Name: dissection and staining of drosophila pupal ovaries
Uploaded: 2018-03-02T14:30:00.000+00:00
Duration: 7 min 35 s
Description: Watch this Scientific Journal Video about Dissection and Staining of Drosophila Pupal Ovaries at JoVE.com

Esta pesquisa foi apoiada pelo National Institutes of Health (RO1 GM079351 de D.K.). Agradecemos Dorothea Godt dela conselhos úteis sobre dissecações de ovário pupal, baseado no seu protocolo original. Agradecemos também a Amy Reilein por sua assistência e comentários sobre o manuscrito.

1. ovíparos
<ol><li>Combine-se aproximadamente dez do sexo masculino e 15 feminino adulto drosófila moscas do genótipo desejado em um frasco normal alimentos ricos em voar suplementados com levedura. Para evitar a superlotação do frasco, permitem que as fêmeas acasaladas para pôr ovos para não mais de 2 – 4 h14.</li>
	<li>Transferi os adultos do frasco para um frasco novo tocando o frasco abertura contra um frasco diferente com comida de mosca. Permitir que os ovos desenvolver-se em larvas em temperatura ambiente por 3 – 4 dias.</li>
</ol>2. seleccionar as larvas femininas
<ol><li>Usando um pincel fino úmido com cerdas macias, faça um movimento de rolamento com o pincel ao longo da parede do frasco para transferir as larvas de terceiro instar errantes do frasco para um copo bem cheio até a borda com 1x tampão fosfato salino (PBS). Lave os restos de comida fora as larvas por transferi-los para outra bem preenchido com PBS 1x.</li>
	<li>Separe as larvas masculinas e femininas, usando fórceps. Identifica as larvas masculinas por um par de testículos grandes, redondos e translúcidos embutido na gordura corporal no lado lateral do corpo aproximadamente dois-terços para baixo de sua extremidade anterior15. Femininas larvas são em média maior, menos translúcida que os machos e têm gônadas muito menores que são mais difíceis de detectar.</li>
	<li>Selecione contra larvas masculinas para coletar larvas femininas. Colete no mínimo dez fêmeas do poço. Coloque as larvas femininas em um separado bem preenchido com PBS 1x.</li>
	<li>Uso de fórceps para transferir as larvas femininas suavemente em um frasco novo de alimentos frescos voar suplementado com fermento.</li>
	<li>Coloque o frasco com as larvas femininos em um local escuro para facilitar pupariation16. Durante todo o dia, monitore as larvas para pupariation. Como cada larva imobiliza e se desenvolve em um prepupa, círculo o prepupa contra o frasco e gravar o tempo aproximado quando ele primeiro forma em um prepupa. Identifica a pré-pupa, a protrusão de espiráculos anteriores e sincronismo de casulo formação17. Permita a pré-pupa desenvolver a ponto de tempo desejado (medido em horas depois da formação do casulo, APF). Nota: Qualquer animal que se submetem a formação do casulo dentro de um intervalo de aproximadamente 10 h pode ser considerado um prepupa. O estágio de pupa começa aproximadamente 12 h após a formação do casulo depois que um interno sofrem metamorfose teve lugar.</li>
</ol>3. preparar pupas para anticorpos
<ol><li>
Forma uma pipeta de vidro fino para ser usado em etapas posteriores para limpar a gordura corporal pupal.
	<ol>
<li>Derreta a ponta de uma pipeta Pasteur de vidro sobre um bico de Bunsen. O vidro derrete, use fórceps para puxar a ponta horizontalmente longe do resto da pipeta para formar uma ponta mais fina.</li>
		<li>Após arrefecimento, quebre uma pequena porção da ponta da pipeta para formar uma abertura circular pura. Anexe um balão para o outro lado da pipeta.</li>
		<li>Carrega a pipeta com PBS 1x.</li>
	</ol>
</li>
	<li>Para colher as pupas, que são coladas na parede do frasco, aplica uma pequena gota de água ao longo da zona de contato entre a pupa e o frasco. Esperar 1 a 2 min para deixar a proteína cola dissolver antes de levantar suavemente a pupa da parede com um pincel fino úmido. Transferi a pupa para um copo bem cheia de 1X PBS. Escreve um único poço por pupa para evitar superlotação do poço.</li>
	<li>Enquanto segurando a extremidade posterior da pupa com um par de pinças, cuidadosamente rasgar a porção anterior do caso com outro par pupa até a cabeça da pupa é visível.</li>
	<li>Segure a ponta mais anterior da cabeça pupal com pinça e puxe delicadamente a pupa fora de seu caso de pupa. Nota: Uma porção do corpo a gordura pode derramar para fora durante este processo.</li>
