Name: dissection and staining of drosophila pupal ovaries
Uploaded: 2018-03-02T14:30:00
Description: Watch this Scientific Journal Video about Dissection and Staining of Drosophila Pupal Ovaries at JoVE.com

Denne forskning blev støttet af National Institutes of Health (RO1 GM079351 til D.K.). Vi takker Dorothea Godt for hendes nyttige råd om puppe æggestokken dissektioner baseret på hendes oprindelige protokol. Vi takker også Amy Reilein for hendes hjælp og kommentarer til manuskriptet.

1. EggLaying
<ol><li>Kombinere cirka ti mand og femten kvindelige voksne Drosophila fluer af den ønskede genotype i et hætteglas med normal rige flyve mad suppleret med gær. For at undgå overbefolkning hætteglasset, tillade skytterne hunner til at lægge æg længere end 2-4 h14.</li>
	<li>Overføre voksne fra hætteglasset ind i en ny hætteglas ved at trykke på hætteglasset åbning mod en forskellige hætteglas med flyve mad. Tillad æg til at udvikle sig til larver ved stuetem...

<ol>
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Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+myeloid+leukemia:+proving+ground+for+cancer+stem+cells.">Chronic myeloid leukemia: proving ground for cancer stem cells.</a> Cell. 119 (3), 314-316 (2004).</li><li>Reilein, A., 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=Alternative+direct+stem+cell+derivatives+defined+by+stem+cell+location+and+graded+Wnt+signaling.">Alternative direct stem cell derivatives defined by stem cell location and graded Wnt signaling.</a> Nat Cell Biol. 19 (5), 433-444 (2017).</li><li>Eliazer, S., Buszczack, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Finding+a+niche:+studies+from+the+Drosophila+ovary.">Finding a niche: studies from the Drosophila ovary.</a> Stem Cell Res Ther. 2 (6), 45 (2011).</li><li>Godt, D., Laski, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+cell+rearrangement+and+cell+recruitment+in+Drosophila+ovary+morphogenesis+and+the+requirement+of+bric+&#224;+brac.">Mechanisms of cell rearrangement and cell recruitment in Drosophila ovary morphogenesis and the requirement of bric &#224; brac.</a> Development. 121 (1), 173-187 (1995).</li><li>King, R. C., Aggarwal, S. K., Aggarwal, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+the+female+Drosophila+reproductive+system.">The development of the female Drosophila reproductive system.</a> J Morphol. 124 (2), 143-166 (1968).</li><li>Vlachos, S., Jangam, S., Conder, R., Chou, M., Nystul, T., Harden, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Pak-regulated+cell+intercalation+event+leading+to+a+novel+radial+cell+polarity+is+involved+in+positioning+of+the+follicle+stem+cell+niche+in+the+Drosophila+ovary.">A Pak-regulated cell intercalation event leading to a novel radial cell polarity is involved in positioning of the follicle stem cell niche in the Drosophila ovary.</a> Development. 142 (1), 82-91 (2015).</li><li>Irizarry, J., Stathopoulos, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FGF+signaling+supports+Drosophila+fertility+by+regulating+development+of+ovarian+muscle+tissues.">FGF signaling supports Drosophila fertility by regulating development of ovarian muscle tissues.</a> Dev Biol. 404 (1), 1-13 (2015).</li><li>Maimon, I., Gilboa, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+and+Staining+of+Drosophila+Larval+Ovaries.">Dissection and Staining of Drosophila Larval Ovaries.</a> J Vis Exp. (51), e2537 (2011).</li><li>Kerkis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Growth+of+the+Gonads+in+DROSOPHILA+MELANOGASTER.">The Growth of the Gonads in DROSOPHILA MELANOGASTER.</a> Genetics. 16 (3), 212-224 (1931).</li><li>Yamanaka, N., 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=Neuroendocrine+Control+of+Drosophila+Larval+Light+Preference.">Neuroendocrine Control of Drosophila Larval Light Preference.</a> Science. 341 (6150), 1113-1116 (2013).</li><li>Bainbridge, S., Bownes, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staging+the+metamorphosis+of+Drosophila+melanogaster.">Staging the metamorphosis of Drosophila melanogaster.</a> Development. 66, 57-80 (1981).</li><li>Courdec, 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=The+bric+&#224;+brac+locus+consists+of+two+paralagous+genes+encoding+BTB/POZ+domain+proteins+and+acts+as+a+homeotic+and+morphogenetic+regulator+of+imaginal+development+in+Drosophila.">The bric &#224; brac locus consists of two paralagous genes encoding BTB/POZ domain proteins and acts as a homeotic and morphogenetic regulator of imaginal development in Drosophila.</a> Development. 129, 2419-2433 (2002).</li><li>Arrese, E. L., Soulages, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=INSECT+FAT+BODY:+ENERGY,+METABOLISM,+AND+REGULATION.">INSECT FAT BODY: ENERGY, METABOLISM, AND REGULATION.</a> Annu Rev Entomol. 55, 207-225 (2011).</li><li>Zhang, Y., Xi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fat+Body+Development+and+its+Function+in+Energy+Storage+and+Nutrient+Sensing+in+Drosophila+melanogaster.">Fat Body Development and its Function in Energy Storage and Nutrient Sensing in Drosophila melanogaster.</a> J Tissue Sci Eng. 6 (1), (2014).</li><li>Wong, L. C., Schedl, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+Drosophila+Ovaries.">Dissection of Drosophila Ovaries.</a> J Vis Exp. (1), e52 (2006).</li></ol>

