IM est soutenu par la Marie Curie réintégration subvention. Ce travail a été soutenu par le Fonds ne Israel Science Grant. 1146-1108, par le Helen et Martin Kimmel Institut de recherche de cellules souches à l&#39;Institut Weizmann des Sciences et de la Fondation de bienfaisance Leir.

1. La ponte des oeufs <ol><li> Cinq jours avant la dissection: permettre femelles fécondées à pondre des oeufs pendant 2-4 heures sur les aliments frais complété avec de la levure. Pour obtenir des larves synchronisée et bien développé, il est important de ne pas avoir surpeuplées cultivés (environ 30 œufs / flacon 25mm). Typiquement, 7-16 femelles sont utilisées par flacon, en fonction de la façon dont ils pondent. </li></ol> 2. Sélection des larves <ol><li> Préparer un plat en verre de 9 ainsi disséquer remplie de milieu de Ringer (NaCl 128mm, 2 mM de KCl, CaCl 2 1,8 mm, 4mm MgCl2, 35.5mm sucrose, 5 mM pH Hepes 6,9). </li><li> Préparer les crépines de cellules dans une plaque de six puits contenant du milieu de Ringer et le placer sur la glace. Sinon, nous utilisons des moules spécialement équipé d&#39;un filet de nylon. </li><li> Choisissez les larves chronométrée des murs flacon à l&#39;aide fines pinces biologiques (forceps) et les placer dans le contenant de Ringer plat dissection. </li><li> Transfert d&#39;une larve femelle pour un bien propre. On distingue les mâles des femelles de par leurs gonades. Testicules des mâles sont facilement identifiés comme grands cercles transparents intégré dans le tiers postérieur du corps gras. Ovaires Femme, situé à la même partie de la graisse du corps, peut être identifiée comme une beaucoup plus petite, claire, de sphères rondes. </li></ol> 3. Dissection de la larve <ol><li> Tenez la larve bas juste derrière le cerveau avec une pince et retirer la tête avec une deuxième paire de pinces. </li><li> Placez la partie restante postérieure sur la face dorsale, avec la trachée vers le bas. </li><li> Tenez la larve par les stigmates postérieurs avec une paire de pinces et de le pousser vers l&#39;intérieur lentement tandis que l&#39;autre paire est coulissant la cuticule arrière jusqu&#39;à environ la moitié de la graisse du corps larvaire sort. </li><li> Maintenez fermement l&#39;extrémité postérieure et l&#39;utilisation de l&#39;autre paire de pinces pour tenir vaguement la cuticule. Doucement et lentement s&#39;éloigner de l&#39;extrémité postérieure de telle sorte que la cuticule et la diapositive intestin attachés à l&#39;écart. A la fin de ce processus, la graisse du corps doit être complètement séparée de l&#39;intestin et la cuticule. Débranchez l&#39;intestin de la partie antérieure du corps gras. Pour rendre la coloration et de montage plus facile, il est important que la graisse du corps reste intact. </li><li> Mouiller une pipette pâturages soigneusement avec un milieu de Ringer, de préférence à partir d&#39;un puits contenant des larves disséquées. Cette aide de la pipette poil et empêche le corps gras de coller à lui. Utilisez la pipette pour transférer les pâturages de la graisse du corps dans le milieu de la sonnerie de la glace froide dans la passoire cellule. </li></ol> 4. Fixation et une coloration Toutes les étapes sont réalisées à température ambiante, sauf pour l&#39;incubation avec l&#39;anticorps d&#39;abord, à 4 ° C. <ol><li> Incuber la graisse du corps dans du formol à 5% en milieu de Ringer. pendant 20 minutes avec une agitation douce. </li><li> Laver pendant 5 minutes avec 1% PBT (1% de Triton X-100 dans du PBS). Répétez cette étape pour 10 minutes et une troisième fois pendant 45 minutes. 