Questo lavoro è stato sostenuto da una borsa di studio a Ehrman HY. H., Jane Coffin Childs Memorial Fund per la borsa di studio post-dottorato di ricerca medica RJJ, NIH Grant F32EY016309 a DV, Fellowship Tesi di New York University di Dean a DJ, NIH GrantR01 EY13010 su CD e una borsa di studio per DFG JR (RI 2208/1- 1). Ringraziamo Nina Vogt e Pamela Boodram per i commenti sul manoscritto.

1. Introduzione In questo video, si descrivono i metodi per dissezioni retiniche e immunoistochimica in tre periodi definiti di sviluppo: il terzo instar larvali, la midpupal e la fase adulta. Anche se il nostro protocollo funziona anche per le altre fasi di pupa (per i dettagli su fasi precedenti, vedi 16), abbiamo scelto lo stadio midpupal, come è ottimale per l&#39;imaging tutte le PR in un unico piano focale e la loro nuclei sono facilmente identificabili, che facilita la visualizzazione di fattore di trascrizione espressione modelli. La dissezione di ritardo pupa retina è molto simile alla dissezione dei retine adulti. In primo luogo, si dimostra come raccogliere larve e pupe nelle fasi desiderati. Poi, ci spiega come ottenere dissezioni ottimali di larvale 17-18 midpupal e retine adulti. In secondo luogo, si dimostra come visualizzare sviluppo PR in queste fasi usando la microscopia confocale e immunoistochimica. Il periodo di sviluppo degli insetti dipende dalla temperatura. Per facilitare la messa in scena riproducibile di larve e pupe, si raccomanda di mettere tutte le scorte di mosca a 25 ° C sotto un hr/12 12 ore ciclo luce / buio. Terzo instar larve: A proposito di 96 ore dopo la deposizione delle uova (a 25 ° C), fine del III alimentazione larve instar stop e vagare sulle pareti della fiala alimentare, dove alla fine pupate. Utilizzare una spazzola morbida o una pinza per rimuovere delicatamente un vagare terzo instar larve dalla parete del flaconcino cibo. 50% pupe (&#39;midpupae&#39;) sono stati osservati circa 48 ore dopo impupamento. Per i tempi esatti, si può girare intorno appena formata pupe bianco con un pennarello sul flaconcino cibo. In alternativa, spostare tutti pupe bianco a un nuovo flacone per ottenere una fiala di messa in scena uniforme (per la messa in scena di pupe in base a criteri morfologici, vedere 19). Dopo 48 ore, con attenzione remove il midpupae dal flacone utilizzando una pinza. Mosche adulte: anestetizzare mosche giovani adulti (2-5 giorni dopo eclosion) su un tappetino di CO 2. Rimuovere testine di stampa utilizzando una pinza e memorizzarli in un bicchiere e servire freddo contenente 1x PBS. 3. Dissezione Procedura (Circa 1-2 ore per Experiment) Dopo aver raccolto il campione, metterli in una goccia di PBS freddo su un piatto dissezione Sylgard. Dissect circa 10-15 retine per genotipo. Si prega di consultare le schede di sicurezza, nonché la salute ambientale del proprio istituto e Office di sicurezza per la corretta manipolazione dei reagenti e delle attrezzature (vedi tabelle alla fine di questo protocollo). 1. Dischi Eye larvali immaginale <ol><li> Individuare l&#39;estremità anteriore della larva, che contiene i ganci bocca. Utilizzare una pinza per premere la parte centrale della larva dolcemente contro la base del piatto Sylgard. Utilizzare another pinza per afferrare i ganci bocca e tirare delicatamente e lentamente dal corpo. Scartare la parte principale del corpo e utilizzare l&#39;apparato boccale per tenere la parte anteriore in modo da rimuovere il tessuto estraneo dai dischi immaginali che rimangono attaccate al cervello. </li><li> Trasferimento cervello con i dischi immaginali afferrando i ganci bocca con una pinza a 1x PBS in un tre ben piatto di vetro. Eseguire le operazioni di fissaggio (passo 4.1.). </li><li> Dopo la fissazione, rimuovere ghiandole salivari, tessuto adiposo e tirare il cervello lontano dagli occhi-antennale disco immaginale. Per migliorare l&#39;accessibilità delle &quot;difficili&quot; anticorpi, la membrana peripodial, che si trova sulla superficie dell&#39;epitelio, può essere rimosso premendo la parte antennale del disco e la membrana peeling via con un perno dissezione. Successivamente, eseguire immunoistochimica (punto 5.1.). </li></ol> 2. 50% ninfosi / Midpupal retine <ol><li> Tenere estremità posteriore del case pupa con una pinza.Rimuovere delicatamente la superficie anteriore del case pupa afferrando la cuticola con altre pinze e tirare lentamente dal pupario. Staccare cuticola residua per esporre la testa. </li><li> Utilizzare i suggerimenti pinze &quot;per perforare il fondo, sacco morbido nella regione dorsale anteriore. Questo fornisce l&#39;accesso alla testa. Delicatamente separare la testa dal torace lentamente succhiando la testa con una pipetta P20. Espellere testa dalla punta della pipetta nel fresco freddo 1x PBS e rimuovere il tessuto in eccesso; afferrare la cuticola testa e rimuovere proboscide. Procedere alla fase di fissaggio (passo 4.1.). </li><li> Pierce il lobo ottico con un perno di dissezione per stabilizzare il tessuto. Inserire l&#39;altro perno dissezione tra la lamina e retina per separare la retina dalla lamina / lobo ottico. Eseguire immunoistochimica (punto 