Name: dissection and mounting of drosophila pupal eye discs
Uploaded: 2014-11-09T15:00:00.000+00:00
Duration: 7 min 48 s
Description: Watch this Scientific Journal Video about Dissection and Mounting of Drosophila Pupal Eye Discs at JoVE.com

We appreciate and would like to thank the Howard Hughes Medical Institute for the HHMI Professor award to U.B. which made this project possible. We thank the college at the University of California, Los Angeles for providing facilities and teaching infrastructure support for this work. The work was also supported with funding from Midwestern University and a generous donation from the Charity Fidelity Gift Fund. We thank John VandenBrooks for comments on the manuscript and Krista Pearman for her technical assistance.

Questa procedura è una procedura di 2 giorni. 1 ° giorno (2 ore + tempo di dissezione) 1. pupa Disco Eye Dissection <ol><li> Selezionare una pupa per la dissezione. NOTA: L&#39;età della pupa essere sezionato sarà determinata dalle esigenze sperimentali. Tuttavia, se l&#39;esame di morfologia cellulare, questo è spesso fatto a 42 ore dopo la formazione puparium (APF) a 25 ° C, che è l&#39;età delle pupe mostrato nel video. Raccogliere bianco pupe (considerato APF 0 ore) con un pennello bagnata e disporli in ordine cronologico (in base al tempo di raccolta) in un nuovo flacone mosca cibo mantenuta a 25 ° C. Sezionare il pupe affinato in questo stesso ordine cronologico di mantenere le differenze di tempo tra pupe più vicino possibile. </li><li> Trasferire la pupa in una goccia di soluzione salina tamponata con fosfato (PBS, pH 7,4, vedi materiali per ricetta) su una piastra di dissezione silicone. </li><li> Tenere la parte superiore del case pupa con una pinza e forare e aprire il lparte ower del caso pupa con un paio di pinze taglienti. Rimuovere la pupa dal caso nel foro appena formata. </li><li> Fare un taglio diagonale con le forbici, a metà del torace, in pupa. Rimuovere i detriti o trasferire la testa pupa di una nuova goccia di PBS. Pulire utilizzando il tubo pipetta soffiante (vedi materiali e la Figura 1 per i dettagli) e aggiungere ulteriori PBS, se necessario. Assicurati di evitare la disidratazione. </li><li> Afferrare saldamente la parte superiore della cuticola testa di pupa con un paio di pinze. Utilizzando la punta del tubo soffiatore, premere delicatamente sul caso pupa per estrudere il cervello e altri tessuti, tra cui i dischi degli occhi, fuori dal bozzolo. Essere sicuri di evitare di afferrare qualsiasi tessuto all&#39;interno della cuticola. </li><li> Utilizzando il tubo soffiatore riempito con PBS, soffiare delicatamente il tessuto grasso corporeo e altri detriti fuori del caso testa di separare in modo chiaro e identificare i dischi del cervello e degli occhi. NOTA: Mantenere il cervello attaccato alla testa caso pupa permetterà ai ricercatori di utilizzare il luicaso ad una &quot;maniglia&quot; per la futura manipolazione del tessuto senza danneggiare i dischi occhi o cervello. Tuttavia, è comune per rimuovere i dischi cervello e degli occhi da quelle della testa e questo non è dannoso per la procedura generale ei risultati. Spesso ci vogliono diversi tentativi di soffiaggio e spingendo per estrarre correttamente i dischi occhi e cervello dall&#39;interno del bozzolo testa e in modo che siano nettamente separate dal tessuto circostante. Il più pulito e più un separato possono ottenere i dischi occhi dal tessuto circostante contribuisce a prevenire i tessuti estranei di fissare ai dischi degli occhi. </li><li> Dopo la dissezione, trasferire immediatamente il tessuto di un piatto di vetro 3 bene su ghiaccio con 0,5 ml di soluzione Fix (vedi materiali). Float il tessuto sulla superficie della soluzione Fix per alcuni minuti (in genere il tempo necessario per sezionare il successivo insieme di dischi oculari). NOTA: Se questo non è il primo disco occhio ad essere sezionato in questo giro di dissezione, completely immergere il disco precedente occhio nella soluzione Fix. Se l&#39;esecuzione di procedure di colorazione speciali (ad esempio, le specie reattive dell&#39;ossigeno, lisosomi, ecc), questo deve essere fatto prima della fissazione. Per fare questo, conservare i dischi occhio sezionato in PBS freddo prima di continuare con il protocollo di colorazione appropriata. </li><li> A seguito di dissezione e la sommersione di tutti i dischi degli occhi viene sezionato, spostare il piatto di vetro 3 oltre a un rocker e rock a temperatura ambiente per 30 minuti. </li></ol> 2. Immunostaining <ol><li> Rimuovere con cautela la soluzione Fix e aggiungere 0,5 ml di PBS con 