	<li>Separar e descartar a metade anterior da pupa da metade posterior até que reste apenas o saco abdominal.</li>
	<li>
Extrai as células de gordura corporal da bolsa abdominal.
	<ol>
<li>Segure o saco abdominal contra o fundo do poço com pinça em uma mão segurando a pipeta de vidro fino repleta de 1X PBS na outra mão.</li>
		<li>Aponte a ponta da pipeta fina em direção a abertura do saco e lentamente Introduza com pipeta 1X PBS no abdómen para lavar as células de gordura corporal em torno dos ovários pupal. Nota: Os ovários são um par de estruturas pequenas, translúcidas, estriados e oblongas que deve permanecer dentro do saco abdominal.</li>
		<li>Lave que o corpo gordura células até pelo menos dois terços do corpo gordura desaparecer ou até que os ovários são visíveis perto da abertura do saco abdominal. É muito importante executar este passo devagar para não lavar os ovários fora do abdômen por acidente.</li>
	</ol>
</li>
	<li>Examine o poço para verificar se os ovários têm derramado durante a lavagem da célula de gordura corporal. Se os ovários não são visíveis no interior do poço, eles devem ter permanecido no abdômen. Se os ovários tem saído, coloque uma nova pupa no poço e repita este passo.</li>
	<li>Transferir o abdômen em uma câmara de lamela clara cheia de buffer de fixação (1X PBS, paraformaldeído 4%) e coloque a tampa na parte superior. Para garantir um número suficiente de ovários suportar a coloração e o processo de montagem, dissecar os ovários mais do que o necessário. Atenção: A reserva de fixação contém paraformaldeído, que é tóxico. Por favor use proteções adequadas, tais como luvas, óculos de segurança, etc.</li>
</ol>4. imuno-histoquímica
<ol><li>Durante todo o processo de coloração, use fórceps para empurre suavemente para baixo qualquer flutuantes ovários para o fundo do poço tampa vidro para garantir que os ovários estão completamente imersos em solução de anticorpo.</li>
	<li>Tiras de fita adesiva sobre a superfície plana de um Nutator e em seguida colocar a lamela septadas na superfície adesiva.</li>
	<li>Incube os ovários em tampão de fixação da etapa 3.7 para 15 min à temperatura ambiente.</li>
	<li>Lave os ovários três vezes em PBS 1x com 1% Triton X-100 para 5, 10 e 45 min (1h no total), respectivamente, para permitir a completa permeabilização.</li>
	<li>Bloquear os ovários no soro de cabra normal de 10% em PBS 1x com 0,5% Triton X-100 por 30 min.</li>
	<li>Incubar os ovários durante a noite em 600 µ l de anticorpo primário de escolha diluído na concentração apropriada em 1X PBS com 0,5% Triton X-100 a 4 ° C.</li>
	<li>Lave os ovários três vezes por 5 min em 1X PBS com 0,5% Triton X-100.</li>
	<li>Incube os ovários para 2h em 600 µ l de anticorpo secundário de escolha diluído na concentração apropriada em 1X PBS com 0,5% Triton X-100 à temperatura ambiente.</li>
	<li>Lave os ovários duas vezes por 5 min em 1X PBS com 0,5% Triton X-100 e uma vez por 5 min em 1X PBS.</li>
</ol>5. dissecando e ovários Pupal de montagem
<ol><li>Coloque uma pequena gota de 1X PBS numa lâmina de microscópio. Uso de fórceps para transferir o saco abdominal da lamela septadas para um copo bem cheio de 1X PBS. Escreve um único poço por pupa para evitar superlotação do poço.</li>
	<li>Separar o saco abdominal e qualquer restante gordura corporal com fórceps. Nota: Os ovários, que são um par de estruturas pequenas, translúcidas, estriados e oblongos, devem ser visíveis no interior do poço. Os ovários são muitas vezes fortemente cercados pela gordura corporal, por isso é importante se certificar toda gordura corporal é completamente rasgada distante.</li>
	<li>Transferi os ovários do poço à gota de 1X PBS no microscópio deslizar segurando o centro dos ovários entre as pontas da pinça. É muito importante usar um aperto firme sem apertar os ovários para não perder ou destruí-los durante a transferência. Adicione algumas gotas de 1X PBS para o slide quando a solução seca ao longo do tempo.</li>
	<li>Uma vez que todos os ovários foram dissecados e transferidos para a lâmina de microscópio, pipetar 40 µ l de meio de montagem sobre uma lamela de 22 x 22 mm e o lugar da lamela suavemente sobre os ovários. Deixe a slide secar durante a noite antes da imagem latente.</li>
</ol>