5.1.). </li></ol> 3. Occhi adulti <ol><li> Afferra il centro della capsula posteriore testa con una pinza e rimuovere le parti della bocca (proboscide), Tiss grassiue e trachee. </li><li> Afferra la cuticola dorsale testa con una pinza e usare l&#39;altra per separare gli occhi di tessuto cerebrale, tirando delicatamente lateralmente. Nella maggior parte dei casi, la lamina, una guaina sottile e trasparente di neuropilo cervello, rimarrà attaccato alla retina. È più facile rimuovere la lamina dopo fissazione. </li><li> Rimuovere le cuticole al margine occhio, ma lascia un piccolo pezzo dietro per facilitare il trasferimento di retine a soluzioni diverse. Effettuare fissaggio (passo 4.1.). </li><li> Rimuovere la lamina afferrandolo da un lato con una pinza e delicatamente tirando lateralmente con le pinze altri senza compromettere la retina. Rimuovere cuticola residuo, in quanto potrebbe ripiegare sulla retina durante il montaggio. Questi due passaggi sono fondamentali per la visualizzazione fotorecettori, che altrimenti oscurato dalla lamina e rimane della cuticola durante il processo di imaging. Procedere alla immunoistochimica (punto 5.1.). </li></ol> 4. Fissazione e Washing (circa 1 ora) <ol><li> Conservare le retine sezionati in 1x PBS in tre ben piatto di vetro immessi sul ghiaccio mentre appena la preparazione del fissativo (diluire 40 microlitri di soluzione di formaldeide al 37% con 360 microlitri di PBS 1x, vortice e memorizzare sul ghiaccio). Si consiglia di procedere con il protocollo di fissazione e di evitare lunghi tempi di conservazione in ghiaccio (&gt; 15 min). </li><li> Sostituire 1x PBS con 200 ml di soluzione di formaldeide 3,7%. Assicurarsi che il tessuto viene immerso in fissativo. Questo è critico, in quanto può non essere altrimenti fissato correttamente. Se necessario, rimuovere le bolle d&#39;aria e trachee con le pinze. </li><li> Incubare su agitatore per 15 min a temperatura ambiente. </li><li> Sostituire il fissativo con 1x PBS e lavare due volte con PBS 1x. Continuare il lavaggio in PBS per 30 minuti (su agitatore, a temperatura ambiente). </li><li> Sostituire PBS con PBST. Risciacquare due volte con PBST. </li><li> Procedere con la rimozione della membrana peripodial dai dischi degli occhi larvali (3.1.3.) E le lamine di midpupal / adulto retine &lt;strong&gt; (3.2.3., 3.3.4.). </li></ol> 5. Immunoistochimica <ol><li> Bloccante: Sostituire PBS con 200 pl del 5% siero normale in PBST e incubare per 20 min retine su agitatore a temperatura ambiente. </li><li> Sostituire la soluzione di saturazione con la soluzione di anticorpo primario. Sigillare il tre ben piatto con parafilm e incubare per una notte su agitatore a temperatura ambiente. </li><li> Sostituire la soluzione con PBST e lavare 3 volte. Continuare lavaggio con PBST per almeno 1 ora su agitatore a temperatura ambiente. </li><li> Trasferimento in soluzione di anticorpo secondario ed incubare in un parafilm-piatto di vetro sigillato bene su agitatore notte a temperatura ambiente. </li><li> Sostituire la soluzione di anticorpi con PBST e lavare tre volte. Continuare lavaggio con PBST per un minimo di un hr su agitatore a temperatura ambiente. I campioni possono essere conservati in PBST per diversi giorni, ma è importante aggiungere PBST fresco almeno una volta al giorno per evitare che si secchi. </li></ol> 6. Montaggio <ol><li> Per il montaggio retine larvali e midpupal, utilizzare il P20 per trasferire retine al centro di un vetrino pulito. Rimuovere PBST eccesso. Utilizzare una pinza per allineare retine per facilitare l&#39;imaging. </li><li> Aggiungere 10 ml di mezzo di montaggio sopra le retine. Coprire delicatamente con un foglio 24x40-1 coperchio e sigillare con smalto trasparente. </li><li> Per montaggio retine adulti, preparare &#39;ponti&#39; 20: Aggiungere due gocce 5 ml di glicerolo al 50% su entrambe le estremità di un lato di vetro e coprire con 22x22-1 vetrini (0,13 a 0,17 mm di spessore). </li><li> Rimuovere il PBST con una pipetta P20, fare in modo che la retina non si seccano. Aggiungere 20 ml di mezzo di montaggio al pozzo. Utilizzare mezzo di montaggio per trasferire retine con il P20 al vetrino &#39;ponte&#39;. </li><li> Utilizzare una pinza per orientare (lenti devono affrontare il vetrino) e allineare retine per facilitare l&#39;imaging. Se necessario, rimuovere le bolle d&#39;aria con il P20. </li><li> Lentamenteposto antiscivolo 24x40-1 Coperchio (0,13 a 0,17 mm di spessore) da un lato in alto e sigillare i bordi con smalto trasparente o nastro adesivo. Conservare i campioni a 4 ° C in una casella di vetrino da microscopio saver (tenere al buio). </li></ol> 7. Risultati rappresentativi Ad esempio per l&#39;applicazione del presente protocollo, si mostrano i risultati rappresentativi per la visualizzazione di differenziazione dei fotorecettori i tre stadi di sviluppo nel video. A larvale eye-antennale disco e una retina midpupal state colorate con anticorpi che etichettano tipi fotorecettrici diversi durante il loro sviluppo (Figura 1A, B) e una retina adulta è stato colorato con due anticorpi Rhodopsin di visualizzare due sottotipi fotorecettrici (Figura 1C).

