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.

Dieses Verfahren ist einen 2-tägigen Verfahren. Tag 1 (2 HR + Sezieren Zeit) 1. Pupal Eye Disc Dissection <ol><li> Wählen Sie eine Puppe für die Präparation. HINWEIS: Das Alter der Puppe zu seziert werden durch die experimentellen Bedürfnisse ermittelt werden. Wenn jedoch die Prüfung der Zellmorphologie, wird dies häufig bei 42 h nach Pupariumbildung (APF) bei 25 ° C, die das Alter der in der Video gezeigt Puppen durchgeführt wird. Sammeln weißen Puppen (als 0 hr APF) mit einem angefeuchteten Pinsel und ordnen sie in chronologischer Reihenfolge (basierend auf Sammelzeit) in einer neuen Fliege Essen Fläschchen bei 25 ° C gehalten. Präparieren Sie die im Alter von Puppen in diesem gleichen chronologischen Reihenfolge, um die Zeitunterschiede zwischen Puppen so nah wie möglich zu halten. </li><li> Übertragen Sie die Puppe in einen Tropfen Phosphat-gepufferte Salzlösung (PBS, pH 7,4, siehe Materialien für Rezept) auf einem Silikon Seziersaal Platte. </li><li> Halten Sie die Spitze der Puppenhülle mit einer Pinzette und Pierce und öffnen Sie die lower Teil der Puppenhülle mit einem Paar von scharfen Pinzette. Entfernen Sie die Puppe von der neu gegründeten Loch Fall. </li><li> Einen Diagonalschnitt mit einer Schere, mid-Thorax, in der Puppe. Entfernen Sie den Fremdkörper oder überweisen Sie den Puppenkopf zu einem neuen Tropfen PBS. Sauber mit der Pipette Blasrohr (siehe Materialien und Abbildung 1 für Details) und zusätzliche PBS nach Bedarf. Achten Sie darauf, um Austrocknung zu vermeiden. </li><li> Sicher zu greifen die Besten der Puppenkopf Kutikula mit einer Pinzette. Mit der Spitze des Blasrohr, schieben auf der Puppenhülle, um das Gehirn und anderen Geweben, einschließlich der Augenscheiben, aus der Puppenhülle zu extrudieren. Achten Sie darauf, um zu vermeiden, greifen jedes Gewebe innerhalb der Nagelhaut. </li><li> Mit dem Blasrohr mit PBS gefüllt, blasen das Fett Körpergewebe und andere Verunreinigungen aus dem Kopf Fall klar zu trennen und identifizieren die Gehirn und Auge-Discs. HINWEIS: Halten Sie das Gehirn an den Puppenkopf Gehäuse angebracht wird es der Forscher auf die Verwendung erad Fall als &quot;Griff&quot; für zukünftige Manipulation des Gewebes, ohne die Augenscheiben oder Gehirn. Jedoch ist es üblich, das Gehirn und Augen Scheiben vom Kopfgehäuse zu entfernen und dies ist nicht schädlich für das Gesamtverfahren und Ergebnisse. Es dauert oft mehrere Versuche von Treib und schob die Augenscheiben und Gehirn aus dem Puppenkopf Fall und so, dass sie sich deutlich von umliegenden Gewebe erfolgreich zu extrahieren. Die saubere und getrennte man die Augenscheiben aus dem umgebenden Gewebe zu helfen zu verhindern äußerem Gewebe von Befestigung an der Augenscheiben. </li><li> Nach Dissektion, sofort das Gewebe übertragen, um einem 3 gut Glasschale auf Eis mit 0,5 ml Fix-Lösung (siehe Materialien). Float das Gewebe auf der Oberfläche der Fixier-Lösung für einige Minuten (typischerweise der Zeit, die für die nächste Reihe von Augen Scheiben zu zerlegen). HINWEIS: Ist das nicht das erste Auge Disk in dieser Runde der Dissektion, completel seziert werdeny tauchen die vorherige Augen Disc in den Fix-Lösung. Wenn für besondere Färbeverfahren (zB reaktive Sauerstoffspezies, Lysosomen, etc.) muss dies vor der Fixierung durchgeführt werden. Um dies zu tun, vor der Fortsetzung mit dem entsprechenden Färbungsprotokoll speichern die seziert Augenscheiben in eiskaltem PBS. </li><li> Nach Präparation und Untertauchen aller Augenscheiben seziert, bewegen Sie den 3 gut Glasschale auf einer Wippe und Rock bei Raumtemperatur für 30 min. </li></ol> 2. Immunfärbung <ol><li> Entfernen Sie vorsichtig das Fix-Lösung und 0,5 ml PBS mit 0,3% Triton X-100 (0,3% PBT, siehe Materialien) und waschen Sie das Gewebe auf einer Wippe für 10 min. Wiederholen Sie diesen Schritt noch dreimal für insgesamt vier Waschvorgängen (je 10 min). HINWEIS: Die sorgfältige Entfernung der Lösung aus der Quelle unter einem Präpariermikroskop zu Gewebeschäden oder Verlust zu verhindern, durchgeführt werden. Die Lösung kann vorsichtig mit einer Pipette angesaugt