<ol>
	<li>Losick, V., Morris, L., Fox, D., Spradling, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+Stem+Cell+Niches:+A+Decade+of+Discovery+Suggests+a+United+View+of+Stem+Cell+Regulation.">Drosophila Stem Cell Niches: A Decade of Discovery Suggests a United View of Stem Cell Regulation.</a> Dev Cell. 21 (1), 159-171 (2011).</li><li>Kirilly, D., Xie, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+ovary:+an+active+stem+cell+community.">The Drosophila ovary: an active stem cell community.</a> Cell Res. 17 (1), 15-25 (2007).</li><li>Dansereau, D. A., Lasko, P. <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+Germline+Stem+Cells+in+Drosophila.">The Development of Germline Stem Cells in Drosophila.</a> Method Mol Biol. 450, 3-26 (2008).</li><li>Pearson, J., L&#243;pez-Onieva, L., Rojas-R&#237;os, P., Gonz&#225;les-Reyes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+Drosophila+stem+cell+biology.">Recent advances in Drosophila stem cell biology.</a> Int J Dev Biol. 53, 1329-1339 (2009).</li><li>Arwert, E. N., Hoste, E., Watt, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+stem+cells,+wound+healing+and+cancer.">Epithelial stem cells, wound healing and cancer.</a> Nat Rev Cancer. 12 (3), 170-180 (2012).</li><li>Barker, 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=Crypt+stem+cells+as+the+cells-of-origin+of+intestinal+cancer.">Crypt stem cells as the cells-of-origin of intestinal cancer.</a> Nature. 457 (7229), 608-611 (2009).</li><li>Daley, G. 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>

Os autores têm sem conflitos de interesse para declarar.

A etapa mais crítica e difícil do presente protocolo envolve a preparação de pupa ovários antes da fixação. Para garantir que os ovários, pequenos e enterrados por células de gordura corporal dentro do abdômen pupal, estão manchados suficientemente com anticorpos, é importante não só rasgar uma abertura grande no saco abdominal com fórceps, mas também extrair as células de gordura corporal que obstruem o ovários de anticorpos. Execução bem sucedida dessa etapa requer a aplicação de pressão sutil so...