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	<li>Ready, D. F., Hanson, T. E., Benzer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+Drosophila+retina,+a+neurocrystalline+lattice.">Development of the Drosophila retina, a neurocrystalline lattice.</a> Dev. Biol. 53, 217-240 (1976).</li><li>Hardie, R. C., D, O. t. t. o. s. o. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+organization+of+the+fly+retina.">Functional organization of the fly retina.</a> Sensory Physiology. 5, 1-79 (1985).</li><li>O'Tousa, J. E. <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+ninaE+gene+encodes+an+opsin.">The Drosophila ninaE gene encodes an opsin.</a> Cell. 40, 839-850 (1985).</li><li>Zuker, C. S., Cowman, A. F., Rubin, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+structure+of+a+rhodopsin+gene+from+D.+melanogaster.">Isolation and structure of a rhodopsin gene from D. melanogaster.</a> Cell. 40, 851-858 (1985).</li><li>Rister, J., Desplan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+retinal+mosaics+of+opsin+expression+in+invertebrates+and+vertebrates.">The retinal mosaics of opsin expression in invertebrates and vertebrates.</a> Dev. Neurobiol. 71, 1212-1226 (2011).</li><li>Chou, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+novel+Drosophila+opsin+reveals+specific+patterning+of+the+R7+and+R8+photoreceptor+cells.">Identification of a novel Drosophila opsin reveals specific patterning of the R7 and R8 photoreceptor cells.</a> Neuron. 17, 1101-1115 (1996).</li><li>Chou, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterning+of+the+R7+and+R8+photoreceptor+cells+of+Drosophila:+evidence+for+induced+and+default+cell-fate+specification.">Patterning of the R7 and R8 photoreceptor cells of Drosophila: evidence for induced and default cell-fate specification.</a> Development. 126, 607-616 (1999).</li><li>Papatsenko, D., Sheng, G., Desplan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+rhodopsin+in+R8+photoreceptors+of+Drosophila:+evidence+for+coordinate+expression+with+Rh3+in+R7+cells.">A new rhodopsin in R8 photoreceptors of Drosophila: evidence for coordinate expression with Rh3 in R7 cells.</a> Development. 124, 1665-1673 (1997).</li><li>Wolff, T., Ready, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+beginning+of+pattern+formation+in+the+Drosophila+compound+eye:+the+morphogenetic+furrow+and+the+second+mitotic+wave.">The beginning of pattern formation in the Drosophila compound eye: the morphogenetic furrow and the second mitotic wave.</a> Development. 113, 841-850 (1991).</li><li>Roignant, J. Y., Treisman, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pattern+formation+in+the+Drosophila+eye+disc.">Pattern formation in the Drosophila eye disc.</a> Int. J. Dev. Biol. 53, 795-804 (2009).</li><li>Tsachaki, M., Sprecher, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+and+developmental+mechanisms+underlying+the+formation+of+the+Drosophila+compound+eye.">Genetic and developmental mechanisms underlying the formation of the Drosophila compound eye.</a> Dev. Dyn. 241, 40-56 (2012).</li><li>Tomlinson, A., Ready, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+differentiation+in+Drosophila+ommatidium.">Neuronal differentiation in Drosophila ommatidium.</a> Dev. Biol. 120, 366-376 (1987).</li><li>Zipursky, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+and+genetic+analysis+of+Drosophila+eye+development:+sevenless,+bride+of+sevenless+and+rough.">Molecular and genetic analysis of Drosophila eye development: sevenless, bride of sevenless and rough.</a> Trends Neurosci. 12, 183-189 (1989).</li><li>Basler, K., Hafen, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specification+of+cell+fate+in+the+developing+eye+of+Drosophila.">Specification of cell fate in the developing eye of Drosophila.</a> Bioessays. 13, 621-631 (1991).