werden; bevorzugen wir eine fein Tipped, Einmaltransferpipette, die die Öffnung abgeflacht ist, um die Wahrscheinlichkeit zu verringern Absaugen Gewebe hatte. </li><li> Am Ende des 4. waschen, entfernen Sie die Wasch- und 0,5 ml Blocklösung (siehe Materialien), um den Brunnen und Inkubation für 30 min. Hinweis: Dieser Zeitrahmen kann nach Ihren Bedürfnissen erweitert werden. </li><li> Am Ende der 30 Minuten Blockzeit, Pipette 10 ul Primärantikörperlösung (in Block-Lösung verdünnt) in die entsprechende Anzahl von Vertiefungen in einer Mikrotiterplatte. HINWEIS: Eine Vertiefung einer Mikrotiterplatte wird über 4 Paar Augenscheiben zum Puppenhülle angebracht oder 8 isolierte Paare von Augenscheiben mit Köpfchen befestigt zubringen. Die primären Antikörper in diesem Protokoll für die repräsentative Daten verwendet werden, sind Anti-Phosphotyrosin (1: 500 Verdünnung) und Anti-Cut (1: 200 Verdünnung). </li><li> Übertragen Sie die sezierten Gewebes in die Mikrotiterplatte Wells mit dem primären Antikörper-Lösung aus der 3 gut Glasschale gefüllt. </li><li> Ortder Mikrotiterplatte auf einem befeuchteten Papiertuch in einem leeren Pipettenspitze Box (oder andere ähnliche Behälter) und bei 4 ° C über Nacht (um eine Austrocknung zu verhindern). </li></ol> Tag 2 (4 h + Montagezeit) <ol start="6"><li> Übertragen Sie das Gewebe aus der Mikrotiterplatte auf ein neues 3 gut Glasschale mit 0,3% PBT (≈0.5 ml) und waschen Sie das Gewebe für 10 Minuten auf der Wippe. Wiederholen Sie diesen Schritt noch dreimal für insgesamt vier Waschvorgängen (je 10 min) </li><li> Entfernen Sie die letzten Wasch und 0,5 ml Block-Lösung zum Brunnen. Block für 30 min. Hinweis: Dieser Zeitrahmen kann nach Ihren Bedürfnissen erweitert werden. </li><li> Entfernen Sie die Block-Lösung und 0,5 ml des Sekundärantikörperlösung zum Brunnen (von fluoreszenzmarkierten entsprechend Ihrer primären Antikörper und Fluoreszenzwellenlänge, in Block-Lösung verdünnt Antikörper hergestellt) und zum Schutz der 3 gut Glasschale von Licht mit einem Deckel oder Aluminium Folie und Inkubation für mindestens 2 Stunden bei RaumTemperatur. HINWEIS: Alle nachfolgenden Schritte sollten in ähnlicher Weise von Lichtexposition geschützt werden. Diese Inkubation kann über Nacht bei 4 ° C erweitert werden, in einem Setup ähnlich wie der primäre Antikörper Inkubation, aber es kann auch in einem 3 gut Glasschale mit Labor-Film oder eine Mikrotiterplatte, wenn Antikörper Erhaltung gewünscht abgedeckt durchgeführt werden. </li><li> Am Ende der Antikörper-Inkubation, entfernen Sie die Sekundärantikörperlösung und 0,5 ml von 0,3% PBT zum Brunnen und Wäsche auf der Wippe für 10 min. Wiederholen Sie diesen Schritt für insgesamt 4 Wäschen (jeweils 10 Minuten). HINWEIS: Wenn DAPI-Färbung gewünscht wird, vor dem Start der ersten Wäsche, fügen 0,5 ul DAPI-Stammlösung (siehe Materialien) zu den 500 ul PBT, die für die erste Wäsche verwendet wird (1: 1000 Verdünnung). </li></ol> 3. Montage <ol><li> Am Ende der letzten Waschung montieren anten / Augenscheiben auf einem Objektträger mit 70% Glycerin oder PBS als Medium, um die br entfernenain und anderen unerwünschten Gewebe in einem Pool auf einer Seite der Folie. </li><li> Das Gewebe vorsichtig bewegen Sie mit hydrostatischen Druck zwischen den Zangen, oder sehr vorsichtig mit einem Zangenspitze, und richten Sie die Augenscheiben in einer Kolonne in Richtung der Mitte des Schlittens. </li><li> Entfernen Sie alle Fremd Glycerin oder PBS aus der ganzen Gewebe (mit Dochtwirkung eines Laborwischtuch oder die Zange arbeiten für diese). </li><li> Legen Sie eine kleine Menge (≈10 ul) Montagemedium entlang einer Seite des Auges Disc Spalte und setzen Sie ein Deckglas auf das Gewebe, um so die Montagemedium auf das Auge Scheiben verteilen. </li><li> Lassen Rutsche sit für 1 bis 2 min, damit das Gewebe / Montagemedium vollständig auszubreiten. </li><li> Wischen Sie zusätzliche Montagemedium weg von den Rändern des Deckglases und verschließen Sie diese mit klarem Nagellack. </li><li> Erfassen Fluoreszenzbilder der Augenscheiben mittels konfokaler Mikroskopie. </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...

