IM è supportato dal Marie Curie di reintegrazione di sovvenzione. Questo lavoro è stato sostenuto dal Fondo non Scienza Israele Grant. 1146-1108, da Helen e Martin Kimmel Istituto per la ricerca sulle cellule staminali presso il Weizmann Institute of Science e la Fondazione Leir beneficenza.

1. La deposizione delle uova <ol><li> Cinque giorni prima della dissezione: permette femmine accoppiate per deporre le uova per 2-4 ore con cibo fresco integrato con il lievito. Per ottenere larve sincronizzato e ben sviluppato, è importante non avere colto sovraffollamento (circa 30 uova / fiala 25mm). Tipicamente, 7-16 femmine sono utilizzati per flaconcino, a seconda di quanto bene laici. </li></ol> 2. Selezione larve <ol><li> Preparare un 9-ben piatto di vetro riempita con mezzo di dissezione Ringer (128mm NaCl, 2mM KCl, CaCl 2 1,8 mm, 4mm MgCl 2, 35.5mm saccarosio, 5mm Hepes pH 6,9). </li><li> Preparare filtri cella in una sei pozzetti contenenti medio di Ringer e posizionarlo sul ghiaccio. In alternativa, possiamo utilizzare stampi appositamente dotato di una rete di nylon. </li><li> Scegli larve tempo dalle pareti fiala usando bene le pinzette biologici (forcipe) e metterli in un piatto contenente sezionare la suoneria di. </li><li> Trasferire una larva femminile per un pozzo pulito. Si distinguono i maschi dalle femmine per la loro gonadi. Testicoli maschili sono facilmente identificabili come grandi ovali chiare incorporato nel terzo posteriore del corpo grasso. Ovaie femminili, che si trova alla stessa parte del grasso corporeo, può essere identificato come un molto più piccolo, chiaro, sfere rotonde. </li></ol> 3. Dissezione di larva <ol><li> Tenere la larva giù appena posteriore al cervello con una pinza e togliere la testa con un secondo paio di pinze. </li><li> Mettere la restante parte posteriore sul lato dorsale, con la trachea verso il basso. </li><li> Tenere la larva dagli spiracoli posteriori con un paio di pinze e spingerlo lentamente verso l&#39;interno, mentre l&#39;altra coppia sta scivolando la cuticola posteriormente fino a circa la metà del grasso corporeo larvale emerge. </li><li> Tengono saldamente l&#39;estremità posteriore e usare l&#39;altra coppia di pinze a tenere liberamente la cuticola. Delicatamente e lentamente tirare via le estremità posteriore in modo che la cuticola e la diapositiva intestino collegato attraverso la fessura. Alla fine di questo processo, il grasso corporeo dovrebbe essere completamente separato dal intestino e la cuticola. Scollegare l&#39;intestino dalla parte anteriore del corpo grasso. Per rendere la colorazione e montaggio più semplice, è importante che il corpo di grasso rimarrà intatto. </li><li> Wet una pipetta pascolo a fondo con medie di Ringer, preferibilmente da un pozzetto contenente larve sezionato. Questo aiuta a cappotto la pipetta e impedisce al corpo di grasso di attaccarsi ad essa. Utilizzare la pipetta pascolo per trasferire il grasso corporeo in media il Ringer ghiacciata nella cella di filtro. </li></ol> 4. Fissazione e colorazione Tutti i passaggi vengono eseguiti a temperatura ambiente, fatta eccezione per l&#39;incubazione con il primo anticorpo, a 4 ° C. <ol><li> Incubare il grasso corporeo in 5% di formaldeide in media di Ringer. per 20 minuti con agitazione. </li><li> Lavare per 5 minuti con 1% PBT (1% Triton X-100 in PBS). Ripetere questo passaggio per 10 minuti e una terza volta per 45 minuti. 