0,3% Triton X-100 (0,3% PBT, vedi materiali) e lavare il tessuto su una sedia a dondolo per 10 minuti. Ripetere questa operazione per altre tre volte per un totale di 4 lavaggi (10 min ciascuna). NOTA: la rimozione accurata della soluzione dal pozzo può essere eseguita sotto un microscopio da dissezione per evitare danni ai tessuti o di perdita. La soluzione può essere aspirato accuratamente con una pipetta; preferiamo un fine-Tipped, pipetta di trasferimento monouso che ha avuto l&#39;apertura appiattita per ridurre la possibilità di aspirare il tessuto. </li><li> Alla fine del quarto lavaggio, rimuovere la pulizia e aggiungere 0,5 ml di soluzione di blocco (vedi materiali) al pozzetto ed incubare per 30 min. NOTA: Questo lasso di tempo può essere esteso in base alle vostre esigenze. </li><li> Alla fine del tempo di blocco 30 min, pipettare 10 ml di soluzione di anticorpo primario (diluito in soluzione Block) nel numero appropriato di pozzetti in una micropiastra. NOTA: Un pozzetto di una micropiastra ospiterà circa 4 coppie di dischi occhi collegati al caso pupa o 8 coppie isolate di dischi occhio con il cervello attaccato. Gli anticorpi primari utilizzati nel presente protocollo per i dati rappresentativi sono anti-fosfotirosina (diluizione 1: 500) e anti-Cut (diluizione 1: 200). </li><li> Trasferire il tessuto sezionato nei pozzetti per micropiastre riempiti con la soluzione di anticorpo primario dal piatto di vetro 3 bene. </li><li> Luogola micropiastra su un tovagliolo di carta bagnato (per evitare la disidratazione) in una scatola vuota pipetta punta (o altro contenitore simile) e conservare a 4 ° C per una notte. </li></ol> 2 ° giorno (4 ore + tempo di montaggio) <ol start="6"><li> Trasferire il tessuto dalla micropiastra in un piatto di vetro 3 bene con 0,3% PBT (≈0.5 ml) e lavare il tessuto per 10 min a bilanciere. Ripetere questa operazione per altre tre volte per un totale di 4 lavaggi (10 min ciascuna) </li><li> Rimuovere l&#39;ultimo lavaggio e aggiungere 0,5 ml di soluzione di blocco al pozzo. Blocco per 30 min. NOTA: Questo lasso di tempo può essere esteso in base alle vostre esigenze. </li><li> Rimuovere la soluzione di blocco e aggiungere 0,5 ml di Soluzione Anticorpi secondaria (a base di anticorpi fluorescente appropriate per il vostro anticorpo primario e lunghezza d&#39;onda fluorescente, diluito in soluzione Block) per il bene e proteggere il piatto di vetro 3 e dalla luce con un coperchio o di alluminio sventare e incubare per almeno 2 ore a cameratemperatura. NOTA: Tutti i passi successivi devono essere protetti in modo analogo da esposizione alla luce. Questo incubazione può essere estesa a tutta la notte a 4 ° C, in una configurazione simile a quella di incubazione dell&#39;anticorpo primario, ma può essere eseguita anche in un piatto di vetro 3 ben coperta con la pellicola di laboratorio, o una micropiastra qualora la conservazione degli anticorpi è desiderato. </li><li> Al termine dell&#39;incubazione anticorpi, rimuovere la Soluzione Anticorpi secondari e aggiungere 0,5 ml di 0,3% PBT al pozzo e lavare il bilanciere per 10 min. Ripetere questo passaggio per un totale di 4 lavaggi (10 minuti ciascuno). NOTA: Se colorazione DAPI si desidera, prima di iniziare il primo lavaggio, aggiungere 0,5 ml di DAPI soluzione madre (vedi materiali) ai 500 ml di PBT che saranno utilizzati per il primo lavaggio (1: 1.000 diluizione). </li></ol> 3. Montaggio <ol><li> Alla fine l&#39;ultimo lavaggio, montare i dischi delle antenne / occhio su un vetrino utilizzando 70% glicerolo o PBS come mezzo per rimuovere il brain e altri tessuti indesiderati in una piscina su un lato della slitta. </li><li> Spostare con attenzione il tessuto con pressione idrostatica tra le pinze, o molto delicatamente con una punta pinza, e allineare i dischi degli occhi in una colonna verso il centro della diapositiva. </li><li> Rimuovere tutti glicerolo estraneo o PBS provenienti da tutto il tessuto (con wicking azione di un laboratorio wipe o la pinza lavorare per questo). </li><li> Mettere una piccola quantità (≈10 ml) di mezzo di montaggio lungo un lato della colonna disco occhi e posizionare un coprioggetto sul tessuto in modo da distribuire la soluzione di montaggio sui dischi oculari. </li><li> Lasciate scivolo sedersi per 1 o 2 minuti per consentire il / mezzo di montaggio tessuto a diffondersi completamente. </li><li> Pulire qualsiasi mezzo di montaggio in più fuori intorno ai bordi del coprioggetto e sigillare con smalto trasparente. </li><li> Cattura immagini fluorescenti dei dischi oculari utilizzando la microscopia confocale. </li></ol>