Det mest vigtige og vanskelige skridt af denne protokol omfatter forberedelse af puppe æggestokke før fiksering. For at sikre, at æggestokkene, små og begravet af fedt kroppens celler inde i puppe maven, farves tilstrækkeligt med antistoffer, er det vigtigt at ikke kun rive en stor åbning i den abdominal sæk med pincet, men også ekstrakt fedt kroppens celler, der hæmmer den æggestokkene fra antistoffer. Vellykket gennemførelse af dette trin kræver anvendelsen af subtil pres på Pasteur afpipetteres pære mens...

Forskning i stamceller ved hjælp af Drosophila æggestokke har meget udvidet siden den første dokumentation af en stamcelle niche1,2,3,4. Efter udviklingen af afstamning sporingen genetiske værktøjer, Drosophila æggestokken dissektioner har været almindeligt anvendt til at undersøge stamceller lineages og signalering veje, der regulerer stamcelle vedligeholdelse, spredning og skæbne i stamceller niche. Kendskab til disse signaling veje kan give indsigt i potentielle årsager til kræft, der stammer fra afvigende stamcelle aktivitet5,6,7. Det har også for nylig blevet vist, at somatiske stamceller i Drosophila æggestokken, kendt som follikel stamceller (FSCs), stærkt ligner mammale tarm stamceller i mange aspekter af deres organisation8. Af denne grund er Drosophila æggestokkene en meget nyttig modelsystem til at undersøge stamceller adfærd.
Mens larve og voksen æggestokkene tilbyder spor til tidlig udvikling af stamceller og sidste stamcelle organisation i niche, henholdsvis, er puppe æggestokken en mellemliggende struktur, hvor kønscelleoverførsel og somatiske celler omorganisere og etablere deres identiteter 9 , 10. selvom adskillige studier har undersøgt aspekter af væv udviklingen i puppe æggestokken10,11,12,13, stadig spørgsmål om differentiering og rumlige organisation af æggestokkene celletyper under puppe udvikling. Navnlig, opstår specifikation af FSCs i denne periode. Denne protokol beskriver en metode til at dissekere og farvning puppe æggestokkene på ønskede tidspunkter – en teknik, der kan bruges i gang kursus eksperimenter, at analysere puppe æggestokken udvikling i detaljer fra den larve til det voksne Stadium.
For at tage højde for den lille størrelse, translucence og utilgængelighed i puppe æggestokkene inden for puppe maven, denne protokol benytter værktøjer såsom en custom-made tynd spids Pasteur pipette til at fjerne abdominal fedt kropsvæv blokerer antistof adgang til den æggestokkene. En klar, chambered daekglas anvendes under antistof farvning tilbyder større synlighed af pupper og en blidere platform for vuggende æggestokkene på en "Nutator." Baseret på en protokol for larver æggestokken dissektioner af Maimon og Gilboas14, en relativt høj koncentration af Triton X-100 har været ansat i de indledende trin i proceduren farvning at maksimere cellemembranen permeabilization og antistof adgang til den æggestokkene celler.