1% Triton X-100 è necessario per perforare l&#39;ovaio larvale e anticorpi permettono di penetrare in esso. </li><li> Blocco con PBTB 0,3% (0,3% Triton X-100 e 1% BSA in PBS) per 1 ora e scuotendo con cautela </li><li> Incubare con l&#39;anticorpo desiderato 1 ° diluito in 0,3% PBTB durante la notte a 4 ° C con agitazione. Questo passo è di solito eseguita in provette da 0,2 ml su un rullo. </li><li> Trasferimento di grasso corporeo indietro filtri cellulare. </li><li> Lavare 3 volte, a 30 minuti ciascuno, con il 0,3% PBTB scuotendo con cautela. </li><li> Blocco con il 0,3% PBTB integrato con asino siero normale al 5% per 1 ora e scuotendo con cautela. </li><li> Incubare con un anticorpo secondario diluito appropriato (secondo le specifiche del produttore) nella soluzione bloccante (0,3% PBTB arricchito con 5% di siero asino normale nello 0,3% PBT) per 2 ore con agitazione. Se l&#39;anticorpo è fluorescente, incubare al buio da questo punto in avanti. Questo passo è di solito eseguita in provette da 0,2 ml su un rullo. </li><li> Lavare 3 volte per 30 minuti ciascuno con 0,3% PBT e scuotendo con cautela. </li></ol> 5. Montaggio <ol><li> Trasferire il grasso corporeo in una provetta eppendorf pulita. Con molta attenzione rimuovere tutti i liquidi lontani e coprire immediatamente con i media Vectashield montaggio. Noi usiamo Vectashield routine, dato che non si indurisce, mentre le ovaie vengono separati dal grasso corporeo. I campioni possono essere memorizzati in supporti di montaggio a 4 ° C per un massimo di una settimana. </li><li> Tagliare l&#39;estremità di un puntale e utilizzarlo per trasferire con attenzione il grasso corporeo, con volume minimo di supporti di montaggio (circa 30 microlitri per 30 millimetri di copertura antiscivolo) su un vetrino da microscopio. </li><li> Utilizzare due titolari pin placcato nichel, tenendo 0,1 mm di diametro perni per diffondere il grasso corporeo. Le ovaie sono situate al terzo posteriore del corpo grasso. Il corpo di grasso che li circonda di solito a forma come un &#39;fiore&#39;. Questo aiuta a identificare la posizione delle ovaie. Sezionare i &#39;fiori&#39; dal resto del corpo il grasso ed eliminare il secondo. </li><li> Togliere il grasso corporeo SurroINANZIAMENTO della gonade attraversando i due perni con attenzione intorno ad esso. Posizionare la gonade isolato sul scivolare via dal residuo di grasso corporeo. </li><li> Coprire con un vetrino e sigillare con smalto. </li><li> Visualizzare direttamente utilizzando un microscopio confocale. Campioni possono essere conservati a 4 ° C per un massimo di tre settimane. </li></ol> 6. Rappresentante Risultati Abbiamo usato il protocollo di sopra di seguire la creazione di linee cellulari diverse all&#39;interno dell&#39;ovaio somatiche, tra cui GSCs e le loro nicchie di somatico. A questo scopo si utilizza anticorpi specifici e indicatori di distinguere tra i diversi tipi di cellule della gonade via di sviluppo. Qui vi mostriamo un esempio di due LL3 ovaie colorati con diverse combinazioni di anticorpi. Figura 1A evidenzia filamento terminale e cellule cappuccio (verde), che insieme formano le cellule somatiche della nicchia. Figura 1B, mostra le cellule Interlacciati (IC, magenta), che direttamente in contatto con le cellule germinali (blu). <img src="/files/ftp_upload/2537/2537fig1.jpg" alt="Figura 1" /> Figura 1. LL3 ovaie. (A) 1B1 anticorpo monoclonale (magenta) delinea le cellule somatiche e le macchie della fusome, un organello intracellulare all&#39;interno di PGC (frecce). La trappola enhancer HH-lacZ (anti β-galattosidasi, verde) è espresso in filamenti terminale. Alla base del filamento di cellule somatiche tappo (punte di freccia) possono essere osservati. (B) anticorpi 1B1 (verde) delinea tutte le cellule somatiche nell&#39;ovaio. Anti-Vasa (blu) etichette tutti i PGC. PGC contattare direttamente le cellule Interlacciati (IC, anti-ingorgo, magenta). Bar (per A e B) è di 20 micron.