Investigação em células estaminais usando Drosophila ovários expandiu-se, amplamente, desde a primeira documentação de uma célula-tronco nicho1,2,3,4. Acompanhar o desenvolvimento de ferramentas de genética linhagem rastreamento, ovário de Drosophila dissecções foram comumente usadas para estudar as linhagens de células-tronco e sinalização de vias que regulam a manutenção de células-tronco, proliferação e destino no nicho de células-tronco. Conhecimento destas vias de sinalização pode produzir insights sobre causas potenciais de cânceres que se originam de células-tronco aberrante atividade5,6,7. Recentemente também mostrou que as células-tronco somáticas no ovário drosófila , conhecidas como células-tronco do folículo (FSCs), fortemente se assemelham a mamíferos células-tronco intestinais em muitos aspectos de sua organização8. Por esta razão, Drosophila ovários são um sistema de modelo altamente útil para estudar o comportamento de células-tronco.
Enquanto os ovários de larvas e adultos oferecem pistas para o desenvolvimento precoce de células-tronco e células-tronco final organização no nicho, respectivamente, o ovário pupal é uma estrutura intermediária em que a linha germinativa e células somáticas reorganizar e estabelecer suas identidades 9 , 10. embora vários estudos examinaram os aspectos do desenvolvimento de tecido no ovário pupal11,10,12,13, perguntas permanecem sobre a diferenciação e organização espacial tipos de células dos ovários durante o desenvolvimento pupal. Em particular, a especificação de FSCs ocorre durante este período. Este protocolo descreve um método para dissecar e coloração pupal ovários em pontos de tempo desejado — uma técnica que pode ser usada em experimentos de curso do tempo que analisam o desenvolvimento do ovário pupal em detalhe de larval para a fase adulta.
Para explicar o tamanho pequeno, translucidez e inacessibilidade do ovário dentro do abdômen pupa pupa, este protocolo utiliza ferramentas como uma feito por pipeta Pasteur com ponta fina para remover o corpo de gordura abdominal tecido obstruindo o acesso de anticorpo para o ovários. Uma lamela clara, septada, usada durante o anticorpo que mancha oferece maior visibilidade das pupas e uma plataforma mais suave para balançar os ovários em um "Nutator". Baseado em um protocolo para dissecções de ovário larval de Maimon e Gilboa14, uma concentração relativamente alta de Triton X-100 foi empregada nas etapas iniciais do processo de coloração para maximizar o acesso de permeabilização e anticorpo de membrana celular para a células dos ovários.

<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&#039;s yeast, cornmeal, molasses, agar, EtOH, penicillin, methyl 4-hydrobenzoate, and propionic acid</td></tr><tr><td>Male and female &lt;em&gt;Drosophila&lt;/em&gt; 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

Ao contrário de ovários de Drosophila adultos, pupal ovários são relativamente difíceis de acessar e examinar devido a suas pequenas tamanho, translucidez e encerra dentro de um caso de pupa. O desafio de dissecação pupal ovários também reside na sua localização física dentro a pupa: os ovários estão rodeados por células de gordura corporal dentro do abdômen pupal, e essas células de gordura deve ser removidas para permitir a adequada anticorpos. Para superar estes desafios, este protocolo utiliza personalizadas pipetas Pasteur para extrair células de gordura corporal do abdome pupal. Além disso, uma lamela septada é usada no lugar de um tubo de microcentrifugadora durante o processo de coloração para melhorar a visibilidade das pupas. No entanto, apesar de estas e outras vantagens das ferramentas utilizadas no presente protocolo, execução bem sucedida destas técnicas ainda pode envolver vários dias de prática devido ao pequeno tamanho dos ovários pupal. As técnicas descritas neste protocolo poderiam ser aplicadas para experimentos de curso do tempo em que os ovários são analisados em vários estágios de desenvolvimento pupal.

Execução bem sucedida deste procedimento deve resultar na mancha do anticorpo claro que revela a estrutura e organização celular de um ovário pupal da drosófila . Imuno-histoquímica descrita no presente protocolo pode ser usado para identificar tipos de células, comumente manchados em ovários de larvas e adultos. As células do caule pupal derivado enxame células18 (esboçado por Fasciclin III em branco) são mostrados na Fig...

