitando delicatamente. (Per agitazione preparazione larvale non è necessario in ogni passo, e può essere considerato dannoso per l&#39;integrità del tessuto cardiaco). </li><li> Lavare i campioni per tre volte per dieci minuti con PBSTx a temperatura ambiente con agitazione continua. </li><li> Per gli adulti, con attenzione tagliare di nuovo i bordi ventrale della cuticola addominale in modo che ciò che rimane è più ellittica e meno arrotondata. Inoltre, con un taglio unico tra l&#39;addome e il torace, attento e pulito separato da entrambi i segmenti del corpo e garantire il minimo danno alla regione anteriore del cuore. Per i cuori larvale, rimuovere con attenzione il grasso corporeo con una pinza sottile. La rimozione del grasso deve essere eseguito con estrema cautela, poiché il cuore larvale è particolarmente fragile e ha un supporto molto poco da altri muscoli o del tessuto connettivo. Non rimuovere i rami tracheali in quanto ciò potrebbe danneggiare il cuore. </li><li> Trasferire la regione tagliata dorsale della cuticola addominale per i bordi, evitando il contatto con il tubo in posizione centrale cardiaco, in una piastra da 96 pozzetti con pozzi che contengono 50-100 ml di anticorpo primario diluito in PBSTx. Luogo non più di 12 esemplari per bene. Incubare a temperatura ambiente per 2 ore con agitazione continua. L&#39;incubazione può essere effettuata anche durante la notte a 4 ° C. </li><li> Rimuovere la soluzione primaria di anticorpi. Lavare i cuori per tre volte per dieci minuti con 100 microlitri PBSTx a temperatura ambiente con agitazione continua. </li><li> Dopo la rimozione di tampone di lavaggio finale, aggiungere 100 ml di anticorpo secondario in PBSTx, integrato con Alexa594-falloidina (1:1000). Incubare per un&#39;ora con agitazione continua a temperatura ambiente. Conservare i campioni coperti per evitare fluoroforo sbiancamento. </li><li> Dopo l&#39;incubazione secondaria, lavare i cuori per tre volte per dieci minuti con 100 ml di PBSTx a temperatura ambiente con agitazione continua. Mantenere i campioni coperto per tutta la fasi di lavaggio. </li><li> Per la rimozione di Triton X-100, lavare i cuori un&#39;ultima volta in 100 ml di PBS per 10 minuti. I campioni possono essere conservati al buio a 4 ° C per diversi giorni prima del montaggio. </li></ol> Cuori di montaggio per adulti <ol><li> Rispettare due 18 x 18 mm per coprire scivola un vetrino da microscopio con 10 l di media Vectashield montaggio. I coprioggetti devono essere distanziati ~ 10-15 mm. Mettere una piccola goccia terzo di montaggio di medie-tra le due lamelle. </li><li> Mettere 20 ml di Vectashield al centro di un vetrino coprioggetto terzo. </li><li> Rimuovere con attenzione ciascuno dei cuori dalla soluzione di lavaggio PBS dai bordi estremi della cuticola, e delicatamente metterli cuore rivolto verso il basso sulla goccia di mezzo di montaggio sul vetrino terzo. Luogo non più di cinque cuori per goccia sul vetrino. </li><li> Controllare al microscopio per garantire che tutti i cuori sono rivolti verso il basso. </li><li> Con attenzione invertire il vetrino che contiene il cuore e rapidamente posto sulla diapositiva che contiene la coppia di coprioggetto in modo che la goccia con tubi cardiaci fonde con la goccia Vectashield tra la coppia coprioggetto. Un &quot;ponte&quot; dovrebbe essere formata da i coprioggetti. I cuori saranno sospesi tra un vetrino e il vetrino da microscopio. </li><li> Controllare al microscopio per garantire che i cuori sono ora rivolte verso l&#39;alto. </li><li> Fissare coprioggetto ai bordi con smalto. </li><li> I cuori sono ora pronti per essere ripreso mediante microscopia a fluorescenza o confocale. </li></ol><ol><li> Mettere una goccia (~ 20 l) Vectashield su un vetrino da microscopio. </li><li> Cura di trasferimento fino a due esemplari larvali nel loro lato Vectashield e orientare dorsale giù usando aghi di tungsteno. </li><li> Individualmente