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H., Doan, C., 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=Germline+stem+cells+anchored+by+adherens+junctions+in+the+Drosophila+ovary+niches.">Germline stem cells anchored by adherens junctions in the Drosophila ovary niches.</a> Science. 296, 1855-1857 (2002).</li><li>Tazuke, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+germline-specific+gap+junction+protein+required+for+survival+of+differentiating+early+germ+cells.">A germline-specific gap junction protein required for survival of differentiating early germ cells.</a> Development. 129, 2529-2539 (2002).</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=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. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+IV+collagens+regulate+BMP+signalling+in+Drosophila.">Type IV collagens regulate BMP signalling in Drosophila.</a> Nature. 455, 72-77 (2008).</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+a+brac.">Mechanisms of cell rearrangement and cell recruitment in Drosophila ovary morphogenesis and the requirement of bric a brac.</a> Development. 121, 173-187 (1995).</li><li>Sahut-Barnola, I., Godt, D., Laski, F. A., Couderc, 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=Drosophila+ovary+morphogenesis:+analysis+of+terminal+filament+formation+and+identification+of+a+gene+required+for+this+process.">Drosophila ovary morphogenesis: analysis of terminal filament formation and identification of a gene required for this process.</a> Developmental biology. 170, 127-135 (1995).</li><li>Song, X., Call, G. B., 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=Notch+signaling+controls+germline+stem+cell+niche+formation+in+the+Drosophila+ovary.">Notch signaling controls germline stem cell niche formation in the Drosophila ovary.</a> 134, 1071-1080 (2007).</li><li>Zhu, C. 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Questo video dimostra un isolamento e il protocollo di colorazione della fine del III instar ovaie larvale. Per eseguire questo protocollo di routine e in modo affidabile, l&#39;attenzione dovrebbe essere prestata ai seguenti punti: <ol><li> Per le larve sincronizzati e ben sviluppato, oltre affollamento deve essere evitato. </li><li> Per evitare la perdita delle ovaie piccole traslucido, assicurarsi di sezionare il corpo di grasso intatto. Questo aiuterà anche a trovare le ovaie in fase di montaggio, in particolar...

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dissection and staining of drosophila larval ovaries

Molti organi dipendono da cellule staminali per il loro sviluppo durante l&#39;embriogenesi e per la manutenzione o la riparazione durante la vita adulta. Capire come le cellule staminali forma, e come interagiscono con l&#39;ambiente è quindi cruciale per la comprensione dello sviluppo, l&#39;omeostasi e malattia. Dell&#39;ovaio del moscerino della frutta Drosophila melanogaster ha servito come modello influente per l&#39;interazione delle cellule staminali della linea germinale (GSCs) con le loro cellule somatiche di supporto (di nicchia) 1, 2. La posizione nota della nicchia e la GSCs, unita alla capacità di manipolare geneticamente, ha permesso ai ricercatori di chiarire una serie di interazioni tra cellule staminali e le loro nicchie 3-12. Nonostante la ricchezza di informazioni sui meccanismi che controllano la manutenzione GSC e differenziazione, relativamente poco si sa su come GSCs e le loro nicchie di somatico forma durante lo sviluppo. Circa 18 nicchie somatiche, il cui cellulare componenti includono filamento terminale e cellule tappo (Figura 1), si formano durante la terza larvale instar 13-17. GSCs originano dalle cellule germinali primordiali (PGC). PGC proliferano in primi stadi larvali, ma in seguito alla formazione della nicchia un sottogruppo di PGC diventa GSCs 7, 16, 18, ​​19. Insieme, le cellule somatiche di nicchia e la GSCs fare una unità funzionale che produce uova per tutta la vita dell&#39;organismo. Molte domande riguardanti la formazione del gruppo GSC rimangono senza risposta. Processi quali il coordinamento tra le cellule precursore per nicchie e precursori di cellule staminali, o la generazione di asimmetria all&#39;interno PGC man mano che diventano GSCs, può essere meglio studiato nella larva. Tuttavia, uno studio metodico di sviluppo dell&#39;ovaio larvale è fisicamente impegnativo. In primo luogo, le ovaie sono piccole larve. Anche al fine di stadi larvali sono solo 100μm di diametro. Inoltre, le ovaie sono trasparenti e sono integrati in un corpo grasso bianco. Qui si descrive passo per passo il protocollo per isolare ovaie da fine del III instar (LL3) larve di Drosophila, seguita dalla colorazione con anticorpi fluorescenti. Vi offriamo alcune soluzioni tecniche a problemi quali localizzare le ovaie, colorazione e lavaggio dei tessuti che non affondare, e fare in modo che gli anticorpi penetrano nel tessuto. Questo protocollo può essere applicato a stadi precedenti larvali e di testicoli larvale pure.