1% de Triton X-100 est nécessaire pour perforer l&#39;ovaire larvaire et permettent des anticorps pour le pénétrer. </li><li> Bloc avec PBTB 0,3% (0,3% de Triton X-100 et 1% de BSA dans du PBS) pendant 1 heure avec agitation douce </li><li> Incuber avec l&#39;anticorps désiré 1 er dilué dans PBTB 0,3% pendant la nuit à 4 ° C avec agitation douce. Cette étape est généralement effectuée dans des tubes de 0,2 ml sur un rouleau. </li><li> Transfert de graisse du corps Retour à crépines cellule. </li><li> Lavez 3 fois, 30 minutes chacune, avec PBTB 0,3% avec une légère agitation. </li><li> Bloc avec PBTB 0,3% supplémenté avec 5% d&#39;âne sérum normal pendant 1 heure avec agitation douce. </li><li> Incuber avec un anticorps secondaire approprié dilué (selon les spécifications du constructeur) dans la solution de blocage (0,3% PBTB supplémentés avec du sérum d&#39;âne 5% de la normale chez 0,3% PBT) pendant 2 heures avec agitation douce. Si l&#39;anticorps est fluorescent, incuber dans l&#39;obscurité partir de ce point. Cette étape est généralement effectuée dans des tubes de 0,2 ml sur un rouleau. </li><li> Lavez 3 fois pendant 30 minutes chacun avec 0,3% PBT avec agitation douce. </li></ol> 5. Montage <ol><li> Transférer la graisse du corps dans un tube propre Eppendorf. Très soigneusement enlever tous les liquides loin et couvrir immédiatement avec les médias Vectashield montage. Nous utilisons régulièrement depuis Vectashield il ne durcit pas alors que les ovaires sont séparés du corps de graisse. Les échantillons peuvent être stockés dans les médias de montage à 4 ° C jusqu&#39;à une semaine. </li><li> Coupez l&#39;extrémité d&#39;une pointe de pipette et de l&#39;utiliser pour transférer soigneusement le corps gras avec un volume minimal de supports de montage (environ 30 ul d&#39;une lamelle de 30mm) sur une lame de microscope. </li><li> Utilisez deux détenteurs de nickel broches plaquées, tenant 0,1 mm de diamètre broches d&#39;étaler la graisse corporelle. Les ovaires sont situés au tiers postérieur du corps gras. La graisse du corps qui les entoure est habituellement la forme d&#39;une «fleur». Cela permet d&#39;identifier l&#39;emplacement des ovaires. Disséquer les «fleurs» du reste du corps gras et jetez celui-ci. </li><li> Retirez la graisse du corps Surroinancement la gonade en croisant les deux broches attentivement autour de lui. Placez la gonade isolé sur la lame de suite à partir du résidu de graisse corporelle. </li><li> Couvrir avec une lamelle et scellez avec du vernis à ongles. </li><li> Visualisez directement en utilisant un microscope confocal. Les échantillons peuvent être conservés à 4 ° C pendant au maximum trois semaines. </li></ol> 6. Les résultats représentatifs Nous avons utilisé le protocole ci-dessus pour suivre la mise en place de plusieurs lignées de cellules somatiques dans l&#39;ovaire, y compris les CSS et leurs niches somatiques. A cet effet, nous utilisons des anticorps spécifiques et des marqueurs pour distinguer les différents types de cellules dans la gonade en développement. Ici, nous montrons un exemple de deux ovaires LL3 colorés avec différentes combinaisons d&#39;anticorps. Figure 1A souligne filaments terminaux et les cellules de la PAC (en vert), qui forment ensemble les cellules somatiques de la niche. Figure 1B, montre les cellules entremêlées (CI, magenta), qui contacter directement les cellules germinales (bleu). <img src="/files/ftp_upload/2537/2537fig1.jpg" alt="Figure 1" /> Figure 1. LL3 ovaires. (A) anticorps monoclonal 1B1 (magenta) expose les cellules somatiques et les taches de la fusome, une organelle intracellulaire dans les PGC (flèches). Le piège enhancer hh-lacZ (anti β-galactosidase, vert) est exprimée en filaments terminaux. A la base des cellules somatiques bouchon filaments (flèches) peuvent être observées. (B) d&#39;anticorps 1B1 (vert) décrit toutes les cellules somatiques dans l&#39;ovaire. Anti-Vasa (bleu) toutes les étiquettes PGC. PGC contacter directement les cellules entremêlées (CI, anti-embouteillages, magenta). Bar (A et B) est de 20 um.