Drosophila melanogaster: Die Felder der Entwicklungs- und Zellbiologie wurden stark von der Modellorganismus beeinflusst. Bei diesem Modell haben Studien des Auges Disc viel Wissen über Signalisierung, Zellbiologie und anderen Bereichen beigetragen. Die späten dritten Larvenstadium Auge Disc wurde ausgiebig untersucht und ist ein leistungsfähiges Modell zu nutzen, da es eine Momentaufnahme aus einer Reihe von Entwicklungsperioden, jede mit ihrer eigenen einzigartigen Signalmoleküle und Prozesse, wie der morphogenetischen Furche fortschreitet über das Auge Disc 8. Es besteht jedoch ein Bedarf für unser Verständnis von Entwicklungsprozessen weiter ausbauen in den Puppenentwicklungsphase. Zwar gab es auf der Puppenaugenscheibe 3-7 wurden Studien, unser Wissen nicht nähern die Breite der Arbeit, die auf dem dritten Larvenaugenscheibe durchgeführt worden ist. Dies liegt zum Teil an die größere Schwierigkeit der Zerlegung des Puppenaugen Disc. Daher ist eine Präsentation derrichtige Methode der Präparation konnte erheblich erweitern Forschung in diesem Bereich. Zwar gibt es Stufen innerhalb Puppenaugenscheibe Entwicklung, die leicht zerlegt werden, insbesondere um die Mitte des Puppenperiode, sind andere Zeiträume viel schwieriger zu sezieren. Das Protokoll stellt ein Verfahren zum Präparieren Puppenaugenscheiben, die universell für alle Puppenentwicklungszeitrahmen verwendet werden kann. Dieses Protokoll kann als eine Alternative zu einem anderen Protokoll 9, die eine einfachere und schnellere Methode zur Augenscheiben von den Zeitpunkten, zu sezieren midpupal zeigt verwendet werden. Dieses Protokoll wurde ursprünglich gefilmt und für die Ausbildung fortgeschrittene Studierende in der UCLA Undergraduate Research Consortium in Functional Genomics (URCFG) 10,11 in der Technik der Puppenaugen Dissektion entwickelt. Viele Studenten waren in der Lage, um dieses Video und Verfahren zu nutzen, um diese anspruchsvolle Technik zu erlernen.

