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 com meio de Ringer (NaCl 128mm, 2mM KCl, CaCl 2 1,8 mm, 4mm MgCl 2, 35.5mM sacarose, pH Hepes 5mM 6.9). </li><li> Prepare filtros célula em uma placa de seis poços contendo meio de Ringer e colocá-lo no gelo. Alternativamente, usamos moldes feitos especialmente equipado com uma tela de náilon. </li><li> Escolha larvas cronometrado de paredes finas frasco utilizando uma pinça biológicos (fórceps) e colocá-los no prato contendo o toque de dissecação. </li><li> Transferência de uma larva fêmea para um poço limpo. Podemos distinguir fêmeas de machos por suas gônadas. Testículos são facilmente identificados como grandes formas ovais claro embutido no terço posterior da gordura corporal. Ovários femininos, localizada na mesma parte da gordura corporal, pode ser identificado como um muito menor, claro, esferas rodada. </li></ol> 3. Dissecção da larva <ol><li> Segure a larva para baixo apenas posterior ao cérebro com uma pinça e retire a cabeça com um segundo par de fórceps. </li><li> Coloque a parte restante posterior no lado dorsal, com a traquéia virada para baixo. </li><li> Segure a larva pelos espiráculos posterior com um par de fórceps e empurrá-lo para dentro lentamente enquanto o outro par está deslizando a cutícula posteriormente até cerca de metade da gordura corporal larval emerge. </li><li> Segure firmemente a extremidade posterior e usar o outro par de pinças para prender vagamente a cutícula. Cuidado e lentamente afastar o fim posterior de modo que a cutícula eo slide intestino ligado através da abertura. No final deste processo, a gordura corporal deve ser completamente separado do intestino e da cutícula. Desligue o intestino da parte anterior do corpo de gordura. Para fazer a coloração e montagem fácil, é importante que a gordura corporal permanecerá intacto. </li><li> Wet uma pipeta pastagens cuidadosamente com média de Ringer, de preferência a partir de um poço que contém larvas dissecadas. Isso ajuda a cobrir a pipeta e impede que a gordura corporal a partir aderindo a ela. Use a pipeta de pastagem para transferir o corpo gordo em meio ao gelo Ringer frio no coador de celular. </li></ol> 4. Fixação e coloração Todas as etapas são realizadas em temperatura ambiente, exceto para a incubação com o anticorpo em primeiro lugar, a 4 ° C. <ol><li> Incubar a gordura corporal em formol 5% em meio Ringer. por 20 minutos com agitação suave. </li><li> Lavar por 5 minutos com PBT de 1% (1% Triton x-100 em PBS). Repita este passo para 10 minutos e uma terceira vez por 45 minutos. 1% Triton X-100 é necessária para perfurar o ovário larval e permitir que os anticorpos para penetrá-la. </li><li> Bloco com PBTB 0,3% (0,3% Triton x-100 e BSA 1% em PBS) por 1 hora com agitação suave </li><li> Incube com o anticorpo desejado 1 ª diluída em PBTB 0,3% durante a noite a 4 ° C com agitação suave. Esta etapa é normalmente realizada em 0,2 ml em tubos de um rolo. </li><li> Transferência de gordura corporal de volta para filtros celular. </li><li> Lavar 3 vezes, 30 minutos cada, com PBTB 0,3% com agitação suave. </li><li> Bloco com PBTB 0,3% suplementada com 5% de soro normal de burro por 1 hora com agitação suave. </li><li> Incube com um anticorpo secundário apropriado diluído (de acordo com a especificação do fabricante) na solução de bloqueio (0,3% PBTB suplementado com soro burro de 5% em PBT 0,3%) por 2 horas com agitação suave. Se o anticorpo é fluorescente, incubar no escuro a partir deste ponto em diante. Esta etapa é normalmente realizada em 0,2 ml em tubos de um rolo. </li><li> Lavar 3 vezes durante 30 minutos cada, com PBT de 0,3% com agitação suave. </li></ol> 5. Montagem <ol><li> Transferir a gordura corporal para um tubo eppendorf limpo. Com muito cuidado remova todos os líquidos longe e cobrir imediatamente com a mídia Vectashield montagem. Nós usamos rotineiramente vectashield uma vez que não endurecer enquanto os ovários estão sendo separada do corpo de gordura. Amostras podem ser armazenadas em meios de montagem a 4 ° C por até uma semana. </li><li> Corte a extremidade de uma ponta de pipeta e usá-lo para transferir cuidadosamente a gordura corporal, com volume mínimo de meios de montagem (cerca de 30 mL de uma tampa de deslizamento, 30 milímetros) em uma lâmina de microscópio. </li><li> Use dois retentores de pinos de níquel chapeados, segurando 0,1 mm de diâmetro pinos para difundir a gordura corporal. Os ovários estão localizados no terço posterior do corpo de gordura. A gordura corporal que os rodeia é geralmente em forma de &quot;flor&quot;. Isso ajuda a identificar a localização dos ovários. Dissecar as &quot;flores&quot; do resto da gordura corporal e descartar o último. </li><li> Remover o surro de gordura corporalINANCIAMENTO da gônada cruzando os dois pinos com cuidado em torno dela. Coloque o gônadas isoladas no slide longe do resíduo de gordura corporal. </li><li> Cubra com uma lamínula e vedação com unha polonês. </li><li> Visualize diretamente usando um microscópio confocal. Amostras podem ser armazenadas a 4 ° C por até três semanas. </li></ol> 6. Resultados representante Temos utilizado o protocolo acima de seguir o estabelecimento de linhagens celulares diversas dentro do ovário somáticas, incluindo GSCS e seus nichos de somática. Para isso, use anticorpos específicos e marcadores para distinguir entre os diferentes tipos de células na gônada em desenvolvimento. Aqui nós mostramos um exemplo de dois ovários LL3 corados com diferentes combinações de anticorpos. Figura 1A destaques filamento terminal e as células da tampa (verde), que juntos formam as células somáticas do nicho. Figura 1B, mostra as células Misturadas (ICs, magenta), que contactam directamente as células germinativas (azul). <img src="/files/ftp_upload/2537/2537fig1.jpg" alt="Figura 1" /> Figura 1. LL3 ovários. (A) anticorpo monoclonal 1B1 (magenta) define células somáticas e manchas do fusoma, uma organela intracelular dentro PGCs (setas). A armadilha enhancer hh-lacZ (anti β-galactosidase, verde) é expressa em filamentos terminais. Na base das células somáticas de filamentos de tampa (pontas de seta) podem ser observados. (B) anticorpo 1B1 (verde) descreve todas as células somáticas no ovário. Anti-Vasa (azul) todos os rótulos PGCs. PGCs contactar directamente as células Misturadas (ICs, anti-jam tráfego magenta). Bar (para A e B) é de 20 mM.