</li><li>Charlton-Perkins, M., Cook, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Building+a+fly+eye:+terminal+differentiation+events+of+the+retina,+corneal+lens,+and+pigmented+epithelia.">Building a fly eye: terminal differentiation events of the retina, corneal lens, and pigmented epithelia.</a> Curr. Top Dev. Biol. 93, 129-173 (2010).</li><li>Walther, R. F., Pichaud, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunofluorescent+staining+and+imaging+of+the+pupal+and+adult+Drosophila+visual+system.">Immunofluorescent staining and imaging of the pupal and adult Drosophila visual system.</a> Nat. Protoc. 1, 2635-2642 (2006).</li><li>Wolff, T., Sullivan, W. e. a. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histological+Techniques+for+the+Drosophila+Eye+Part+I:+Larva+and+Pupa.">Histological Techniques for the Drosophila Eye Part I: Larva and Pupa.</a> Drosophila Protocols. , (2000).</li><li>Wolff, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+techniques+for+pupal+and+larval+Drosophila+eyes.">Dissection techniques for pupal and larval Drosophila eyes.</a> CSH Protoc. 2007, pdb prot4715 (2007).</li><li>Bainbridge, S. P., Bownes, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staging+the+metamorphosis+of+Drosophila+melanogaster.">Staging the metamorphosis of Drosophila melanogaster.</a> J. Embryol. Exp. Morphol. 66, 57-80 (1981).</li><li>Morante, J., Desplan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+and+staining+of+Drosophila+optic+lobes+at+different+stages+of+development.">Dissection and staining of Drosophila optic lobes at different stages of development.</a> Cold Spring Harb Protoc. , 652-656 (2011).</li><li>Williamson, W. R., Hiesinger, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+Developing+and+Adult+Drosophila+Brains+and+Retinae+for+Live+Imaging.">Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging.</a> J. Vis. Exp. (37), e1936 (2010).</li><li>Stowers, R. S., Schwarz, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetic+method+for+generating+Drosophila+eyes+composed+exclusively+of+mitotic+clones+of+a+single+genotype.">A genetic method for generating Drosophila eyes composed exclusively of mitotic clones of a single genotype.</a> Genetics. 152, 1631-1639 (1999).</li><li>Newsome, T. P., Asling, B., Dickson, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+Drosophila+photoreceptor+axon+guidance+in+eye-specific+mosaics.">Analysis of Drosophila photoreceptor axon guidance in eye-specific mosaics.</a> Development. 127, 851-860 (2000).</li><li>Sood, P., Johnston, R. J., Kussell, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stochastic+De-repression+of+Rhodopsins+in+Single+Photoreceptors+of+the+Fly+Retina.">Stochastic De-repression of Rhodopsins in Single Photoreceptors of the Fly Retina.</a> PLoS Comput. Biol. 8, e1002357 (2012).</li><li>Johnston, 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=Interlocked+feedforward+loops+control+cell-type-specific+Rhodopsin+expression+in+the+Drosophila+eye.">Interlocked feedforward loops control cell-type-specific Rhodopsin expression in the Drosophila eye.</a> Cell. 145, 956-968 (2011).</li><li>Jukam, D., Desplan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binary+regulation+of+Hippo+pathway+by+Merlin/NF2,+Kibra,+Lgl,+and+Melted+specifies+and+maintains+postmitotic+neuronal+fate.">Binary regulation of Hippo pathway by Merlin/NF2, Kibra, Lgl, and Melted specifies and maintains postmitotic neuronal fate.</a> Dev. Cell. 21, 874-887 (2011).</li><li>Vasiliauskas, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feedback+from+rhodopsin+controls+rhodopsin+exclusion+in+Drosophila+photoreceptors.">Feedback from rhodopsin controls rhodopsin exclusion in Drosophila photoreceptors.</a> Nature. 479, 108-112 (2011).</li><li>Kumar, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Building+an+ommatidium+one+cell+at+a+time.">Building an ommatidium one cell at a time.</a> Dev. Dyn. 241, 136-149 (2012).</li></ol>