<table>
	<tbody>
		<tr>
			<td>Dumont #5 Forceps, biology</td>
			<td>Fine Scientific Tools</td>
			<td>11252-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc Lab-Tek Chambered Coverglass</td>
			<td>Thermo Fisher Scientific</td>
			<td>155383</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection microscope</td>
			<td>Nikon</td>
			<td>SMZ-10A</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal Microscope</td>
			<td>Carl Zeiss</td>
			<td>LSM 700</td>
			<td></td>
		</tr>
		<tr>
			<td>Analysis software</td>
			<td>Carl Zeiss</td>
			<td>Zen</td>
			<td></td>
		</tr>
		<tr>
			<td>9 Depression Glass Spot Plates</td>
			<td>Pyrex</td>
			<td>7220-85</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipet</td>
			<td>Fisher Scientific</td>
			<td>13-678-6B</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipet bulb</td>
			<td>Various vendors</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bunsen burner</td>
			<td>Various vendors</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherfinest Premium Frosted Microscope Slides</td>
			<td>Thermo Fisher Scientific</td>
			<td>12-544-2</td>
			<td></td>
		</tr>
		<tr>
			<td>22 x 22 mm glass coverslips No 1</td>
			<td>VWR</td>
			<td>48366-067</td>
			<td></td>
		</tr>
		<tr>
			<td>Dapi Fluoromount-G</td>
			<td>SouthernBiotech</td>
			<td>0100-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Double-sided tape</td>
			<td>Scotch</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nutator</td>
			<td>Clay Adams</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fine brush #0, #3-#5</td>
			<td>Various vendors</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gilson Pipetman Starter Kit</td>
			<td>Thomas Scientific</td>
			<td>F167300</td>
			<td>Contains p20, p200, p1000 pipettors</td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td>Dilute to 4% paraformaldehyde in 1x PBS</td>
		</tr>
		<tr>
			<td>Triton</td>
			<td>Sigma-Aldrich</td>
			<td>9002-93-1</td>
			<td></td>
		</tr>
		<tr>
			<td>10x PBS</td>
			<td>Ambion</td>
			<td>AM9624</td>
			<td>Dilute to 1x PBS</td>
		</tr>
		<tr>
			<td>Normal Goat Serum</td>
			<td>Jackson ImmunoResearch</td>
			<td>5000121</td>
			<td>Dilute to 10% normal goat serum in PBST with 0.5% Triton concentration</td>
		</tr>
		<tr>
			<td>Primary antibodies (in protocol: 7G10 anti-Fasciclin III diluted 1:250, rabbit anti-phosphohistone H3 diluted 1:1000)</td>
			<td>Various vendors (in protocol: Developmental Studies Hybridoma Bank, Millipore)</td>
			<td></td>
			<td>Dilute in PBST with 0.5% Triton concentration</td>
		</tr>
		<tr>
			<td>Secondary antibodies (in protocol: Alexa-546, FITC-conjugated anti-rabbit serum)</td>
			<td>Various vendors (in protocol: Molecular Probes, Jackson ImmunoResearch Laboratories, Inc.)</td>
			<td></td>
			<td>Dilute in PBST with 0.5% Triton concentration</td>
		</tr>
		<tr>
			<td>Fly vials</td>
			<td>Denville Scientific</td>
			<td>V9406</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton Balls, For Wide Vials</td>
			<td>Genesee Scientific</td>
			<td>51-102W</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast, Bakers Dried Active</td>
			<td>MP Biomedicals</td>
			<td>101400</td>
			<td></td>
		</tr>
		<tr>
			<td>Fly food</td>
			<td>Produced in laboratory</td>
			<td></td>
			<td>Mixture of water, brewer&#39;s yeast, cornmeal, molasses, agar, EtOH, penicillin, methyl 4-hydrobenzoate, and propionic acid</td>
		</tr>
		<tr>
			<td>Male and female Drosophila flies (genotype used in protocol: yw; P[Fz3-RFP, w+]/TM2)</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