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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=Male+and+female+Drosophila+germline+stem+cells:+two+versions+of+immortality.">Male and female Drosophila germline stem cells: two versions of immortality.</a> Science. 316, 402-404 (2007).</li><li>Li, L., 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=Stem+cell+niche:+structure+and+function.">Stem cell niche: structure and function.</a> Annual review of cell and developmental biology. 21, 605-631 (2005).</li><li>Chen, D., McKearin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+circuitry+controlling+a+stem+cell+niche.">Gene circuitry controlling a stem cell niche.</a> Curr Biol. 15, 179-184 (2005).</li><li>Kai, T., 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=An+empty+Drosophila+stem+cell+niche+reactivates+the+proliferation+of+ectopic+cells.">An empty Drosophila stem cell niche reactivates the proliferation of ectopic cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 100, 4633-4638 (2003).</li><li>Kai, T., 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=Differentiating+germ+cells+can+revert+into+functional+stem+cells+in+Drosophila+melanogaster+ovaries.">Differentiating germ cells can revert into functional stem cells in Drosophila melanogaster ovaries.</a> Nature. 428, 564-569 (2004).</li><li>Lopez-Onieva, L., Fernandez-Minan, A., Gonzalez-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=Jak/Stat+signalling+in+niche+support+cells+regulates+dpp+transcription+to+control+germline+stem+cell+maintenance+in+the+Drosophila+ovary.">Jak/Stat signalling in niche support cells regulates dpp transcription to control germline stem cell maintenance in the Drosophila ovary.</a> Development. 135, 533-540 (2008).</li><li>Song, X., Zhu, 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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<td>NaCl</td>
<td>&nbsp;</td>
<td>J.T.Baker</td>
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<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>
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<td>J.T.Baker</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Hepes</td>
<td>&nbsp;</td>
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<td>PBS</td>
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<td>Albumin Bovine Fraction V</td>
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<td>Dumont biology tweezers 5 dumstar polished</td>
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<td>Nickel plated pin holder</td>
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<td>s.s minutien pins 0.1mm diam, 10mm long</td>
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<td>Corning</td>
<td>2940-223</td>
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<td>Vectashield</td>
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<td>Cell strainer</td>
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<td>FAL352350</td>
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<td>1B1 antibody</td>
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<td>Developmental Studies Hybridoma Bank</td>
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<td>Anti-Traffic Jam</td>
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<td>Laboratory of Dr. Dorothea Godt</td>
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<td>Anti-Vasa</td>
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<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