Nessun conflitto di interessi dichiarati.

1. Risoluzione dei problemi Nella nostra esperienza, dissezioni richiedono una pratica (fino a diverse settimane) e sono facilitate da raggiungere una posizione comoda a mano 21 con appoggio i gomiti e gli avambracci sul tavolo e con le dita in contatto con il piatto dissezione. Dita In questo modo, solo il pollice, indice e medio compiere movimenti sottili. Rimozione della lamina senza danneggiare i fotorecettori è probabilmente il passo più difficile....

<table border="1"><tr><td></td><td></td><td></td><td> Reagente </td></tr><tr><td> Tampone fosfato salino (PBS1x, pH 7.4) </td><td> Sigma </td><td></td><td> Preparare soluzione di 10x 20. Diluire con acqua distillata per ottenere 1x PBS e conservare a temperatura ambiente. Raffreddare in ghiaccio prima dissezioni. </td></tr><tr><td> Triton-X 100 </td><td> Sigma </td><td> 9002-93-1 </td><td> Attenzione: Irritante Indossare guanti. Preparare 50 ml 1xPBS con 0,3% Triton X-100 (PBST). Conservare a temperatura ambiente. </td></tr><tr><td> Soluzione di formaldeide al 37% </td><td> Fisher Scientific </td><td> F75P1GAL </td><td> Attenzione: Tossico, probabile cancerogeno umano Indossare guanti. Prima della fase di fissazione, di fresco preparare 3,7% soluzione in una cappa di diluizione con PBS, negozio su ghiaccio. </td></tr><tr><td> 5% di siero di cavallo normale </td><td> Jackson Immuno Research </td><td> 008-000-001 </td><td> Preparare 5% v / v di diluizione in PBST. Conservare a quattro gradi. </td></tr><tr><td> Gli anticorpi primari e secondari (ad esempio, Donkey anti-pecore Alexa Fluor 488, Donkey-coniglio anti Alexa Fluor 555, Donkey anti-topo Alexa Fluor 647) </td><td> Probes Invitrogen Molecular </td><td> A11015 A31572 A31571 </td><td> Diluire 1:800 anticorpi secondari in PBST e conservare a quattro gradi. </td></tr><tr><td> Alexa Fluor 488 falloidina </td><td></td><td> A12379 </td><td> Diluire 1:100 in soluzione anticorpo secondario. </td></tr><tr><td> Slowfade Oro Antifade reagente </td><td> Probes Invitrogen Molecular </td><td> S36936 </td><td> Mezzo di montaggio. Conservare a -20 gradi. </td></tr><tr><td> Glycerol </td><td> Fisher Scientific </td><td> G31-1 </td><td> Per il montaggio. Preparare 10 ml di 50% di diluizione con acqua distillata, conservare a temperatura ambiente. </td></tr><tr><td> CO 2 </td><td></td><td></td><td> Per anestetizzare mosche adulte. </td></tr><tr><td></td><td></td><td></td><td> Attrezzatura </td></tr><tr><td> Due pinze taglienti (Dumont # 55) </td><td> Strumenti Scienza Belle </td><td> 11255-20 </td><td></td></tr><tr><td> Sylgard 184 Kit di silicone elastomero </td><td> Dow Corning </td><td></td><td> Preparare piatto dissezione Sylgard compilando un piatto di plastica con la miscela Sylgard Petri. </td></tr><tr><td> Tre piatti e vetro dissezione </td><td> Fisher Scientific </td><td> 21-379 </td><td></td></tr><tr><td> Two perni minutien dissezione (0,1 mm di diametro) e due pinholders (12 cm) </td><td> Strumenti per le scienze Belle </td><td> 26002-10 </td><td> Inserire un pin minutien in ciascuno dei due pinholders e piegare uno dei perni per formare un gancio. </td></tr><tr><td> Microscopio vetrini coprioggetto (22x22-24x40-1 e 1) </td><td> Fisher Scientific </td><td> 12-542A </td><td></td></tr><tr><td> Vetrini portaoggetto (25x75x1.0 mm; predepurata) </td><td> Fisher Scientific </td><td> 12-550-143 </td><td></td></tr><tr><td> 1,5 ml microcentrifuga tubi </td><td></td><td></td><td></td></tr><tr><td> Cancellare smalto o nastro adesivo </td><td></td><td></td><td></td></tr><tr><td> Agitatore orbitale </td><td> Bellco </td><td></td><td></td></tr><tr><td> Far scorrere il porta finestra </td&gt; <td> Fisher Scientific </td><td></td><td></td></tr><tr><td> Parafilm </td><td> Bemis </td><td> PM996 </td><td></td></tr><tr><td> Pennello sottile </td><td></td><td></td><td></td></tr><tr><td> P20 e P200 micropipette e suggerimenti </td><td></td><td></td><td></td></tr><tr><td> Dissezione microscopio </td><td></td><td></td><td></td></tr><tr><td> Piccolo secchio con ghiaccio </td><td></td><td></td><td> Per raffreddare le lastre di vetro e, retine sezionati e soluzioni. </td></tr></table>

dissection and immunohistochemistry of larval, pupal and adult drosophila retinas

L&#39;occhio composto di Drosophila melanogaster è composto da circa 750 ommatidi (occhi unità). Ogni ommatidium è composto da circa 20 cellule, inclusa la lente-secernenti coni, cellule del pigmento, una cella a setole e otto fotorecettori (PR) R1-R8 2. Il PRS hanno le strutture di microvillar, le rhabdomeres, che contengono pigmenti sensibili alla luce, i rodopsine (sd). Le rhabdomeres di sei PR (R1-R6) formano un trapezio e contengono Th1 3 4. Le rhabdomeres di R7 e R8 sono posizionati in tandem nel centro del trapezio e condividono lo stesso percorso della luce. R7 e R8 PR stocasticamente esprimono diverse combinazioni di Rhs in due sottotipi principali 5: Nel sottotipo la &#39;p&#39;, in p RH3 R7s è accoppiato con RH5 in p R8s, mentre nel sottotipo del &#39;y&#39;, Rh4 in y R7s è associato RH6 in y R8S 6 7 8. Prime caratteristiche di PR e sviluppo di ommatidi inizia nella larvale eye-antennali disco immaginale, un monostrato di cellule epiteliali. Un&#39;ondata di differenziazione spazza tutto il disco 9 e avvia l&#39;assemblaggio di cellule indifferenziate in ommatidi 10-11. La R8 &#39;cella fondatore&#39; è specificato prima e recluta R1-6 e poi R7 12-14. Successivamente, durante lo sviluppo pupale, PR differenziazione porta a vasti cambiamenti morfologici 15, tra cui la formazione di rhabdomere, sinaptogenesi e di espressione alla fine rh. In questo protocollo, si descrivono i metodi per dissezioni retiniche e immunoistochimica in tre periodi definiti di sviluppo della retina, che possono essere applicati per affrontare una serie di questioni concernenti la formazione della retina e percorsi di sviluppo. Qui, usiamo questi metodi per visualizzare la differenziazione graduale PR a livello di singola cellula per intero monte larvale, midpupal e adulti retine ( <sTrong&gt; Figura 1).