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

Dissection and Staining of Drosophila Pupal Ovaries

O ovário de Drosophila é um sistema excelente modelo para estudar o desenvolvimento de nicho de células-tronco. Embora métodos para dissecando larvas e adultos ovários foram publicados, dissecções de ovário pupal requerem diferentes técnicas que não tenham sido publicadas em detalhe. Aqui nós esboçamos um protocolo para dissecar, coloração e montagem ovários pupal.

Dissecção e coloração dos ovários Pupal drosófila

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

dissection-and-staining-of-drosophila-pupal-ovaries

Research

JoVE Journal

Developmental Biology

Dissection and Staining of Drosophila Pupal Ovaries

9.3K visualizações.  Columbia University. O ovário de Drosophila é um sistema excelente modelo para estudar o desenvolvimento de nicho de células-tronco. Embora métodos para dissecando larvas e adultos ovários foram publicados, dissecções de ovário pupal requerem diferentes técnicas que não tenham sido publicadas em detalhe. Aqui nós esboçamos um protocolo para dissecar, coloração e montagem ovários pupal.

Vídeo: Dissection and Staining of Drosophila Pupal Ovaries

Dissection of Drosophila Ovaries

Dissecção da Drosophila Ovários

Biologia

Upright Imaging of Drosophila Egg Chambers

Drosophila melanogaster oogenesis provides an ideal context for studying varied developmental processes since the ovary is relatively simple in architecture, is well-characterized, and is amenable to genetic analysis. Each egg chamber consists of germ-line cells surrounded by a single epithelial layer of somatic follicle cells. Subsets of follicle cells undergo differentiation during specific stages to become several different cell types. Standard techniques primarily allow for a lateral view of egg chambers, and therefore a limited view of follicle cell organization and identity. The upright imaging protocol describes a mounting technique that enables a novel, vertical view of egg chambers with a standard confocal microscope. Samples are first mounted between two layers of glycerin jelly in a lateral (horizontal) position on a glass microscope slide. The jelly with encased egg chambers is then cut into blocks, transferred to a coverslip, and flipped to position egg chambers upright. Mounted egg chambers can be imaged on either an upright or an inverted confocal microscope. This technique enables the study of follicle cell specification, organization, molecular markers, and egg development with new detail and from a new perspective.

Upright Imaging of Drosophila Egg Chambers

The upright imaging method described in this protocol allows for the detailed visualization of the poles of a developing Drosophila melanogaster egg. This end-on view provides a new perspective into the arrangements and morphologies of multiple cell types in the follicular epithelium.

Imagem Vertical de<em&gt; Drosophila</em&gt; Câmaras de ovo

Drosophila melanogaster oogenesis provides an ideal context for studying varied developmental processes since the ovary is relatively simple in ...

Biologia do Desenvolvimento

Visualizing the Effects of Oxidative Damage on Drosophila Egg Chambers using Live Imaging

Live imaging of Drosophila melanogaster ovaries has been instrumental in understanding a variety of basic cellular processes during development, including ribonucleoprotein particle movement, mRNA localization, organelle movement, and cytoskeletal dynamics. There are several methods for live imaging that have been developed. Due to the fact that each method involves dissecting individual ovarioles placed in media or halocarbon oil, cellular damage due to hypoxia and/or physical manipulation will inevitably occur over time. One downstream effect of hypoxia is to increase oxidative damage in the cells. The purpose of this protocol is to use live imaging to visualize the effects of oxidative damage on the localization and dynamics of subcellular structures in Drosophila ovaries after induction of controlled cellular damage. Here, we use hydrogen peroxide to induce cellular oxidative damage and give examples of the effects of such damage on two subcellular structures, mitochondria and Clu bliss particles. However, this method is applicable to any subcellular structure. The limitations are that hydrogen peroxide can only be added to aqueous media and would not work for imaging that uses halocarbon oil. The advantages are that hydrogen peroxide is readily available and inexpensive, acts quickly, its concentrations can be modulated, and oxidative damage is a good approximation of damage caused by hypoxia as well as general tissue damage due to manipulation.