trascinare i campioni fuori dal mezzo di montaggio e allineare ciascuno in parallelo. </li><li> Mettere un coprioggetti ai lati opposti del campione. Utilizzando pinze, mettere un coprioggetto terza cima, prima che un lato del vetrino posteriore e abbassandolo fino al coprioggetto anteriore in modo da formare un ponte. Forze capillari causerà un flusso di Vectashield da anteriore a posteriore che aiuta il corretto allineamento del cuore larvale. </li><li> Sistemare tutti coprioggetto in posizione, ai bordi, con smalto e con attenzione riempire lo spazio tra i coprioggetti con 20-30 Vectashield microlitri. </li><li> Sigillare con smalto e conservare a 4 ° C, o per la conservazione a lungo termine a -20 ° C. </li></ol> Rappresentante Risultati Quando viene eseguito correttamente, tutti i componenti e dei relativi tessuti del vaso dorsale deve rimanere intatto ed essere facilmente visualizzati. La fluorescenza di fondo dovrebbe essere minimo. Per gli adulti, la ventrale longitudinale macchie strato muscolare molto bene e produce un segnale di sostanziale (Figura 1 e Figura 2). I cardiomiociti base circolare tuttavia, tendono a non produrre come un segnale forte come quella dello strato sovrastante ventrale. I miociti del anteriore &quot;camera conica&quot; del cuore adulto contengono una notevole quantità di materiale contrattile e, di conseguenza, questa regione appare come il parente più robusto per il resto del tubo cardiaco. Il cuore larvale presenta una disposizione a spirale miofibrillare simile a quella dei cardiomiociti adulti contrattile (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: Inizio. Un microscopio a fluorescenza che mostra il tubo intero cardiaca di un adulto Drosophila melanogaster che esprime miosina-GFP. L&#39;immagine è stata scattata con un microscopio a fluorescenza Zeiss Z1 Imager dotato di un modulo Apotome scorrevole. Miosina-GFP viene evidenziato in verde, actina è macchiata di AlexaFluor ® 594 falloidina (rosso) e α-actinina è etichettato con anti-α-actinina anticorpo (blu). Si noti la procedura preparativa permetta il recupero di campioni di cuore con strutture ben conservate. CC = camera conica; AM = muscolo Alary; V = valvola interna; VLL = ventrale strato muscolare longitudinale. Fondo. Una regione del tubo cardiaco adulto dal CC attraverso il terzo segmento addominale del cuore appena sotto lo strato ventrale longitudinale che mostra la disposizione a spirale miofibrilla dei cardiomiociti contrattile. Si prega di <a href="https://www.jove.com/files/ftp_upload/1423/1423fig1a.jpg">cliccare qui</a> per una versione più grande di Figura 1a, e <a href="https://www.jove.com/files/ftp_upload/1423/1423fig1b.jpg">qui</a> per una versione più grande di figura 1b <img border="0" src="/files/ftp_upload/1423/1423fig2tmb.jpg" alt="Figura 2" /> Figura 2: Rappresentante pile confocale di una porzione anteriore del cuore adulto colorate con AlexaFluor ® 594 falloidina (rosso) e un anti-Pericardin (collagene di tipo IV) (verde) di anticorpi. Pericardin è associato con il cuore lungo la superficie ventrale, probabilmente provenienti dalla miofibrille longitudinalmente orientata dello strato muscolare ventrale. Si prega di <a href="https://www.jove.com/files/ftp_upload/1423/1423fig2.jpg">cliccare qui</a> per una versione più grande della figura 2. <img border="0" src="/files/ftp_upload/1423/1423fig3tmb.jpg" alt="Figura 3" /> Figura 3: Un microscopio a fluorescenza del segmento A7 del cuore corretta di una larva di terza fase Drosophila. L&#39;immagine è stata scattata con un microscopio a fluorescenza Zeiss Z1 Imager dotato di un modulo Apotome scorrevole. Actina è etichettato con AlexaFluor ® 594 falloidina (verde) e α-actinina è macchiato con anti-α-actinina anticorpi (rosso). Si prega di <a href="https://www.jove.com/files/ftp_upload/1423/1423fig3.jpg">cliccare qui</a> per una versione più grande di 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>