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

Dissection and Staining of Drosophila Larval Ovaries

Come nicchie e le cellule staminali si formano durante lo sviluppo è una questione importante con implicazioni pratiche. Nel<em&gt; Drosophila</emOvaio&gt;, germe di cellule staminali e la loro linea di nicchie somatiche formano durante lo sviluppo larvale. Questo video mostra come sezionare, macchia e montare gonadi femminili dalla fine del III instar (LL3)<em&gt; Drosophila</em&gt; Larve.

Dissezione e colorazione di Drosophila Larvali ovaie

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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Biology

Dissection and Staining of Drosophila Larval Ovaries

26.1K Views.  Weizmann Institute of Science. Come nicchie e le cellule staminali si formano durante lo sviluppo è una questione importante con implicazioni pratiche. Nel Drosophila, germe di cellule staminali e la loro linea di nicchie somatiche formano durante lo sviluppo larvale. Questo video mostra come sezionare, macchia e montare gonadi femminili dalla fine del III instar (LL3) Drosophila Larve.

Video: Dissection and Staining of Drosophila Larval Ovaries

Dissection of Drosophila Ovaries

Dissezione di ovaie Drosophila

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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.

Dissezione del sistema nervoso centrale larvali 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 ...

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 larvali NMJ 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.

Drosophila larvali NMJ Immunoistochimica

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.

Dissezione di vivere Drosophila Embrioni: Metodi semplificati per lo screening Collezioni Mutant dalla colorazione anticorpale

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

Dissection of Oenocytes from Adult Drosophila melanogaster

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides protection against desiccation and other environmental challenges. Several of these cuticular hydrocarbon (CHC) compounds also function as semiochemical signals, and as such mediate pheromonal communications between members of the same species, or in some instances between different species, and influence behavior. Specialized cells referred to as oenocytes are regarded as the primary site for CHC synthesis. However, relatively little is known regarding the involvement of the oenocytes in the regulation of the biosynthetic, transport, and deposition pathways contributing to CHC output. Given the significant role that CHCs play in several aspects of insect biology, including chemical communication, desiccation resistance, and immunity, it is important to gain a greater understanding of the molecular and genetic regulation of CHC production within these specialized cells. The adult oenocytes of D. melanogaster are located within the abdominal integument, and are metamerically arrayed in ribbon-like clusters radiating along the inner cuticular surface of each abdominal segment. In this video article we demonstrate a dissection technique used for the preparation of oenocytes from adult D. melanogaster. Specifically, we provide a detailed step-by-step demonstration of (1) how to fillet prepare an adult Drosophila abdomen, (2) how to identify the oenocytes and discern them from other tissues, and (3) how to remove intact oenocyte clusters from the abdominal integument. A brief experimental illustration of how this preparation can be used to examine the expression of genes involved in hydrocarbon synthesis is included. The dissected preparation demonstrated herein will allow for the detailed molecular and genetic analysis of oenocyte function in the adult fruit fly.

Dissection of Oenocytes from Adult Drosophila melanogaster

In insects, the oenocytes produce cuticular hydrocarbon compounds. These compounds protect against desiccation and facilitate chemical communication. Here we demonstrate a dissection technique used to isolate the oenocytes from adult Drosophila melanogaster, and illustrate how this preparation can be utilized to study genes involved in hydrocarbon synthesis.

Dissezione di Oenocytes da adulti Drosophila melanogaster</em

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides ...

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.

Dissezione di cuore in larvale, Zebrafish giovanile e degli adulti, 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.

Sonicazione-facilitato immunofluorescenza colorazione di ritardo-fase embrionale e larvale<em&gt; Drosophila</em&gt; Tessuti<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.

Dissezione e Immunostaining di immaginale dischi da<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.

Dissezione e Montaggio di<em&gt; Drosophila</em&gt; pupa Dischi Eye

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