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

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Dissection and Staining of Drosophila Larval Ovaries

26.1K visualizações.  Weizmann Institute of Science. Como nichos e células-tronco se formam durante o desenvolvimento é uma questão importante com implicações práticas. No Drosophila 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) Drosophila.

Vídeo: Dissection and Staining of Drosophila Larval Ovaries

Dissection of Drosophila Ovaries

Dissecção da Drosophila Ovários

Biologia

Dissection of Larval CNS in Drosophila Melanogaster

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

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

Dissecção do CNS Larval em Drosophila melanogaster

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

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 larval Dissection MNJ

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

Drosophila Larval NMJ Immunohistochemistry

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 31 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. Immunohistochemistry can be used to clearly image the components of the NMJ. In this article, we demonstrate how to use antibody staining to visualize the Drosophila larval NMJ.

Drosophila Larval NMJ Immunohistochemistry

This protocol demonstrates how to perform immunohistochemistry on dissected Drosophila larva.

Drosophila larval MNJ Imunohistoquímica

Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining

Drosophila embryos between stages 14 and 17 of embryonic development can be readily dissected to generate "fillet" preparations. In these preparations, the central nervous system runs down the middle, and is flanked by the body walls. Many different phenotypes have been examined using such preparations. In most cases, the fillets were generated by dissection of antibody-stained fixed whole-mount embryos. These "fixed dissections" have some disadvantages, however. They are time-consuming to execute, and it is difficult to sort mutant (GFP-negative) embryos from stocks in which mutations are maintained over GFP balancer chromosomes. Since 2002, our group has been conducting deficiency and ectopic expression screens to identify ligands for orphan receptors. In order to do this, we developed streamlined protocols for live embryo dissection and antibody staining of collections containing hundreds of balanced lines. We have concluded that it is considerably more efficient to examine phenotypes in large collections of stocks by live dissection than by fixed dissection. Using the protocol described here, a single trained individual can screen up to 10 lines per day for phenotypes, examining 4-7 mutant embryos from each line under a compound microscope. This allows the identification of mutations conferring subtle, low-penetrance phenotypes, since up to 70 hemisegments per line are scored at high magnification with a 40X water-immersion lens.

Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining

We describe a streamlined protocol for generating "fillet" preparations of Drosophila embryos of specific genotypes. This protocol allows efficient execution of a variety of genetic screens. It also allows excellent visualization of structures in the late embryo.

Dissecação ao vivo de Drosophila Embriões: Métodos ágeis para Screening Coleções Mutant pela coloração de anticorpos

Drosophila embryos between stages 14 and 17 of embryonic development can be readily dissected to generate "fillet" preparations.  In these preparations, ...

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work examining post-embryonic through adult cardiac development has been limited7-10. Examining the changing morphology of the maturing and aging heart are restricted by the lack of techniques available for staging and isolating juvenile and adult hearts. In order to analyze heart development over the fish's lifespan, we dissect zebrafish hearts at numerous stages and photograph them for further analysis11. The morphological features of the heart can easily be quantified and individual hearts can be further analyzed by a host of standard methods. Zebrafish grow at variable rates and maturation correlates better with fish size than age, thus, post-fixation, we photograph and measure fish length as a gauge of fish maturation. This protocol explains two distinct, size dependent dissection techniques for zebrafish, ranging from larvae 3.5mm standard length (SL) with hearts of 100&mu;m ventricle length (VL), to adults, with SL of 30mm and VL 1mm or larger. Larval and adult fish have quite distinct body and organ morphology. Larvae are not only significantly smaller, they have less pigment and each organ is visually very difficult to identify. For this reason, we use distinct dissection techniques.
We used pre-dissection fixation procedures, as we discovered that hearts dissected directly after euthanization have a more variable morphology, with very loose and balloon like atria compared with hearts removed following fixation. The fish fixed prior to dissection, retain in vivo morphology and chamber position (data not shown). In addition, for demonstration purposes, we take advantage of the heart (myocardial) specific GFP transgenic Tg(myl7:GFP)twu34 (12), which allows us to visualize the entire heart and is particularly useful at early stages in development when the cardiac morphology is less distinct from surrounding tissues. Dissection of the heart makes further analysis of the cell and molecular biology underlying heart development and maturation using in situ hybridization, immunohistochemistry, RNA extraction or other analytical methods easier in post-embryonic zebrafish. This protocol will provide a valuable technique for the study of cardiac development maturation and aging.

