Name: dissection and staining of drosophila larval ovaries
Uploaded: 2011-05-13T12:00:00
Description: Watch this Scientific Journal Video about Dissection and Staining of Drosophila Larval Ovaries at JoVE.com

IM é apoiado pela Marie Curie conceder re-integração. Este trabalho foi financiado pelo Fundo de Ciência Israel não Grant. 1146-1108, pelo e Helen Kimmel Martin Institute for Stem Cell Research no Instituto Weizmann de Ciência ea Fundação Charitable Leir.

1. Postura de ovos <ol><li> Cinco dias antes da dissecação: permitir que as fêmeas põem ovos acoplado a por 2-4 horas em alimentos frescos suplementados com levedura. Para obter larvas sincronizados e bem desenvolvidas, é importante não ter superlotadas cultivadas (cerca de 30 ovos / frasco 25mm). Tipicamente, 7-16 fêmeas são usados ​​por frasco, dependendo de quão bem eles estabelecem. </li></ol> 2. Seleção de larvas <ol><li> Prepare um prato de vidro de 9 dissecar bem cheio c...

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=decapentaplegic+is+essential+for+the+maintenance+and+division+of+germline+stem+cells+in+the+Drosophila+ovary.">decapentaplegic is essential for the maintenance and division of germline stem cells in the Drosophila ovary.</a> Cell. 94, 251-260 (1998).</li><li>Xie, T., Spradling, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+niche+maintaining+germ+line+stem+cells+in+the+Drosophila+ovary.">A niche maintaining germ line stem cells in the Drosophila ovary.</a> Science. , 290-328 (2000).</li><li>Guo, Z., Wang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+glypican+Dally+is+required+in+the+niche+for+the+maintenance+of+germline+stem+cells+and+short-range+BMP+signaling+in+the+Drosophila+ovary.">The glypican Dally is required in the niche for the maintenance of germline stem cells and short-range BMP signaling in the Drosophila ovary.</a> Development. 136, 3627-3635 (2009).</li><li>Wang, X., Harris, R. E., Bayston, L. J., Ashe, H. 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Este vídeo demonstra um isolamento e protocolo de coloração da final do terceiro instar larval ovários. Para executar este protocolo rotineiramente e de forma confiável, a atenção deve ser dada aos seguintes pontos: <ol><li> Para as larvas sincronizados e bem desenvolvida, a superlotação deve ser evitada. </li><li> Para evitar a perda dos ovários pequenos translúcido, certifique-se de dissecar a gordura corporal intacta. Isso também irá ajudar a localizar os ovários na fase de montagem, particularmente...

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		<th>Comment</th>
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<td>NaCl</td>
<td>&nbsp;</td>
<td>J.T.Baker</td>
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<td>Kcl</td>
<td>&nbsp;</td>
<td>Merck</td>
<td>&nbsp;</td>
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<td>CaCl2</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>&nbsp;</td>
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<td>MgCl2</td>
<td>&nbsp;</td>
<td>Merck</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Sucrose</td>
<td>&nbsp;</td>
<td>J.T.Baker</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Hepes</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>PBS</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Triton X-100</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Albumin Bovine Fraction V</td>
<td>&nbsp;</td>
<td>MP Biomedicals</td>
<td>160069</td>
<td>&nbsp;</td>
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<td>Dumont biology tweezers 5 dumstar polished</td>
<td>&nbsp;</td>
<td>FST Fine Science Tools</td>
<td>11295-10</td>
<td>&nbsp;</td>
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<td>Nickel plated pin holder</td>
<td>&nbsp;</td>
<td>FST Fine Science Tools</td>
<td>26018-17</td>
<td>&nbsp;</td>
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<td>s.s minutien pins 0.1mm diam, 10mm long</td>
<td>&nbsp;</td>
<td>FST Fine Science Tools</td>
<td>26002-10</td>
<td>&nbsp;</td>
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<td>9 well plates 85X100 mm, 22mm o.d.x7mm deep</td>
<td>&nbsp;</td>
<td>CORNING</td>
<td>7220-85</td>
<td>&nbsp;</td>
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<td>Stereo Microscope MZ 16.5 with a standard transmitted light base TL ST</td>
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<td>Menzel Glaser GmbH</td>
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<td>Corning</td>
<td>2940-223</td>
<td>&nbsp;</td>
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<td>Mounting media</td>
<td>&nbsp;</td>
<td>Vectashield</td>
<td>H-1200</td>
<td>&nbsp;</td>
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<td>Cell strainer</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>FAL352350</td>
<td>&nbsp;</td>
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<td>1B1 antibody</td>
<td>&nbsp;</td>
<td>Developmental Studies Hybridoma Bank</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Anti-Traffic Jam</td>
<td>&nbsp;</td>
<td>Laboratory of Dr. Dorothea Godt</td>
<td>&nbsp;</td>
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<td>Anti-Vasa</td>
<td>&nbsp;</td>
<td>Laboratory of Dr. Ruth Lehmann</td>
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<td>Anti &beta;-Galactosidase</td>
<td>&nbsp;</td>
<td>Cappel</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Secondary Antibodies</td>
<td>&nbsp;</td>
<td>Jackson ImmunoResearch</td>
<td>&nbsp;</td>
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dissection and staining of drosophila larval ovaries

