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 border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Phosphate-buffered saline (PBS, pH 7.4)</td>
			<td></td>
			<td></td>
			<td>80g NaCl, 2g&#160; KCl, 14.4g Na2HPO4, 2.4g KH2PO4, Bring volume to 1 l, adjust the pH to 7.4, autoclave or filter sterilize, dilute to 1X PBS with autoclaved ddH2O 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.&#160;</td>
		</tr>
		<tr>
			<td>Fix Solution</td>
			<td></td>
			<td></td>
			<td>(&#8776;4% Formaldehyde in PBS) 50 &#956;l of 37% Formaldehyde, 450 &#956;l 1XPBS, make fresh before use</td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>Rockland antibodies &#38; assays</td>
			<td>B304</td>
			<td>Aliquot in 1 ml volumes and store at -80C</td>
		</tr>
		<tr>
			<td>Block Solution</td>
			<td></td>
			<td></td>
			<td>10% NGS in PBT.&#160; This can be made and stored at 4 &#176;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 ddH2O.</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 ddH2O.</td>
		</tr>
		<tr>
			<td colspan="4">Equipment</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 &#176;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><a name="RANGE!A19">Vannas-type Micro Scissors, Straight, 5mm blade</a></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 colspan="4">Tubing dimensions given are inner diameter (ID) x outer diameter (OD) x wall thickness in inches</td>
		</tr>
		<tr>
			<td>PVC tubing (1/8 x 3/16 x 1/32)</td>
			<td>Nalgene</td>
			<td>8000-0010</td>
			<td rowspan="3">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>
		</tr>
		<tr>
			<td>Tygon Silicone tubing (1/32 x 3/32 x 1/32)</td>
			<td>Saint Gobain Performance Plastics</td>
			<td>ABW00001</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

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Biology

Dissection and Mounting of Drosophila Pupal Eye Discs

12.0K 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

Dissection of Drosophila Ovaries

Dissezione di ovaie Drosophila

Ricerca

Biologia

Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins.  Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity.  In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining.  In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS.  Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS.  If the dissection is performed carefully the CNS will remain attached to the cuticle.  We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides.  We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS.  The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

Dissezione del sistema nervoso centrale larvali in Drosophila melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

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

Drosophila Larval NMJ Dissection

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 35 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. In order to access the NMJ for study, it is necessary to carefully dissect each larva. In this article we demonstrate how to properly dissect Drosophila larvae for study of the NMJ by removing all internal organs while leaving the body wall intact. This technique is suitable to prepare larvae for imaging, immunohistochemistry, or electrophysiology.

This protocol demonstrates how to dissect Drosophila larvae in preparation for immunohistochemistry and/or imaging of the neuromuscular junction.

Drosophila larvali NMJ Dissection

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use ...

Dissection and Staining of Drosophila Larval Ovaries

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells form, and how they interact with their environment is therefore crucial for understanding development, homeostasis and disease. The ovary of the fruit fly Drosophila melanogaster has served as an influential model for the interaction of germ line stem cells (GSCs) with their somatic support cells (niche) 1, 2. The known location of the niche and the GSCs, coupled to the ability to genetically manipulate them, has allowed researchers to elucidate a variety of interactions between stem cells and their niches 3-12.
Despite the wealth of information about mechanisms controlling GSC maintenance and differentiation, relatively little is known about how GSCs and their somatic niches form during development. About 18 somatic niches, whose cellular components include terminal filament and cap cells (Figure 1), form during the third larval instar 13-17. GSCs originate from primordial germ cells (PGCs). PGCs proliferate at early larval stages, but following the formation of the niche a subgroup of PGCs becomes GSCs 7, 16, 18, 19. Together, the somatic niche cells and the GSCs make a functional unit that produces eggs throughout the lifetime of the organism.
Many questions regarding the formation of the GSC unit remain unanswered. Processes such as coordination between precursor cells for niches and stem cell precursors, or the generation of asymmetry within PGCs as they become GSCs, can best be studied in the larva. However, a methodical study of larval ovary development is physically challenging. First, larval ovaries are small. Even at late larval stages they are only 100&mu;m across. In addition, the ovaries are transparent and are embedded in a white fat body. Here we describe a step-by-step protocol for isolating ovaries from late third instar (LL3) Drosophila larvae, followed by staining with fluorescent antibodies. We offer some technical solutions to problems such as locating the ovaries, staining and washing tissues that do not sink, and making sure that antibodies penetrate into the tissue. This protocol can be applied to earlier larval stages and to larval testes as well.