Qui vi presentiamo un protocollo utile per la preparazione e la colorazione dorsale Drosophila melanogaster nave e dei relativi tessuti per l&#39;imaging tramite microscopia a fluorescenza o confocale. Forniamo un resoconto sintetico dei passi da raffinate e comunemente impiegati nel nostro laboratorio per la colorazione efficace che permette ben risolta nella diagnostica per immagini in situ di larve e adulti tubi cuore Drosophila. Altri hanno descritto metodi simili in forma abbrevi...

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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>Sigma-Aldrich&reg;</td>
<td>E4378</td>
<td>&nbsp;</td>
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<td>Formaldehyde, 10%, methanol free, Ultra Pure</td>
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<td>Polysciences Inc.</td>
<td>50-00-0</td>
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<td>Triton-X-100</td>
<td>Reagent</td>
<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

Il<em&gt; Drosophila melanogaster</em&gt; Vaso dorsale, o cuore, è una struttura tubolare costituito da un singolo strato contrattile dei cardiomiociti, le cellule del pericardio che si allineano lungo ogni lato del muro di cuore, i muscoli Alary di supporto e, negli adulti, uno strato di cellule muscolari longitudinale ventrale. La casa contrattile delle fibre conservato componenti del citoarchitettura tra fasci muscolari densamente delle miofibrille e dei complessi di proteine ​​del citoscheletro / submembranous, che interagiscono con omologhe componenti della matrice extracellulare. Qui si descrive un protocollo per la fissazione e la marcatura fluorescente di particolari elementi del miocardio dal cuore delle larve sezionato e semi-intatte adulti<em&gt; Drosophila</em&gt;. In particolare, abbiamo dimostrato l&#39;etichettatura dei sarcomerica F-actina e di α-actinina nei cuori larvale. Inoltre, noi dobbiamo fare l&#39;etichettatura di F-α-actina e miosina in actinina-GFP che esprimono le mosche adulte e di α-actinina e pericardin, una matrice di collagene di tipo IV extracellulare, nel cuore selvaggio di tipo adulto. Particolare attenzione viene data a una strategia di montaggio per semi-intatti i cuori degli adulti che riduce al minimo la manipolazione ed ottimizza la possibilità di mantenere l&#39;integrità dei tubi e dei tessuti cardiaci associati. Questi preparati sono adatti per l&#39;imaging tramite microscopia a fluorescenza e confocale. In generale, questa procedura consente di analisi attenta e dettagliata delle caratteristiche strutturali del cuore da un potente sistema geneticamente modello trattabili.

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

Fluorescent Labeling of Drosophila Heart Structures

Qui si descrive un protocollo di base per la marcatura fluorescente di elementi diversi di tubi cuore da larve e adulti<em&gt; Drosophila melanogaster</em&gt;. Questi esemplari sono particolarmente adatti per l&#39;imaging mediante microscopia a fluorescenza o confocale. Questa tecnica permette una dettagliata analisi strutturale delle caratteristiche del cuore di un organismo modello potente.

Etichette fluorescenti di Drosophila Cuore Strutture

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 Views.  San Diego State University. Qui si descrive un protocollo di base per la marcatura fluorescente di elementi diversi di tubi cuore da larve e adulti Drosophila melanogaster. Questi esemplari sono particolarmente adatti per l'imaging mediante microscopia a fluorescenza o confocale. Questa tecnica permette una dettagliata analisi strutturale delle caratteristiche del cuore di un organismo modello potente.

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.

Etichettatura Cellule staminali con le tinture fluorescenti per la rilevazione non invasiva con 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 ...

Ricerca

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.

Visualizzazione di Live Drosophila Gliali-giunzione neuromuscolare con tinture fluorescenti

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.

Visualizzare il cuore pulsante di 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.

Il monitoraggio delle funzioni cardiaca in larvali Drosophila melanogaster Per gli Studi fisiologici

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.

Preparazione di embrioni per la microscopia elettronica del Drosophila Tubo cuore embrionale

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.

Preparazione dei cromosomi di Drosophila Zucche politene per l&#39;etichettatura di anticorpi

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.

Etichettatura fluorescente di COS-7 Esprimendo SNAP-tag proteine ​​di fusione per l&#39;imaging cellulare dal vivo

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.

Tutta la Monte RNA fluorescente<em&gt; In situ</em&gt; L&#39;ibridazione di<em&gt; Drosophila</em&gt; Embrioni

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.

Intravitale Microscopia per le strutture Imaging subcellulari in topi vivi Esprimere proteine ​​fluorescenti

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 preparazione e imaging longitudinale della funzione cardiaca in vivo utilizzando Optical Coherence Microscopy (OCM)

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