The objective of this protocol is to use live imaging to visualize the effects of oxidative damage on the localization and dynamics of subcellular structures in Drosophila ovaries.

Visualizando os efeitos do dano oxidativo nas câmaras de ovos de Drosophila usando imagens ao vivo

Live imaging of Drosophila melanogaster ovaries has been instrumental in understanding a variety of basic cellular processes during development, including ...

Confocal and Super-Resolution Imaging of Polarized Intracellular Trafficking and Secretion of Basement Membrane Proteins During Drosophila Oogenesis

The basement membrane (BM) - a specialized sheet of extracellular matrix present at the basal side of epithelial cells - is critical for the establishment and maintenance of epithelial tissue morphology and organ morphogenesis. Moreover, the BM is essential for tissue modeling, serving as a signaling platform, and providing external forces to shape tissues and organs. Despite the many important roles that the BM plays during normal development and pathological conditions, the biological pathways controlling the intracellular trafficking of BM-containing vesicles and how basal secretion leads to the polarized deposition of BM proteins are poorly understood. The follicular epithelium of the Drosophila ovary is an excellent model system to study the basal deposition of BM membrane proteins, as it produces and secretes all major components of the BM. Confocal and super-resolution imaging combined with image processing in fixed tissues allows for the identification and characterization of cellular factors specifically involved in the intracellular trafficking and deposition of BM proteins. This article presents a detailed protocol for staining and imaging BM-containing vesicles and deposited BM using endogenously tagged proteins in the follicular epithelium of the Drosophila ovary. This protocol can be applied to address both qualitative and quantitative questions and it was developed to accommodate high-throughput screening, allowing for the rapid and efficient identification of factors involved in the polarized intracellular trafficking and secretion of vesicles during epithelial tissue development.

Confocal and Super-Resolution Imaging of Polarized Intracellular Trafficking and Secretion of Basement Membrane Proteins During Drosophila Oogenesis

The basement membrane is essential for tissue and organ morphogenesis during development. To better understand the mechanisms leading to proper placement of this structure, the protocol presented describes methods to visualize and characterize the intracellular trafficking and secretion of basement membrane proteins in epithelial cells using confocal and super-resolution microscopy.

Imagem confocal e super-resolução de tráfico intracelular polarizado e secreção de proteínas de membrana de porão durante Drosophila Oogenesis

The basement membrane (BM) - a specialized sheet of extracellular matrix present at the basal side of epithelial cells - is critical for the establishment ...

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.

Dissecção e coloração de Drosophila Larval Ovários

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 Imunohistoquímica Abdomen Pupal

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

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.

Dissecção e Imunocoloração do Imaginário discos de<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 ...

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Fluorescence microscopy is an indispensable tool for the direct assessment of mitochondrial structure and function in live cells and for studying the mitochondrial structure-function relationship, which is primarily modulated by the molecules governing fission and fusion events between mitochondria. This paper describes and demonstrates specific methods for studying mitochondrial structure and function in live as well as in fixed tissue in the model organism Drosophila melanogaster. The tissue of choice here is the Drosophila ovary, which can be isolated and made amenable for ex vivo live confocal microscopy. Furthermore, the paper describes how to genetically manipulate the mitochondrial fission protein, Drp1, in Drosophila ovaries to study the involvement of Drp1-driven mitochondrial fission in modulating the mitochondrial structure-function relationship. The broad use of such methods is demonstrated in already-published as well as in novel data. The described methods can be further extended towards understanding the direct impact of nutrients and/or growth factors on the mitochondrial properties ex vivo. Given that mitochondrial dysregulation underlies the etiology of various diseases, the described innovative methods developed in a genetically tractable model organism, Drosophila, are anticipated to contribute significantly to the understanding of the mechanistic details of the mitochondrial structure-function relationship and to the development of mitochondria-directed therapeutic strategies.