Dissection and Staining of Drosophila Larval Ovaries

How niches and stem cells form during development is an important question with practical implications. In the Drosophila ovary, germ line stem cells and their somatic niches form during larval development. This video demonstrates how to dissect, stain and mount female gonads from late third instar (LL3) Drosophila larvae.

Dissezione e colorazione di Drosophila Larvali ovaie

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells ...

Drosophila Pupal Abdomen Immunohistochemistry

The Drosophila pupal abdomen is an established model system for the study of epithelial morphogenesis and the development of sexually dimorphic morphologies 1-3. During pupation, which spans approximately 96 hours (at 25 &deg;C), proliferating populations of imaginal cells replace the larval epidermis to generate the adult abdominal segments. These imaginal cells, born during embryogenesis, exist as lateral pairs of histoblast nests in each abdominal segment of the larvae. Four pairs of histoblast nests give rise to the adult dorsal cuticle (anterior and posterior dorsal nests), the ventral cuticle (ventral nests) and the spiracles associated with each segment (spiracle nests) 4. Upon puparation, these diploid cells (distinguishable by size from the larger polyploid larval epidermal cells- LECs) begin a stereotypical process of proliferation, migration and replacement of the LECs. Various molecular and genetic tools can be employed to investigate the contributions of genetic pathways involved in morphogenesis of the adult abdomen. Ultimate adult phenotypes are typically analyzed following dissection of adult abdominal cuticles. However, investigation of the underlying molecular processes requires immunohistochemical analyses of the pupal epithelium, which present unique challenges. Temporally dynamic morphogenesis and the interactions of two distinct epithelial populations (larval and imaginal) generate a fragile tissue prone to excessive cell loss during dissection and subsequent processing. We have developed methods of dissection, fixation, mounting and imaging of the Drosophila pupal abdominem epithelium for immunohistochemical studies that generate consistent high quality samples suitable for confocal or standard fluorescent microscopy.

Drosophila Pupal Abdomen Immunohistochemistry

Antibody staining of the Drosophila pupae can enhance genetic analyses of adult abdominal developmental genetics. We present our protocol for dissection, fixation and antibody staining of staged Drosophila pupal abdomen.

Drosophila pupa Addome Immunoistochimica

The Drosophila pupal abdomen is an established model system for the study of epithelial morphogenesis and the development of sexually dimorphic ...

Imaging Through the Pupal Case of Drosophila melanogaster

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled analysis of cell and developmental biology from a variety of methodological angles. Live imaging is an emerging method for observing dynamic cell processes, such as cell division or cell motility. Having isolated mutations in uncharacterized putative cell cycle proteins it became essential to observe mitosis in situ using live imaging. Most live imaging studies in Drosophila have focused on the embryonic stages that are accessible to manipulation and observation because of their small size and optical clarity. However, in these stages the cell cycle is unusual in that it lacks one or both of the gap phases. By contrast, cells of the pupal wing of Drosophila have a typical cell cycle and undergo a period of rapid mitosis spanning about 20 hr of pupal development. It is easy to identify and isolate pupae of the appropriate stage to catch mitosis in situ. Mounting intact pupae provided the best combination of tractability and durability during imaging, allowing experiments to run for several hours with minimal impact on cell and animal viability. The method allows observation of features as small as, or smaller than, fly chromosomes. Adjustment of microscope settings and the details of mounting, allowed extension of the preparation to visualize membrane dynamics of adjacent cells and fluorescently labeled proteins such as tubulin. This method works for all tested fluorescent proteins and can capture submicron scale features over a variety of time scales. While limited to the outer 20 &#181;m of the pupa with a conventional confocal microscope, this approach to observing protein and cellular dynamics in pupal tissues in vivo may be generally useful in the study of cell and developmental biology in these tissues.