Muitos órgãos depende de células-tronco para o seu desenvolvimento durante a embriogênese e para a manutenção ou reparação durante a vida adulta. Compreensão de como as células-tronco forma, e como eles interagem com seu ambiente é crucial para a compreensão da homeostase, desenvolvimento e doença. O ovário da mosca da fruta Drosophila melanogaster tem servido como um modelo influente para a interação de células-tronco germinativas (GSCS) com suas células somáticas de apoio (nicho) 1, 2. O local conhecido do nicho e as GSCS, aliada à capacidade de manipular geneticamente eles, tem permitido aos pesquisadores elucidar uma série de interações entre células-tronco e seus nichos 3-12. Apesar da riqueza de informações sobre mecanismos de controle de manutenção GSC e diferenciação, relativamente pouco se sabe sobre como GSCS e seus nichos de forma somática durante o desenvolvimento. Cerca de 18 nichos somáticas, cujos componentes incluem celulares filamento terminal e as células da tampa (Figura 1), formam-se durante o terceiro instar larval 13-17. GSCS originam de células germinativas primordiais (PGCs). PGCs proliferam no início fases de larva, mas após a formação do nicho um subgrupo de PGCs torna-se GSCS 7, 16, 18, ​​19. Juntas, as células somáticas e de nicho a GSCS fazer uma unidade funcional que produz ovos durante toda a vida do organismo. Muitas perguntas sobre a formação da unidade GSC permanecem sem resposta. Processos tais como a coordenação entre as células precursoras de nichos e de precursores de células-tronco, ou a geração de assimetria dentro PGCs como eles se tornam GSCS, pode ser melhor estudados na larva. No entanto, um estudo metódico do desenvolvimento larval ovário é fisicamente desafiador. Primeira, os ovários das larvas são pequenas. Mesmo em final de estágios larvais são apenas 100μm de diâmetro. Além disso, os ovários são transparentes e estão embutidos em um corpo de gordura branca. Aqui nós descrevemos um protocolo passo a passo para isolar ovários de final do terceiro instar as larvas Drosophila (LL3), seguido por coloração com anticorpos fluorescentes. Nós oferecemos algumas soluções técnicas para problemas como a localização dos ovários, coloração e lavagem de tecidos que não afundam, e certificando-se que os anticorpos penetrar no tecido. Este protocolo pode ser aplicado a fases anteriores larval e testículos larval também.

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

Dissection and Staining of Drosophila Larval Ovaries

Como nichos e células-tronco se formam durante o desenvolvimento é uma questão importante com implicações práticas. No<em&gt; Drosophila</em&gt; Ovário, células-tronco germinativas e somáticas nichos sua forma durante o desenvolvimento larval. Este vídeo demonstra como dissecar, mancha e montar gônadas femininas do final de terceiro ínstar (LL3)<em&gt; Drosophila</emLarvas&gt;.

Dissecção e coloração de Drosophila Larval Ovários

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Dissection and Staining of Drosophila Larval Ovaries

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