<ol>
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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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Cette vidéo montre un isolement et de protocole de coloration des ovaires fin du troisième stade larvaire. Pour effectuer ce protocole régulièrement et de manière fiable, une attention devrait être accordée aux points suivants: <ol><li> Pour les larves synchronisée et bien développé, le surpeuplement doit être évitée. </li><li> Pour éviter la perte des ovaires petits translucides, assurez-vous de disséquer le corps gras intacte. Cela aidera également à localiser les ovaires au stade de montage, en p...

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<td>NaCl</td>
<td>&nbsp;</td>
<td>J.T.Baker</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Kcl</td>
<td>&nbsp;</td>
<td>Merck</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>CaCl2</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>&nbsp;</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>
<td>&nbsp;</td>
<td>Leica</td>
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<td>Menzel Glaser GmbH</td>
<td>798</td>
<td>&nbsp;</td>
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<td>&nbsp;</td>
<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>
<td>&nbsp;</td>
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<td>Anti-Vasa</td>
<td>&nbsp;</td>
<td>Laboratory of Dr. Ruth Lehmann</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Anti &beta;-Galactosidase</td>
<td>&nbsp;</td>
<td>Cappel</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<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

De nombreux organes dépendent de cellules souches pour leur développement au cours de l&#39;embryogenèse et pour l&#39;entretien ou la réparation pendant la vie adulte. Comprendre comment les cellules souches forme, et comment ils interagissent avec leur environnement est donc crucial pour comprendre le développement, l&#39;homéostasie et la maladie. L&#39;ovaire de la mouche des fruits Drosophila melanogaster a servi de modèle influent pour l&#39;interaction des cellules germinales souches (CSS) avec leurs cellules de soutien somatiques (de niche) 1, 2. L&#39;endroit connu de la niche et le CSS, couplée à la capacité de les manipuler génétiquement, a permis aux chercheurs d&#39;élucider une variété d&#39;interactions entre cellules souches et leurs niches 3-12. Malgré la richesse de l&#39;information sur l&#39;entretien des mécanismes de contrôle de la CGC et de la différenciation, on sait relativement peu sur la façon dont les CSS et leurs niches somatiques forment pendant le développement. Environ 18 créneaux somatiques, dont les filaments cellulaires comprennent des composants du terminal et cellules de la coiffe (Figure 1), la forme au cours du troisième stade larvaire 13-17. CSS proviennent des cellules germinales primordiales (PGC). PGC prolifèrent au premiers stades larvaires, mais après la formation de la niche un sous-groupe de PGC devient CSS 7, 16, 18, ​​19. Ensemble, les cellules somatiques de niche et les CSS font une unité fonctionnelle qui produit des oeufs durant toute la vie de l&#39;organisme. Beaucoup de questions concernant la formation de l&#39;unité de la CGC restent sans réponse. Des procédés tels que la coordination entre les cellules précurseurs des niches et des précurseurs de cellules souches, ou la génération de l&#39;asymétrie dans les PGC à mesure qu&#39;ils deviennent les CSS, peut être mieux étudié dans la larve. Cependant, une étude méthodique de développement de l&#39;ovaire larvaire est difficile physiquement. Premièrement, les ovaires des larves sont petites. Même à la fin de stades larvaires, ils ne sont que 100 microns de diamètre. En outre, les ovaires sont transparentes et sont intégrés dans un corps blanc de graisse. Nous décrivons ici un protocole étape par étape pour isoler les ovaires de la fin du troisième stade larvaire (LL3) larves de drosophile, suivie d&#39;une coloration avec des anticorps fluorescents. Nous offrons des solutions techniques aux problèmes tels que la localisation des ovaires, la coloration et le lavage des tissus qui ne coulent pas, et faire en sorte que les anticorps pénètrent dans les tissus. Ce protocole peut être appliqué à des stades antérieurs de larves et de testicules larvaires ainsi.

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

Dissection and Staining of Drosophila Larval Ovaries

Comment les niches et les cellules souches forment pendant le développement est une question importante avec des implications pratiques. Dans le<em&gt; Drosophile</emL&#39;ovaire&gt;, les cellules germinales souches et leurs niches somatiques se forment pendant le développement larvaire. Cette vidéo montre comment disséquer, aux taches et monter les gonades femelles de la fin du troisième stade larvaire (LL3)<em&gt; Drosophile</emLarves&gt;.

Dissection et la coloration des Drosophile Ovaires larvaires

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 vues.  Weizmann Institute of Science. Comment les niches et les cellules souches forment pendant le développement est une question importante avec des implications pratiques. Dans le Drosophile

Vidéo: Dissection and Staining of Drosophila Larval Ovaries

Dissection of Drosophila Ovaries

Dissection des ovaires chez la drosophile

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Biologie

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.

Dissection de SNC larvaire chez 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.

Drosophile larvaire JNM Dissection

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.

Drosophile larvaire JNM immunohistochimie

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.

Dissection en direct de Drosophile Embryons: méthodes simplifiées pour le dépistage de collections de mutants par immunofluorescence

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.

Dissection du cœur chez les larves, le poisson zèbre juvéniles et adultes, 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.

Sonication facilitée immunofluorescence de phase tardive embryonnaire et larvaire<em&gt; Drosophila</em&gt; tissus<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.

Dissection et Immunocoloration Imaginal disques 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.

Dissection et de montage de<em&gt; Drosophila</em&gt; pupes disques yeux

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.

Mesure de larves activité dans le<em&gt; Drosophila</em&gt; Moniteur d&#39;activité

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

Methods Article

Dissection et la coloration des Drosophile Ovaires larvaires