Name: fluorescent labeling of drosophila heart structures
Uploaded: 2009-10-13T23:30:00
Description: Watch this Scientific Journal Video about Fluorescent Labeling of Drosophila Heart Structures at JoVE.com

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...

<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>
<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>
<tr>
<td>Triton-X-100</td>
<td>Reagent</td>
<td>Sigma-Aldrich&reg;</td>
<td>9002-93-1</td>
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<td>Alexa Fluor&reg; 594 phalloidin</td>
<td>Reagent</td>
<td>Invitrogen&trade;</td>
<td>A12381</td>
<td>&nbsp;</td>
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<td>Vectashield&reg; Mounting Medium for Fluorescence with DAPI</td>
<td>Reagent</td>
<td>Vector Laboratories, Inc.</td>
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<td>Tungsten pins</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.

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