dissection and staining of drosophila pupal ovaries

I modsætning til voksne Drosophila æggestokke, puppe æggestokkene er relativt vanskeligt at få adgang til og undersøge på grund af deres lille størrelse, translucence og omslutter inden en puppe sagen. Udfordringen at dissekere puppe æggestokkene ligger også i deres fysiske placering i puppe: æggestokkene er omgivet af fedt kroppens celler inde i puppe maven, og disse fedtceller skal fjernes for at give mulighed for korrekt antistoffarvning. For at overvinde disse udfordringer, udnytter denne protokol tilpassede Pasteur pipets hen til uddrag fedt kroppens celler fra puppe maven. Desuden, en chambered daekglas er brugt i stedet for et microcentrifuge rør under farvning processen for at forbedre synligheden af pupper. Men trods disse og andre fordele af de værktøjer, der anvendes i denne protokol, vellykket gennemførelse af disse teknikker kan stadig indebære flere dage i praksis på grund af den lille størrelse af puppe æggestokke. De teknikker, der er skitseret i denne protokol kunne anvendes til tid kursus eksperimenter, hvor æggestokkene er analyseret i forskellige faser af pupal udvikling.

Vellykket gennemførelse af denne procedure bør resultere i klare antistoffarvning, der afslører den struktur og cellulære organisation af en Drosophila puppe æggestokken. Immunhistokemi skitseret i denne protokol kan bruges til at identificere celletyper almindeligt farves i larve og voksen æggestokke. Celler puppe stængelens afledt af swarm celler18 (beskrevet af Fasciclin III i hvid) er vist i figur 3. Ud over at frem...

Watch this Scientific Journal Video about Dissection and Staining of Drosophila Pupal Ovaries at JoVE.com

Dissection and Staining of Drosophila Pupal Ovaries

Drosophila æggestokken er en fremragende modelsystem til at undersøge stamceller niche udvikling. Om metoder til dissekere larve og voksen æggestokke er blevet offentliggjort, kræver puppe æggestokken dissektioner forskellige teknikker, der ikke er blevet offentliggjort i detaljer. Her skitsere vi en protokol til dissekere, farvning og montering puppe æggestokke.

Dissektion og farvning af Drosophila puppe æggestokke

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a ...

Research

JoVE Journal

Developmental Biology

Dissection and Staining of Drosophila Pupal Ovaries

Live-imaging of the Drosophila Pupal Eye

Live-imaging of the Drosophila Pupal Eye

Levende-billeddannelse af<em&gt; Drosophila</em&gt; Puppe Eye

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

Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles

Kultur og Co-kultur Mouse æggestokke og ovariefollikler

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together ...

Dissection and Culture of Mouse Embryonic Kidney

Dissektion og kultur af mus embryonisk nyre

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney ...

A Rapid and Efficient Method to Dissect Pupal Wings of Drosophila Suitable for Immunodetections or PCR Assays

A Rapid and Efficient Method to Dissect Pupal Wings of Drosophila Suitable for Immunodetections or PCR Assays

En hurtig og effektiv metode til at dissekere puppe vinger af Drosophila velegnet til Immunodetections eller PCR Assays

Wing development in Drosophila melanogaster is an ideal model for studying morphogenesis at tissue level. These appendages develop from a group of cells ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Dissektion og immunfluorescent farvning af champignon krop og fotoreceptor neuroner i voksen Drosophila melanogaster hjerner

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Whole Mount Immunofluorescence and Follicle Quantification of Cultured Mouse Ovaries

Hele Mount immunofluorescens og follikel kvantificering af kulturperler mus æggestokke

Research in the field of mammalian reproductive biology often involves evaluating the overall health of ovaries and testes. Specifically, in females, ...

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

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

Dissektion af Drosophila puppe nethinden for immunhistokemi, vestlige analyse og RNA isolering

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

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

Optøning, dyrkning og Cryopreserving Drosophila cellelinjer

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the ...

Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages

Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages

Billeddannelse og analyse af vævsorientering og vækstdynamik i udvikling af Drosophila Epithelia under pupalfaser

Within multicellular organisms, mature tissues and organs display high degrees of order in the spatial arrangements of their constituent cells. A ...

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

Præparat af Drosophila Larve og Pupal Testikler til analyse af celledeling i Levende, Intakt væv

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with ...