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

A clear, standardized method for dissection and isolation of the zebrafish heart at multiple developmental stages are described. Annotation and quantification techniques are also discussed.

Dissecção do coração em larval, Zebrafish Juvenil e Adulto, Danio rerio</em

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work ...

Sonication-facilitated Immunofluorescence Staining of Late-stage Embryonic and Larval Drosophila Tissues In Situ

Studies performed in Drosophila melanogaster embryos and larvae provide crucial insight into developmental processes such as cell fate specification and organogenesis. Immunostaining allows for the visualization of developing tissues and organs. However, a protective cuticle that forms at the end of embryogenesis prevents permeation of antibodies into late-stage embryos and larvae. While dissection prior to immunostaining is regularly used to analyze Drosophila larval tissues, it proves inefficient for some analyses because small tissues may be difficult to locate and isolate. Sonication provides an alternative to dissection in larval Drosophila immunostaining protocols. It allows for quick, simultaneous processing of large numbers of late-stage embryos and larvae and maintains in situ morphology. After fixation in formaldehyde, a sample is sonicated. Sample is then subjected to immunostaining with antigen-specific primary antibodies and fluorescently labeled secondary antibodies to visualize target cell types and specific proteins via fluorescence microscopy. During the process of sonication, proper placement of a sonicating probe above the sample, as well as the duration and intensity of sonication, is critical. Additonal minor modifications to standard immunostaining protocols may be required for high quality stains. For antibodies with low signal to noise ratio, longer incubation times are typically necessary. As a proof of concept for this sonication-facilitated protocol, we show immunostains of three tissue types (testes, ovaries, and neural tissues) at a range of developmental stages.

Sonication-facilitated Immunofluorescence Staining of Late-stage Embryonic and Larval Drosophila Tissues In Situ

Immunostaining is an effective technique for visualizing specific cell types and proteins within tissues. By utilizing sonication, the protocol described here alleviates the need to dissect Drosophila melanogaster tissues from late-stage embryos and larvae before immunostaining. We provide an efficient methodology for the immunostaining of formaldehyde-fixed whole mount larvae.

Facilitou-Sonication Imunofluorescência Coloração de Late-estágio embrionário e larval<em&gt; Drosophila</em&gt; Tecidos<em&gt; In Situ</em

Studies performed in Drosophila melanogaster embryos and larvae provide crucial insight into developmental processes such as cell fate specification and ...

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 ...

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.

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.

Dissecção e montagem de<em&gt; Drosophila</em&gt; pupais Discos Eye

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes.  It contains approximately 800 ...

Measurement of Larval Activity in the Drosophila Activity Monitor

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This protocol repurposes an instrument often used to measure adult activity, the TriKinetics Drosophila activity monitor (MB5 Multi-Beam Activity Monitor) to study larval activity. The instrument can monitor the movements of animals in 16 individual 8 cm glass assay tubes, using 17 infrared detection beams per tube. Logging software automatically saves data to a computer, recording parameters such as number of moves, times sensors were triggered, and animals&#8217; positions within the tubes. The data can then be analyzed to represent overall locomotion and/or position preference as well as other measurements. All data are easily accessible and compatible with basic graphing and data manipulation software. This protocol will discuss how to use the apparatus, how to operate the software and how to run a larval activity assay from start to finish.

Measurement of Larval Activity in the Drosophila Activity Monitor

This report describes a method for measuring Drosophila larval activity using the TriKinetics Drosophila Activity Monitor. The device employs infrared beams to detect movements of up to 16 individual animals. Data can be analyzed to represent motion parameters including rates and the positions of the animals within the assay chambers.

Medição da Actividade larval no<em&gt; Drosophila</em&gt; Monitor de Atividade

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This ...