<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.

Als Beispiel für die Verwendung dieses Protokolls, Ergebnisse darstellt midpupal (42 Std APF bei 25 ° C) Augenscheiben mit unterschiedlichen Antikörpern immungefärbt sind in Abbildung 2 Durch Verwendung eines gegen Phosphotyrosinreste gerichteten Antikörper präsentiert, kann die Membran der Zellen (Abbildung 2A). Dies kann verwendet werden, um die regelmäßige Anordnung der Ommatidien Zellen im Puppenauge identifizieren folgenden endgültigen Strukturierungsprozessen, die vor der midpupal Stufe a...

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.

Dissection und Montage<em&gt; Drosophila</em&gt; 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 ...

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

Research

JoVE Journal

Biology

Dissection and Mounting of Drosophila Pupal Eye Discs

12.1K Aufrufe.  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.

Serie: Dissection and Mounting of Drosophila Pupal Eye Discs

Dissection of Drosophila Ovaries

Dissection von Drosophila Ovarien

Forschung

Biologie

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.

Dissection der Imaginalscheiben vom 3. Instar Drosophila-Larven

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 Larven 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.

Dissection und Färbung von Drosophila Larven Ovarien

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 Puppenstadium Abdomen Immunhistochemie

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 Durch die Puppenhülle von<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.

Präparation und Immunfärbung von Imaginalscheiben aus<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-Kultur von Drosophila Pupal Hoden und Single männlichen Keim Online-Zysten: Dissection, Imaging und pharmakologische Behandlung

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.

Dissection und Flach Montage des Dreistachliger Stichling Branchiogene 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 ...

Methods for Staging Pupal Periods and Measurement of Wing Pigmentation of Drosophila guttifera

Diversified species of Drosophila (fruit fly) provide opportunities to study mechanisms of development and genetic changes responsible for evolutionary changes. In particular, the adult stage is a rich source of morphological traits for interspecific comparison, including wing pigmentation comparison. To study developmental differences among species, detailed observation and appropriate staging are required for precise comparison. Here we describe protocols for staging of pupal periods and quantification of wing pigmentation in a polka-dotted fruit fly, Drosophila guttifera. First, we describe the method for detailed morphological observation and definition of pupal stages based on morphologies. This method includes a technique for removing the puparium, which is the outer chitinous case of the pupa, to enable detailed observation of pupal morphologies. Second, we describe the method for measuring the duration of defined pupal stages. Finally, we describe the method for quantification of wing pigmentation based on image analysis using digital images and ImageJ software. With these methods, we can establish a solid basis for comparing developmental processes of adult traits during pupal stages.

Methods for Staging Pupal Periods and Measurement of Wing Pigmentation of Drosophila guttifera

Protocols for staging pupal periods and measurement of wing pigmentation of Drosophila guttifera are described. Staging and quantification of pigmentation provide a solid basis for studying developmental mechanisms of adult traits and enable interspecific comparison of trait development.

Methoden zur Inszenierung Pupal Perioden und Messung der Flügel Pigmentierung der Drosophila guttifera

Diversified species of Drosophila (fruit fly) provide opportunities to study mechanisms of development and genetic changes responsible for evolutionary ...