Imaging Through the Pupal Case of Drosophila melanogaster

This paper demonstrates the use of a fast scanning confocal microscope to image cell behavior directly through the puparium. By leaving the pupal case intact, this method allows observation and measurement of dynamic cell processes at a stage of Drosophila development that is difficult to study directly.

Imaging attraverso il caso pupa di<em&gt; Drosophila melanogaster</em

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled ...

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 ...

Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

During spermatogenesis in mammals and in Drosophila melanogaster, male germ cells develop in a series of essential developmental processes. This includes differentiation from a stem cell population, mitotic amplification, and meiosis. In addition, post-meiotic germ cells undergo a dramatic morphological reshaping process as well as a global epigenetic reconfiguration of the germ line chromatin&#8212;the histone-to-protamine switch.
Studying the role of a protein in post-meiotic spermatogenesis using mutagenesis or other genetic tools is often impeded by essential embryonic, pre-meiotic, or meiotic functions of the protein under investigation. The post-meiotic phenotype of a mutant of such a protein could be obscured through an earlier developmental block, or the interpretation of the phenotype could be complicated. The model organism Drosophila melanogaster offers a bypass to this problem: intact testes and even cysts of germ cells dissected from early pupae are able to develop ex vivo in culture medium. Making use of such cultures allows microscopic imaging of living germ cells in testes and of germ-line cysts. Importantly, the cultivated testes and germ cells also become accessible to pharmacological inhibitors, thereby permitting manipulation of enzymatic functions during spermatogenesis, including post-meiotic stages.
The protocol presented describes how to dissect and cultivate pupal testes and germ-line cysts. Information on the development of pupal testes and culture conditions are provided alongside microscope imaging data of live testes and germ-line cysts in culture. We also describe a pharmacological assay to study post-meiotic spermatogenesis, exemplified by an assay targeting the histone-to-protamine switch using the histone acetyltransferase inhibitor anacardic acid. In principle, this cultivation method could be adapted to address many other research questions in pre- and post-meiotic spermatogenesis.

Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

This protocol describes the dissection and cultivation of intact testes and germ-line cysts from Drosophila melanogaster pupae. This method allows microscopic observation of spermatogenesis ex vivo. Furthermore, we describe a pharmacological assay of the effect of inhibitors on specific stages of germ-cell development in pupal testes.

Ex vivo Cultura della Drosophila pupa testicolo e singolo maschio cisti germinale: Dissezione, Imaging, e trattamento farmacologico

During spermatogenesis in mammals and in Drosophila melanogaster, male germ cells develop in a series of essential developmental processes. This includes ...

Dissection and Flat-mounting of the Threespine Stickleback Branchial Skeleton

The posterior pharyngeal segments of the vertebrate head give rise to the branchial skeleton, the primary site of food processing in fish. The morphology of the fish branchial skeleton is matched to a species' diet. Threespine stickleback fish (Gasterosteus aculeatus) have emerged as a model system to study the genetic and developmental basis of evolved differences in a variety of traits. Marine populations of sticklebacks have repeatedly colonized countless new freshwater lakes and creeks. Adaptation to the new diet in these freshwater environments likely underlies a series of craniofacial changes that have evolved repeatedly in independently derived freshwater populations. These include three major patterning changes to the branchial skeleton: reductions in the number and length of gill raker bones, increases in pharyngeal tooth number, and increased branchial bone lengths. Here we describe a detailed protocol to dissect and flat-mount the internal branchial skeleton in threespine stickleback fish. Dissection of the entire three-dimensional branchial skeleton and mounting it flat into a largely two-dimensional prep allows for the easy visualization and quantification of branchial skeleton morphology. This dissection method is inexpensive, fast, relatively easy, and applicable to a wide variety of fish species. In sticklebacks, this efficient method allows the quantification of skeletal morphology in genetic crosses to map genomic regions controlling craniofacial patterning.

The branchial skeleton, including gill rakers, pharyngeal teeth, and branchial bones, serves as the primary site of food processing in most fish. Here we describe a protocol to dissect and flat-mount this internal skeleton in threespine sticklebacks. This method is also applicable to a variety of other fish species.

Dissezione e Flat-montaggio del Threespine Stickleback branchiale scheletro

The posterior pharyngeal segments of the vertebrate head give rise to the branchial skeleton, the primary site of food processing in fish. The morphology ...