Watch this Scientific Journal Video about Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas at JoVE.com

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

Il<em&gt; Drosophila</emRetina&gt; è un reticolo cristallino composto da un piccolo numero di tipi cellulari che sono generati in modo stereotipato<sup&gt; 1</sup&gt;. La sua riconducibilità a sofisticate analisi genetica permette lo studio di complessi programmi di sviluppo. Questo protocollo descrive le dissezioni e immunoistochimica di retina in tre distinte fasi di sviluppo, con particolare attenzione alla differenziazione dei fotorecettori.

Dissezione e immunoistochimica di larvale, pupa e adulto<em&gt; Drosophila</em&gt; Retine

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including ...

dissection-immunohistochemistry-larval-pupal-adult-drosophila

Research

JoVE Journal

Neuroscience

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

25.1K Views.  New York University. Il Drosophila è un reticolo cristallino composto da un piccolo numero di tipi cellulari che sono generati in modo stereotipato 1. La sua riconducibilità a sofisticate analisi genetica permette lo studio di complessi programmi di sviluppo. Questo protocollo descrive le dissezioni e immunoistochimica di retina in tre distinte fasi di sviluppo, con particolare attenzione alla differenziazione dei fotorecettori.

Video: Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

Mapping and Application of Enhancer-trap Flippase Expression in Larval and Adult Drosophila CNS

The Gal4/ UAS binary method is powerful for gene and neural circuitry manipulation in Drosophila. For most neurobiological studies, however, Gal4 expression is rarely tissue-specific enough to allow for precise correlation of the circuit with behavioral readouts. To overcome this major hurdle, we recently developed the FINGR method to achieve a more restrictive Gal4 expression in the tissue of interest. The FINGR method has three components: 1) the traditional Gal4/UAS system; 2) a set of FLP/FRT-mediated Gal80 converting tools; and 3) enhancer-trap FLP (ET-FLP). Gal4 is used to define the primary neural circuitry of interest. Paring the Gal4 with a UAS-effector, such as UAS-MJD78Q or UAS-Shits, regulates the neuronal activity, which is in turn manifested by alterations in the fly behavior. With an additional UAS-reporter such as UAS-GFP, the neural circuit involved in the specific behavior can be simultaneously mapped for morphological analysis. For Gal4 lines with broad expression, Gal4 expression can be restricted by using two complementary Gal80-converting tools: tubP&gt;Gal80&gt; ('flip out') and tubP&gt;stop&gt;Gal80 ('flip in'). Finally, investigators can turn Gal80 on or off, respectively, with the help of tissue-specific ET-FLP. In the flip-in mode, Gal80 will repress Gal4 expression wherever Gal4 and ET-FLP intersect. In the flip-out mode, Gal80 will relieve Gal4 repression in cells in which Gal4 and FLP overlap. Both approaches enable the restriction of the number of cells in the Gal4-defined circuitry, but in an inverse pattern. The FINGR method is compatible with the vast collection of Gal4 lines in the fly community and highly versatile for traditional clonal analysis and for neural circuit mapping. In this protocol, we demonstrate the mapping of FLP expression patterns in select ET-FLPx2 lines and the effectiveness of the FINGR method in photoreceptor cells. The principle of the FINGR method should also be applicable to other genetic model organisms in which Gal4/UAS, Gal80, and FLP/FRT are used.

Mapping and Application of Enhancer-trap Flippase Expression in Larval and Adult Drosophila CNS

We describe a Flippase-induced intersectional Gal80/Gal4 repression (FINGR) method, allowing tissue-specific FLP to determine Gal80 expression patterns. Wherever Gal4 and FLP overlap, Gal4 expression is turned on (Gal80 flipped out) or off (Gal80 flipped in). The FINGR method is versatile for clonal analysis and neural circuit mapping.

Mappatura e applicazione di Enhancer-trappola Espressione Flippase in larvali e adulti Drosophila SNC

The Gal4/ UAS binary method is powerful for gene and neural circuitry manipulation in Drosophila. For most neurobiological studies, however, Gal4 ...

Ricerca

Neuroscienze

Single Drosophila Ommatidium Dissection and Imaging

The fruit fly Drosophila melanogaster has made invaluable contributions to neuroscience research and has been used widely as a model for neurodegenerative diseases because of its powerful genetics1. The fly eye in particular has been the organ of choice for neurodegeneration research, being the most accessible and life-dispensable part of the Drosophila nervous system. However the major caveat of intact eyes is the difficulty, because of the intense autofluorescence of the pigment, in imaging intracellular events, such as autophagy dynamics2, which are paramount to understanding of neurodegeneration.
We have recently used the dissection and culture of single ommatidia3 that has been essential for our understanding of autophagic dysfunctions in a fly model of Dentatorubro-Pallidoluysian Atrophy (DRPLA)3, 4.
We now report a comprehensive description of this technique (Fig. 1), adapted from electrophysiological studies5, which is likely to expand dramatically the possibility of fly models for neurodegeneration. This method can be adapted to image live subcellular events and to monitor effective drug administration onto photoreceptor cells (Fig. 2). If used in combination with mosaic techniques6-8, the responses of genetically different cells can be assayed in parallel (Fig. 2).