<ol>
	<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 (2), 217-240 (1976).</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 (3), 841-850 (1991).</li><li>Carthew, R. W. <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.">Pattern formation in the Drosophila eye.</a> Curr Opin Genet Dev. 17 (4), 309-313 (2007).</li><li>Bao, S., Cagan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preferential+adhesion+mediated+by+Hibris+and+Roughest+regulates+morphogenesis+and+patterning+in+the+Drosophila+eye.">Preferential adhesion mediated by Hibris and Roughest regulates morphogenesis and patterning in the Drosophila eye.</a> Dev cell. 8 (6), 925-935 (2005).</li><li>Grzeschik, N. A., Knust, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IrreC/rst-mediated+cell+sorting+during+Drosophila+pupal+eye+development+depends+on+proper+localisation+of+DE-cadherin.">IrreC/rst-mediated cell sorting during Drosophila pupal eye development depends on proper localisation of DE-cadherin.</a> Development. 132 (9), 2035-2045 (2005).</li><li>Sprecher, S. 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=Switch+of+rhodopsin+expression+in+terminally+differentiated+Drosophila+sensory+neurons.">Switch of rhodopsin expression in terminally differentiated Drosophila sensory neurons.</a> Nature. 454 (7203), 533-537 (2008).</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 (4), 851-858 (1985).</li><li>Voas, M. G., Rebay, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+integration+during+development:+insights+from+the+Drosophila+eye.">Signal integration during development: insights from the Drosophila eye.</a> Dev Dyn. 229 (1), 162-175 (2004).</li><li>Hsiao, H. Y., et al. <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+immunohistochemistry+of+larval,+pupal+and+adult+Drosophila+retinas.">Dissection and immunohistochemistry of larval, pupal and adult Drosophila retinas.</a> J Vis Exp. , 4347 (2012).</li><li>Call, G. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomewide+clonal+analysis+of+lethal+mutations+in+the+Drosophila+melanogaster+eye:+comparison+of+the+X+chromosome+and+autosomes.">Genomewide clonal analysis of lethal mutations in the Drosophila melanogaster eye: comparison of the X chromosome and autosomes.</a> Genetics. 177 (2), 689-697 (2007).</li><li>Chen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery-based+science+education:+functional+genomic+dissection+in+Drosophila+by+undergraduate+researchers.">Discovery-based science education: functional genomic dissection in Drosophila by undergraduate researchers.</a> PLoS Biol. 3 (2), 59 (2005).</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, (2007).</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, 1936 (2010).</li><li>Xu, T., 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=Analysis+of+genetic+mosaics+in+developing+and+adult+Drosophila+tissues.">Analysis of genetic mosaics in developing and adult Drosophila tissues.</a> Development. 117 (4), 1223-1237 (1993).</li><li>Duffy, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GAL4+system+in+Drosophila:+a+fly+geneticist's+Swiss+army+knife.">GAL4 system in Drosophila: a fly geneticist's Swiss army knife.</a> Genesis. 34 (1-2), 1-15 (2002).</li></ol>

The authors have nothing to disclose.

While it appears that the process is simple and easy to perform, in reality, this technique requires a great deal of practice to master. Routinely, we start students off by learning to dissect and mount third instar eye discs12, which are much easier to work with. This practice helps to develop an appropriate dissection position of the arms, hands and fingers13 so that manipulation of the forceps under the dissecting microscope is stable, easy and experienced. In essence, the practice period shou...