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Specific methods for studying mitochondrial structure and function in live and fixed Drosophila ovaries are described and demonstrated in this paper.

Estudar mitocondrial estrutura e função em<em&gt; Drosophila</em&gt; ovários

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial ...

Dissection and Lipid Droplet Staining of Oenocytes in Drosophila Larvae

Lipids are essential for animal development and physiological homeostasis. Dysregulation of lipid metabolism results in various developmental defects and diseases, such as obesity and fatty liver. Usually, lipids are stored in lipid droplets, which are the multifunctional lipid storage organelles in cells. Lipid droplets vary in size and number in different tissues and under different conditions. It has been reported that lipid droplets are tightly controlled through regulation of its biogenesis and degradation. In Drosophila melanogaster, the oenocyte is an important tissue for lipid metabolism and has been recently identified as a human liver analogue regarding lipid mobilization in response to stress. However, the mechanisms underlying the regulation of lipid droplet metabolism in oenocytes remain elusive. To solve this problem, it is of utmost importance to develop a reliable and sensitive method to directly visualize lipid droplet dynamic changes in oenocytes during development and under stressful conditions. Taking advantage of the lipophilic BODIPY 493/503, a lipid droplet-specific fluorescent dye, described here is a detailed protocol for the dissection and subsequent lipid droplet staining in the oenocytes of Drosophila larvae in response to starvation. This allows for qualitative analysis of lipid droplet dynamics under various conditions by confocal microscopy. Furthermore, this rapid and highly reproducible method can also be used in genetic screens for indentifying novel genetic factors involving lipid droplet metabolism in oenocytes and other tissues.

Dissection and Lipid Droplet Staining of Oenocytes in Drosophila Larvae

Presented here are detailed methods for the dissection and lipid droplet staining of oenocytes in Drosophila larvae using BODIPY 493/503, a lipid droplet-specific fluorescent dye.

Cotocação e gota lipídica manchando de enocitos nas larvas de Drosophila

Lipids are essential for animal development and physiological homeostasis. Dysregulation of lipid metabolism results in various developmental defects and ...

Dissecting, Fixing, and Visualizing the Drosophila Pupal Notum

The pupae of Drosophila melanogaster are immobile for several days during metamorphosis, during which they develop a new body with a thin transparent adult integument. Their immobility and transparency make them ideal for in vivo live imaging experiments. Many studies have focused on the dorsal epithelial monolayer of the pupal&#160;notum because of its accessibility and relatively large size. In addition to the studies of epithelial mechanics and development, the notum has been an ideal tissue to study wound healing. After an injury, the entire epithelial repair process can be captured by live imaging over 6-12 h. Despite the popularity of the notum for live imaging, very few published studies have utilized fixed notum samples. Fixation and staining are common approaches for nearly all other Drosophila tissues, taking advantage of the large repertoire of simple cellular stains and antibodies. However, the pupal notum is fragile and prone to curling and distortion after removal from the body, making it challenging to complement live imaging. This protocol offers a straightforward method for fixing and staining the pupal notum, both intact and after laser-wounding. With this technique, the ventral side of the pupa is glued down to a coverslip to immobilize the pupa, and the notum is carefully removed, fixed, and stained. The notum epithelium is mounted on a slide or between two coverslips to facilitate imaging from the tissue&#39;s dorsal or ventral side.

Dissecting, Fixing, and Visualizing the Drosophila Pupal Notum

The present protocol details the preparation and visualization of fixed tissue of the Drosophila pupal notum. It can be used for either intact or wounded tissue, and the original architecture of the tissue is preserved. The procedures for dissecting, fixing, and staining are all described in this article.

Dissecando, consertando e visualizando o Drosophila Pupal Notum

The pupae of Drosophila melanogaster are immobile for several days during metamorphosis, during which they develop a new body with a thin transparent ...

Dissecção e coloração dos ovários Pupal drosófila

Resumo

Explore mais vídeos

Capítulos neste vídeo