Single Drosophila Ommatidium Dissection and Imaging

The limiting factor in the use of the adult Drosophila eye to study neurodegeneration and cell biology is the difficult imaging of intracellular processes. We describe the dissection of single ommatidia to generate a bona-fide primary neuronal cell culture, which can be subject to drug treatment and advanced imaging.

Singolo Drosophila Ommatidium Dissection e Imaging

The fruit fly Drosophila melanogaster has made invaluable contributions to neuroscience research and has been used widely as a model for neurodegenerative ...

Dissection of Adult Mouse Utricle and Adenovirus-mediated Supporting-cell Infection

Hearing loss and balance disturbances are often caused by death of mechanosensory hair cells, which are the receptor cells of the inner ear. Since there is no cell line that satisfactorily represents mammalian hair cells, research on hair cells relies on primary organ cultures. The best-characterized in vitro model system of mature mammalian hair cells utilizes organ cultures of utricles from adult mice (Figure 1) 1-6. The utricle is a vestibular organ, and the hair cells of the utricle are similar in both structure and function to the hair cells in the auditory organ, the organ of Corti. The adult mouse utricle preparation represents a mature sensory epithelium for studies of the molecular signals that regulate the survival, homeostasis, and death of these cells.
Mammalian cochlear hair cells are terminally differentiated and are not regenerated when they are lost. In non-mammalian vertebrates, auditory or vestibular hair cell death is followed by robust regeneration which restores hearing and balance functions 7, 8. Hair cell regeneration is mediated by glia-like supporting cells, which contact the basolateral surfaces of hair cells in the sensory epithelium 9, 10. Supporting cells are also important mediators of hair cell survival and death 11. We have recently developed a technique for infection of supporting cells in cultured utricles using adenovirus. Using adenovirus type 5 (dE1/E3) to deliver a transgene containing GFP under the control of the CMV promoter, we find that adenovirus specifically and efficiently infects supporting cells. Supporting cell infection efficiency is approximately 25-50%, and hair cells are not infected (Figure 2). Importantly, we find that adenoviral infection of supporting cells does not result in toxicity to hair cells or supporting cells, as cell counts in Ad-GFP infected utricles are equivalent to those in non-infected utricles (Figure 3). Thus adenovirus-mediated gene expression in supporting cells of cultured utricles provides a powerful tool to study the roles of supporting cells as mediators of hair cell survival, death, and regeneration.

Mechanosensory hair cells are the receptor cells of the inner ear. The best-characterized in vitro model system of mature mammalian hair cells utilizes organ cultures of utricles from adult mice. We present the dissection of the adult mouse utricle, and we demonstrate adenovirus-mediated infection of supporting cells in cultured utricles.

Dissezione di utricolo topo adulto e adenovirus-mediata non protagonista infezione delle cellule

Hearing loss and balance disturbances are often caused by death of mechanosensory hair cells, which are the receptor cells of the inner ear.  Since there ...

Functional Analysis of the Larval Feeding Circuit in Drosophila

The serotonergic feeding circuit in Drosophila melanogaster larvae can be used to investigate neuronal substrates of critical importance during the development of the circuit. Using the functional output of the circuit, feeding, changes in the neuronal architecture of the stomatogastric system can be visualized. Feeding behavior can be recorded by observing the rate of retraction of the mouth hooks, which receive innervation from the brain. Locomotor behavior is used as a physiological control for feeding, since larvae use their mouth hooks to traverse across an agar substrate. Changes in feeding behavior can be correlated with the axonal architecture of the neurites innervating the gut. Using immunohistochemistry it is possible to visualize and quantitate these changes. Improper handling of the larvae during behavior paradigms can alter data as they are very sensitive to manipulations. Proper imaging of the neurite architecture innervating the gut is critical for precise quantitation of number and size of varicosities as well as the extent of branch nodes. Analysis of most circuits allow only for visualization of neurite architecture or behavioral effects; however, this model allows one to correlate the functional output of the circuit with the impairments in neuronal architecture.

Functional Analysis of the Larval Feeding Circuit in Drosophila

The feeding circuit in Drosophila melanogaster larvae serves a simple yet powerful model that allows changes in feeding rate to be correlated with alterations in the stomatogastric neural circuitry. This circuit is composed of central serotonergic neurons that send projections to the mouth hooks as well as the foregut.

Analisi funzionale del circuito di alimentazione larvale in Drosophila</i

The serotonergic  feeding circuit in Drosophila  melanogaster larvae can be used to investigate neuronal substrates of  critical importance during the ...

Live Imaging of Drosophila Larval Neuroblasts

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given their relevance to development and disease, understanding the mechanisms that govern asymmetric stem cell division has been a robust area of study. Because they are genetically tractable and undergo successive rounds of cell division about once every hour, the stem cells of the Drosophila central nervous system, or neuroblasts, are indispensable models for the study of stem cell division. About 100 neural stem cells are located near the surface of each of the two larval brain lobes, making this model system particularly useful for live imaging microscopy studies. In this work, we review several approaches widely used to visualize stem cell divisions, and we address the relative advantages and disadvantages of those techniques that employ dissociated versus intact brain tissues. We also detail our simplified protocol used to explant whole brains from third instar larvae for live cell imaging and fixed analysis applications.