I campi di biologia dello sviluppo e delle cellule sono stati notevolmente influenzati dalla organismo modello: Drosophila melanogaster. All&#39;interno di questo modello, gli studi del disco occhi hanno contribuito una grande quantità di conoscenze in materia di segnalazione, biologia cellulare e in altri settori. Il terzo disco in ritardo stadio larvale occhio è stato ampiamente studiato ed è un potente modello di utilizzare, in quanto dà una fotografia istantanea di una serie di periodi di sviluppo, ciascuno con le proprie molecole e dei processi di segnalazione unica, come il solco morfogenetica progredisce attraverso il disco occhio 8. Tuttavia, vi è la necessità di ampliare ulteriormente la nostra comprensione dei processi di sviluppo nella fase di pupa dello sviluppo. Mentre ci sono stati studi sul disco occhio pupa 3-7, la nostra conoscenza non si avvicina l&#39;ampiezza del lavoro che è stato eseguito il terzo disco instar occhio. Ciò è dovuto, in parte, alla maggiore difficoltà di sezionare il disco dell&#39;occhio pupa. Pertanto, una presentazione delcorretto metodo di dissezione potrebbe espandere notevolmente la ricerca in questo settore. Mentre ci sono fasi di sviluppo all&#39;interno di pupa disco occhio che sono facilmente sezionati, in particolare intorno alla metà del periodo di pupa, altri periodi di tempo sono molto più difficili da sezionare. Questo protocollo rappresenta un metodo per sezionare i dischi degli occhi pupa che può essere usato universalmente per tutte le pupe tempi di sviluppo. Questo protocollo può essere utilizzato in alternativa a un altro protocollo 9, che mostra un metodo semplice e veloce per sezionare i dischi occhio dai punti temporali midpupal. Questo protocollo è stato originariamente girato e sviluppato per la formazione di studenti universitari avanzati in Undergraduate Research Consortium UCLA in Genomica Funzionale (URCFG) 10,11 nella tecnica di pupa dissezione dell&#39;occhio. Molti studenti universitari sono stati in grado di utilizzare questo video e metodo per imparare questa tecnica impegnativa.

<table><tbody><tr><td>Phosphate-buffered saline (PBS, pH 7.4)</td><td></td><td></td><td>80 g NaCl, 2g&amp;nbsp; KCl, 14.4 g Na&lt;sub&gt;2&lt;/sub&gt;HPO4, 2.4 g KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, Bring volume to 1 &amp;#956;l, adjust the pH to 7.4, autoclave or filter sterilize, dilute to 1x PBS with autoclaved ddH&lt;sub&gt;2&lt;/sub&gt;O before using.</td></tr><tr><td>Triton X-100</td><td>Promega</td><td>H5142</td><td>Caution: Irritant! Wear gloves.</td></tr><tr><td>0.3% PBT</td><td></td><td></td><td>1.5 ml of Triton X-100, 500 ml 1X PBS.</td></tr><tr><td>37% formaldehyde solution</td><td>Fisher Scientific</td><td>F75P1GAL</td><td>Caution: Toxic, probable human carcinogen! Wear gloves.&amp;nbsp;</td></tr><tr><td>Fix Solution</td><td></td><td></td><td>(&amp;asymp;4% Formaldehyde in PBS) 50 &amp;mu;l of 37% Formaldehyde, 450 &amp;mu;l 1x PBS, make fresh before use</td></tr><tr><td>Normal goat serum</td><td>Rockland antibodies &amp;amp; assays</td><td>B304</td><td>Aliquot in 1 ml volumes and store at -80 &amp;#176;C</td></tr><tr><td>Block Solution</td><td></td><td></td><td>10% NGS in PBT.&amp;nbsp; This can be made and stored at 4 &amp;deg;C for a few days prior to use.</td></tr><tr><td>DAPI stock solution</td><td>Life Technologies</td><td>D3571</td><td>For coutnerstaining nuclei. Prepare a 1 mg/ml solution with ddH&lt;sub&gt;2&lt;/sub&gt;O.</td></tr><tr><td>VectaShield Mounting Medium</td><td>Vector Labs</td><td>H-1000</td><td>Mounting medium</td></tr><tr><td>Glycerol</td><td>Sigma</td><td>G5516</td><td>For mounting. Prepare 70% dilution with ddH&lt;sub&gt;2&lt;/sub&gt;O.</td></tr><tr><td>Equipment</td><td></td><td></td><td></td></tr><tr><td>Nutating mixer</td><td>VWR</td><td>82007-202</td><td>Used to rock tissue in 3 well glass dish</td></tr><tr><td>SylGard 182 Silicone Elastomer Kit</td><td>Krayden</td><td>NC9897184</td><td>Used to make silicone dissection dish</td></tr><tr><td>Silicone dissecting dish</td><td></td><td></td><td>Mix Sylgard elastomer kit (above) according to directions gently (to avoid bubbles). Pour mixture into Petri dish (any size). Allow SylGard to cure overnight in 37 &amp;deg;C incubator.</td></tr><tr><td>3 well glass dish</td><td>Corning</td><td>7220-85</td><td>The 3 well variety of these are no longer available, this is the 9 well product.</td></tr><tr><td>72 well microwell minitray</td><td>Nunc</td><td>438733</td><td></td></tr><tr><td>Sharp forceps (Dumont #55)</td><td>Fine Science Tools</td><td>11255-20</td><td></td></tr><tr><td>Vannas-type Micro Scissors, Straight, 5mm blade</td><td>Ted Pella</td><td>1346</td><td></td></tr><tr><td>100 mm Borosilicate glass capillaries</td><td>World Precision Instruments</td><td>1B100-4</td><td>Pull with needle puller to make fine point tip that allows a small stream of PBS to flow.</td></tr><tr><td>Disposable Transfer Pipets, Fine Tip</td><td>Samco Scientific</td><td>231</td><td></td></tr><tr><td>Tubing dimensions given are inner diameter (ID) x outer diameter (OD) x wall thickness in inches</td><td></td><td></td><td></td></tr><tr><td>PVC tubing (1/8 x 3/16 x 1/32)</td><td>Nalgene</td><td>8000-0010</td><td>Use these with pulled needle to assemble the blower tube as shown in Figure 2.</td></tr><tr><td>Tygon Silicone tubing (3/32 x 5/32 x 1/32)</td><td>Saint Gobain Performance Plastics</td><td>ABW00004</td><td></td></tr><tr><td>Tygon Silicone tubing (1/32 x 3/32 x 1/32)</td><td>Saint Gobain Performance Plastics</td><td>ABW00001</td><td></td></tr></tbody></table>

dissection and mounting of drosophila pupal eye discs

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes. It contains approximately 800 separate eye units, termed ommatidia1. Each ommatidium contains eight neuronal photoreceptors that develop from undifferentiated cells following the passage of the morphogenetic furrow in the third larval instar2. Following the sequential differentiation of the photoreceptors, non-neuronal cells develop, including cone and pigment cells, along with mechanosensory bristle cells3. Final differentiation processes, including the structured arrangement of all the ommatidial cell types, programmed cell death of undifferentiated cell types and rhodopsin expression, occurs through the pupal phase4-7. This technique focuses on manipulating the pupal eye disc, providing insight and instruction on how to dissect the eye disc during the pupal phase, which is inherently more difficult to perform than the commonly dissected third instar eye disc. This technique also provides details on immunostaining to allow the visualization of various proteins and other cell components.

Come esempio dell&#39;uso di questo protocollo, risultati illustrano midpupal (42 ore APF a 25 ° C) dischi occhio immunostained con anticorpi differenti sono rappresentati nella figura 2. Utilizzando un anticorpo diretto contro residui fosfotirosina, la membrana delle cellule può essere osservati (Figura 2A). Questo può essere utilizzato per identificare la disposizione regolare di celle ommatidial nell&#39;occhio pupa seguenti processi patterning finali che si verificano prima della fase midpupal. U...

Watch this Scientific Journal Video about Dissection and Mounting of Drosophila Pupal Eye Discs at JoVE.com

Dissection and Mounting of Drosophila Pupal Eye Discs

The goal of this technique is to enable researchers to perform dissection, immunostaining and mounting of pupal eye discs from Drosophila melanogaster of any age.

Dissezione e Montaggio di<em&gt; Drosophila</em&gt; pupa Dischi Eye

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes.  It contains approximately 800 ...

dissection-and-mounting-of-drosophila-pupal-eye-discs

Research

JoVE Journal

Biology

Dissection and Mounting of Drosophila Pupal Eye Discs

12.1K Views.  University of California at Los Angeles (UCLA). The goal of this technique is to enable researchers to perform dissection, immunostaining and mounting of pupal eye discs from Drosophila melanogaster of any age.

Video: Dissection and Mounting of Drosophila Pupal Eye Discs

Live-imaging of the Drosophila Pupal Eye

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track during live cell imaging. These processes include: retinal rotation, cell growth and organismal movement. Additionally, irregularities in the topology of the epithelium, including subtle bumps and folds often introduced as the pupa is prepared for imaging, make it challenging to acquire in-focus images of more than a few ommatidia in a single focal plane. The workflow outlined here remedies these issues, allowing easy analysis of cellular processes during Drosophila pupal eye development. Appropriately-staged pupae are arranged in an imaging rig that can be easily assembled in most laboratories. Ubiquitin-DE-Cadherin:GFP and GMR-GAL4&#8211;driven UAS-&#945;-catenin:GFP are used to visualize cell boundaries in the eye epithelium 1-3. After deconvolution is applied to fluorescent images captured at multiple focal planes, maximum projection images are generated for each time point and enhanced using image editing software. Alignment algorithms are used to quickly stabilize superfluous motion, making individual cell behavior easier to track.

Live-imaging of the Drosophila Pupal Eye

This protocol presents an efficient method for imaging the live Drosophila pupal eye neuroepithelium. This method compensates for tissue movement and uneven topology, enhances visualization of cell boundaries through the use of multiple GFP-tagged junction proteins, and uses an easily-assembled imaging rig.

Live-imaging del<em&gt; Drosophila</em&gt; Pupa Eye

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track ...

Ricerca

Biologia dello sviluppo

Dissection of Imaginal Discs from 3rd Instar Drosophila Larvae

This protocol demonstrates the dissection technique used for isolating imaginal discs from drosophila larvae at the 3rd instar stage. Methods for fixing the tissue after saying and removing the wing, leg, and eye discs are demonstrated directly on a microscope slide for subsequent visualization.

Dissezione di dischi immaginale da larve 3 Instar Drosophila

Biologia

Long-term Live Imaging of Drosophila Eye Disc

Live imaging provides the ability to continuously track dynamic cellular and developmental processes in real time. Drosophila larval imaginal discs have been used to study many biological processes, such as cell proliferation, differentiation, growth, migration, apoptosis, competition, cell-cell signaling, and compartmental boundary formation. However, methods for the long-term ex vivo culture and live imaging of the imaginal discs have not been satisfactory, despite many efforts. Recently, we developed a method for the long-term ex vivo culture and live imaging of imaginal discs for up to 18 h. In addition to using a high insulin concentration in the culture medium, a low-melting agarose was also used to embed the disc to prevent it from drifting during the imaging period. This report uses the eye-antennal discs as an example. Photoreceptor R3/4-specific m&#948;0.5-Ga4 expression was followed to demonstrate that photoreceptor differentiation and ommatidial rotation can be observed during a 10 h live imaging period. This is a detailed protocol describing this simple method.

Long-term Live Imaging of Drosophila Eye Disc

This protocol describes a detailed method for the long-term ex vivo culture and live imaging of a Drosophila imaginal disc. It demonstrates photoreceptor differentiation and ommatidial rotation within the 10 h period of live imaging of the eye disc. The protocol is simple and does not require expensive setup.

A lungo termine Imaging in diretta di<em&gt; Drosophila</em&gt; Disco Eye

Live imaging provides the ability to continuously track dynamic cellular and developmental processes in real time. Drosophila larval imaginal discs have ...

Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons. While recent progress has been made to describe the live migration of glial cells in the developing pupal wing (1), studies of Drosophila glial cell migration have typically involved the examination of fixed tissue. Live microscopic analysis of motile cells offers the ability to examine cellular behavior throughout the migratory process, including determining the rate of and changes in direction of growth. Paired with use of genetic tools, live imaging can be used to determine more precise roles for specific genes in the process of development. Previous work by Silies et al. (2) has described the migration of glia originating from the optic stalk, a structure that connects the developing eye and brain, into the eye imaginal disc in fixed tissue. Here we outline a protocol for examining the live migration of glial cells into the Drosophila eye imaginal disc. We take advantage of a Drosophila line that expresses GFP in developing glia to follow glial cell progression in wild type and in mutant animals.

Here we describe a protocol to examine the migration of glial cells into the developing Drosophila eye using live microscopic analysis paired with GFP tagged glial cells.

Vivere Imaging della migrazione cellule gliali nella Eye Disco Drosophila immaginale

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons.  While ...

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including lens-secreting cone cells, pigment cells, a bristle cell and eight photoreceptors (PRs) R1-R8 2. The PRs have specialized microvillar structures, the rhabdomeres, which contain light-sensitive pigments, the Rhodopsins (Rhs). The rhabdomeres of six PRs (R1-R6) form a trapezoid and contain Rh1 3 4. The rhabdomeres of R7 and R8 are positioned in tandem in the center of the trapezoid and share the same path of light. R7 and R8 PRs stochastically express different combinations of Rhs in two main subtypes5: In the 'p' subtype, Rh3 in pR7s is coupled with Rh5 in pR8s, whereas in the 'y' subtype, Rh4 in yR7s is associated with Rh6 in yR8s 6 7 8.
Early specification of PRs and development of ommatidia begins in the larval eye-antennal imaginal disc, a monolayer of epithelial cells. A wave of differentiation sweeps across the disc9 and initiates the assembly of undifferentiated cells into ommatidia10-11. The 'founder cell' R8 is specified first and recruits R1-6 and then R7 12-14. Subsequently, during pupal development, PR differentiation leads to extensive morphological changes 15, including rhabdomere formation, synaptogenesis and eventually rh expression.
In this protocol, we describe methods for retinal dissections and immunohistochemistry at three defined periods of retina development, which can be applied to address a variety of questions concerning retinal formation and developmental pathways. Here, we use these methods to visualize the stepwise PR differentiation at the single-cell level in whole mount larval, midpupal and adult retinas (Figure 1).

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

The Drosophila retina is a crystal-like lattice composed of a small number of cell types that are generated in a stereotyped manner 1. Its amenability to sophisticated genetic analysis allows the study of complex developmental programs. This protocol describes dissections and immunohistochemistry of retinas at three discrete developmental stages, with a focus on photoreceptor differentiation.

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 ...

Neuroscienze

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

The Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes during development. In this paper, we present a reliable protocol for the dissection of the delicate Drosophila pupal retina. Our surgical approach utilizes readily-available microdissection tools to open pupae and precisely extract eye-brain complexes. These can be fixed, subjected to immunohistochemistry, and retinas then mounted onto microscope slides and imaged if the goal is to detect cellular or subcellular structures. Alternatively, unfixed retinas can be isolated from brain tissue, lysed in appropriate buffers and utilized for protein gel electrophoresis or mRNA extraction (to assess protein or gene expression, respectively). Significant practice and patience may be required to master the microdissection protocol described, but once mastered, the protocol enables relatively quick isolation of mainly undamaged retinas.

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

This paper presents a surgical method for dissecting Drosophila pupal retinas along with protocols for the processing of tissue for immunohistochemistry, western analysis, and RNA-extraction.&#160;

Dissezione della drosofila Pupal Retina per immunoistochimica, analisi occidentale e isolamento del RNA

The Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes during development. In this paper, we present a ...

A Novel RFP Reporter to Aid in the Visualization of the Eye Imaginal Disc in Drosophila

The Drosophila eye is a powerful model system for studying areas such as neurogenesis, signal transduction and neurodegeneration. Many of the discoveries made using this system have taken advantage of the spatiotemporal nature of photoreceptor differentiation in the developing eye imaginal disc. To use this system it is first necessary for the researcher to learn to identify and dissect the eye disc. We describe a novel RFP reporter to aid in the identification of the eye disc and the visualization of specific cell types in the developing eye. We detail a methodology for dissection of the eye imaginal disc from third instar larvae and describe how the eye-RFP reporter can aid in this dissection. This eye-RFP reporter is only expressed in the eye and can be visualized using fluorescence microscopy either in live tissue or after fixation without the need for signal amplification. We also show how this reporter can be used to identify specific cells types within the eye disc. This protocol and the use of the eye-RFP reporter will aid researchers using the Drosophila eye to address fundamentally important biological questions.

A Novel RFP Reporter to Aid in the Visualization of the Eye Imaginal Disc in Drosophila

We describe a novel red fluorescent protein (RFP) reporter that is expressed specifically in the Drosophila eye. We detail a methodology for dissection of the eye imaginal disc and how this reporter can be used to aid in the dissection and identification of specific cell types in the developing eye.

Un Reporter Novel RFP per aiutare nella visualizzazione del Disco Eye immaginale in Drosophila</em

The Drosophila eye is a powerful model system for studying areas such as neurogenesis, signal transduction and neurodegeneration. Many of the discoveries ...

Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis

Drosophila has long been used as model system to study development, mainly due to the ease with which it is genetically tractable. Over the years, a plethora of mutant strains and technical tricks have been developed to allow sophisticated questions to be asked and answered in a reasonable amount of time. Fundamental insight into the interplay of components of all known major signaling pathways has been obtained in forward and reverse genetic Drosophila studies. The fly eye has proven to be exceptionally well suited for mutational analysis, since, under laboratory conditions, flies can survive without functional eyes. Furthermore, the surface of the insect eye is composed of some 800 individual unit eyes (facets or ommatidia) that form a regular, smooth surface when looked at under a dissecting microscope. Thus, it is easy to see whether a mutation might affect eye development or growth by externally looking for the loss of the smooth surface ('rough eye' phenotype; Fig. 1) or overall eye size, respectively (for examples of screens based on external eye morphology see e.g.1). Subsequent detailed analyses of eye phenotypes require fixation, plastic embedding and thin-sectioning of adult eyes.
The Drosophila eye develops from the so-called eye imaginal disc, a bag of epithelial cells that proliferate and differentiate during larval and pupal stages (for review see e.g. 2). Each ommatidium consists of 20 cells, including eight photoreceptors (PR or R-cells; Fig. 2), four lens-secreting cone cells, pigment cells ('hexagon' around R-cell cluster) and a bristle. The photoreceptors of each ommatidium, most easily identified by their light sensitive organelles, the rhabdomeres, are organized in a trapezoid made up of the six "outer" (R1-6) and two "inner" photoreceptors (R7/8; R8 [Fig. 2] is underneath R7 and thus only seen in sections from deeper areas of the eye). The trapezoid of each facet is precisely aligned with those of its neighbors and the overall anteroposterior and dorsoventral axes of the eye (Fig. 3A). In particular, the ommatidia of the dorsal and ventral (black and red arrows, respectively) halves of the eye are mirror images of each other and correspond to two chiral forms established during planar cell polarity signaling (for review see e.g. 3).
The method to generate semi-thin eye sections (such as those presented in Fig. 3) described here is slightly modified from the one originally described by Tomlinson and Ready4. It allows the morphological analysis of all cells except for the transparent cone cells. In addition, the pigment of R-cells (blue arrowheads in Fig. 2 and 3) can be used as a cell-autonomous marker for the genotype of a R-cell, thus genetic requirements of genes in a subset of R-cells can readily be determined5,6.

Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis

A standard approach to prepare adult Drosophila eyes for semi-thin sectioning and light microscopic analysis is presented here. The protocol can be used for gross morphological analysis of eye defects, or with the indicated adjustments can be used to determine genetic requirements of genes in specific cell types of the eye (e.g. clonal analysis of photoreceptors) or for electron microscopic analysis.

Preparazione di adulti Drosophila Occhi di sezionamento sottile e analisi microscopica

Drosophila has long been used as model system to study development, mainly due to the ease with which it is genetically tractable. Over the years, a ...

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called imaginal discs. These discs give rise to a high percentage of adult structures that are found within the adult fly. Here we describe a protocol that has been optimized to recover these discs and prepare them for analysis with antibodies, transcriptional reporters and protein traps. This procedure is best suited for thin tissues like imaginal discs, but can be easily modified for use with thicker tissues such as the larval brain and adult ovary. The written protocol and accompanying video will guide the reader/viewer through the dissection of third instar larvae, fixation of tissue, and treatment of imaginal discs with antibodies. The protocol can be used to dissect imaginal discs from younger first and second instar larvae as well. The advantage of this protocol is that it is relatively short and it has been optimized for the high quality preservation of the dissected tissue. Another advantage is that the fixation procedure that is employed works well with the overwhelming number of antibodies that recognize Drosophila proteins. In our experience, there is a very small number of sensitive antibodies that do not work well with this procedure. In these situations, the remedy appears to be to use an alternate fixation cocktail while continuing to follow the guidelines that we have set forth for the dissection steps and antibody incubations.

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

The adult structures of Drosophila are derived from sac-like structures called imaginal discs. Analysis of these discs provides insight into many developmental processes including tissue determination, compartment boundary establishment, cell proliferation, cell fate specification, and planar cell polarity. This protocol is used to prepare imaginal discs for light/fluorescent microscopy.

Dissezione e Immunostaining di immaginale dischi da<em&gt; Drosophila melanogaster</em

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called ...

Dissection of the Eye-Brain Complex from Fly Pupae: A Method to Isolate Retinal Tissue

Source: DeAngelis, M. W., Johnson, R. I. <a href="https://www.jove.com/video/59299/" target="_blank">Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation</a>. J. Vis. Exp.&#160;(2019).
This video describes how to dissect and isolate the eye-brain complex from Drosophila pupae. The featured protocol demonstrates the procedure yielding high-quality tissue, compatible with, for example, immunostaining, as well as, other gene expression experiments.

Dissezione del complesso occhio-cervello da pupe di mosca: un metodo per isolare il tessuto retinico

Source: DeAngelis, M. W., Johnson, R. I. Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation. J. Vis. ...

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Live-imaging del

Dissezione di dischi immaginale da larve 3 Instar Drosophila

A lungo termine Imaging in diretta di

Vivere Imaging della migrazione cellule gliali nella Eye Disco Drosophila immaginale

Dissezione e immunoistochimica di larvale, pupa e adulto

Dissezione della drosofila Pupal Retina per immunoistochimica, analisi occidentale e isolamento del RNA

Un Reporter Novel RFP per aiutare nella visualizzazione del Disco Eye immaginale in Drosophila

Preparazione di adulti Drosophila Occhi di sezionamento sottile e analisi microscopica

Dissezione e Immunostaining di immaginale dischi da

Dissezione del complesso occhio-cervello da pupe di mosca: un metodo per isolare il tessuto retinico