Live Imaging of Drosophila Larval Neuroblasts

This protocol details a streamlined method used to conduct live cell imaging in the context of an intact larval brain. Live cell imaging approaches are invaluable for the study of asymmetric neural stem cell divisions as well as other neurogenic and developmental processes, consistently uncovering mechanisms that were previously overlooked.

Immagini dal vivo di<em&gt; Drosophila</em&gt; larvali neuroblasti

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given ...

Acquisition of High-Quality Digital Video of Drosophila Larval and Adult Behaviors from a Lateral Perspective

Drosophila melanogaster is a powerful experimental model system for studying the function of the nervous system. Gene mutations that cause dysfunction of the nervous system often produce viable larvae and adults that have locomotion defective phenotypes that are difficult to adequately describe with text or completely represent with a single photographic image. Current modes of scientific publishing, however, support the submission of digital video media as supplemental material to accompany a manuscript. Here we describe a simple and widely accessible microscopy technique for acquiring high-quality digital video of both Drosophila larval and adult phenotypes from a lateral perspective. Video of larval and adult locomotion from a side-view is advantageous because it allows the observation and analysis of subtle distinctions and variations in aberrant locomotive behaviors. We have successfully used the technique to visualize and quantify aberrant crawling behaviors in third instar larvae, in addition to adult mutant phenotypes and behaviors including grooming.

Acquisition of High-Quality Digital Video of Drosophila Larval and Adult Behaviors from a Lateral Perspective

Here we describe a simple and widely accessible microscopy technique to acquire high-quality digital video of Drosophila adult and larval mutant phenotypes from a lateral perspective.

Acquisizione di alta qualità video digitale di<em&gt; Drosophila</em&gt; Comportamenti larvali e adulti da una prospettiva laterale

Drosophila melanogaster is a powerful experimental model system for studying the function of the nervous system. Gene mutations that cause dysfunction of ...

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.

We present a method to isolate the adult organ of Corti as three intact cochlear turns (apex, middle, and base). We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies. Together these techniques allow the study of hair cells, supporting cells, and other cell types found in the cochlea.

Tutta Monte dissezione e immunofluorescenza del topo adulto Coclea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a ...

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat open surfaces is complex,&#160;including: pausing, turning, and meandering. This complexity in the repertoire of movement hinders detailed analysis of the events occurring during a single crawl stride cycle. To overcome this obstacle, linear agarose channels were made that constrain larval behavior to straight, sustained, rhythmic crawling. In principle, because agarose channels and the Drosophila larval body are both optically clear, the movement of larval structures labeled by genetically-encoded fluorescent probes can be monitored in intact, freely-moving larvae. In the past, larvae were placed in linear channels and crawling at the level of whole organism, segment, and muscle were analyzed1. In the future, larvae crawling in channels can be used for calcium imaging to monitor neuronal activity. Moreover, these methods can be used with larvae of any genotype and with any researcher-designed channel. Thus the protocol presented below is widely applicable for studies using the Drosophila larva as a model to understand motor control.

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

The Drosophila larva is a powerful model system to study neural control of behavior. This publication describes the use of linear agarose channels to elicit sustained bouts of linear crawling and methods to quantify the dynamics of larval structures during repetitive crawling behavior.

Utilizzando canali lineari Agarose per studiare<em&gt; Drosophila</em&gt; Comportamento larvale Crawling

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

Un semplice passo-dissezione Protocollo per tutto il montaggio Preparazione per adulti<em&gt; Drosophila</em&gt; Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs provides a tissue-level understanding of energy utilization that cannot be captured when analyzing primary cells and cell lines. While this analysis is ex vivo, it allows for the measurement from a number of specialized cells working together to perform a function in one tissue and more closely models the in vivo organ. Metabolic reprogramming has been observed in many neurological diseases, including neoplasia, and neurodegenerative diseases. This protocol was developed to assist the D. melanogaster community&#39;s investigation of metabolism in neurological disease models using a commercially available metabolic analyzer. Measuring metabolism of whole brains in the metabolic analyzer is challenging due to the geometry of the brain. This analyzer requires samples to remain at the bottom of a 96-well plate. Cell samples and tissue punches can adhere to the surface of the cell plate or utilize spheroid plates, respectively. However, the spherical, three-dimensional shape of D. melanogaster brains prevents the tissue from adhering to the plate. This protocol requires a specially designed and manufactured micro-tissue restraint that circumvents this problem by preventing any movement of the brain while still allowing metabolic measurements from the analyzer&#39;s two solid-state sensor probes. Oxygen consumption and extracellular acidification rates are reproducible and sensitive to a treatment with metabolic inhibitors. With a minor optimization, this protocol can be adapted for use with any whole tissue and/or model system, provided that the sample size does not exceed the chamber generated by the restraint. While basal metabolic measurements and an analysis after a treatment with mitochondrial inhibitors are described within this protocol, countless experimental conditions, such as energy source preference and rearing environment, could be interrogated.

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

We present a protocol for measuring oxygen consumption and extracellular acidification in Drosophila melanogaster larval and adult brains. A metabolic analyzer is utilized with an adapted and optimized protocol. Micro-tissue restraints are a critical component of this protocol and were designed and created specifically for their use in this analysis.

Analisi metabolica di Drosophila melanogaster Larval e cervello adulto

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs ...