Grazie a Marie Birne, Areti Tsiola, Jaymie Estevez e Arelys Uribe. Questo lavoro è stato sostenuto in parte da CUNY Research Foundation e Howard Hughes Medical Institute programma estivo di sovvenzione per gli studenti universitari (SPUR) e NIH RO3 41702-00-01. La ricerca è stata sostenuta anche dal Queens College e alcuni degli esperimenti sono stati fatti su apparecchi dal Fondo Core for Imaging, Biologia Cellulare e Molecolare al Queens College.

Zebrafish sono state sollevate utilizzando protocolli standard 13 nel Fondo Queens College di animali a 28 ° C. Coppie o singoli pesci accoppiamenti gruppo dovrebbe essere preformato e gli embrioni risultanti puliti e conservati in un incubatore 28 ° C. Salute dei pesci dovrebbero essere esaminati ogni giorno. A 5 giorni di fecondazione post (DPF), il pesce deve essere separata in vasche ad una densità 10-15 pesce ed è entrato in funzione il vivaio di pesci. Per il protocollo presentato qui zebrafish sono stati raccolti attraverso le fasi di larva adulto: 15, 30, 45, 60, 90, 180 dpf. 1. Zebrafish raccolta soggetto e la fissazione <ol><li> Rimuovere il pesce dal serbatoio habitat tramite rete e mettere in Tricaine 0,2% per anestetizzare. </li><li> Mettere i pesci anestetizzati in acqua ghiacciata per 15 minuti per eutanasia. </li><li> Fai fresca paraformaldeide 4% (PFA) in 1x PBS (PBS). </li><li> Incubare i pesci raccolti in PFA 4% per 1-2 ore a temperatura ambiente poi la notte a 4 ° C in una capsula di Petri avvolto in parafilm. Ciò mantiene il piatto di pesce e rende molto più facile dopo la dissezione. </li><li> Dopo la fissazione, lavare due volte in PBS con Tween (PBT). Pesce può essere mantenuto in PBT a 4 ° C per un massimo di 10 giorni. </li></ol> 2. Misura di pesce e la dissezione <ol><li> Pesce singolo posto in mezzo piastra di Petri riempita con PBS fresco e appoggiarla sul lato destro. </li><li> Misurare la lunghezza del pesce dal muso alla base della coda (peduncolo caudale), non comprese le pinne di coda, con una pinza digitale millimetri (figura 1). Questa è la lunghezza standard (SL). </li><li> Ogni pesce può essere assegnato un numero / combinazione di lettere per consentire un singolo pesce e il cuore risulta sezionato per essere monitorati per una successiva analisi. Dati, tra cui il numero dei genitori magazzino, età alla fissazione, misure SL, sono stati inseriti in un foglio elettronico e organizzato sulla base di queste etichette. </li><li> Prima della dissezione, etichetta strisce tubo PCR con numero di tracking assegnato e riempire con PBT o altre soluzioni appropriate per ulteriori analisi. </li><li> Orientare il lato ventrale pesce in piastra di Petri, mentre la stabilizzazione del corpo con una pinza tenendo la testa tra gli occhi e le branchie (Figura 2). </li></ol> Pesci più piccoli (inferiori a 12 millimetri SL): <ol start="4"><li> a) Con pinze taglienti, o un micro-ago in un supporto perno, rimuovere i muscoli pettorali e le pinne dal corpo per rivelare il cuore. Il movimento costante del forcipe durante la dissezione può causare correnti nel PBT che si muovono i pesci. Ciò è particolarmente problematico per i pesci più piccoli, quindi un micro-ago può essere usato per rimuovere i muscoli pettorali e le pinne e aprire la cavità del corpo. </li><li> a) delicatamente usare il forcipe per raccogliere il cuore fuori della cavità da sotto l&#39;atrio. In alternativa, in alcuni pesci si può essere in grado di rimuovere il cuore con delicatezza tirando fuori il bulbo e il resto del cuore seguirà. Fare attenzione della posizione dell&#39;atrio se si usa questa tecnica come si può facilmente danneggiarsi. Se vi è il tessuto in più che viene fornito con il cuore, che sta bene e può essere cancellato dopo. </li><li> a) Se il pesce contiene un marcatore fluorescente cardiaco come Tg (myl7: GFP) twu34, utilizzare la portata fluorescenti per la dissezione e verificare l&#39;asportazione del cuore da parte fioritura (Figura 3). </li><li> a) Una volta che il cuore è stato rimosso dalla cavità, usare le pinze per tenere il cuore e un micro-ago per rimuovere supplementari non tessuto cardiaco e la fodera epicardico dall&#39;esterno del cuore. </li></ol> I pesci più grandi (più grandi di 12mm SL): <ol start="4"><li> b) Utilizzando primavera trattati micro-forbici, fare tre incisioni di meno di 1 millimetro in profondità: 1 - taglio trasversale attraverso le branchie 2 - taglio trasversale al ventre anteriore 3 - taglio sagittale sul lato ventrale collega i due tagli (Figura 4). </li><li> b) Utilizzando pinze taglienti rimuovere i muscoli pettorali e le pinne dal corpo di aprire la cavità del corpo e rivelare il tessuto argenteo del pericardio (Figura 5A). Rimuovere questo tessuto pericardico e il cuore diventa visibile (Figura 5B). </li><li> b) Usare la micro-forbici per tagliare l&#39;arteria collegata al bulbo arterioso, che si trova superiore al cuore. Una volta che questa arteria è tagliato, mettere le punte pinza sotto l&#39;atrio, usare la pinza per raccogliere il cuore fuori della cavità. Se vi è il tessuto in più che viene fornito con il cuore, che va bene e può essere rimosso in seguito. </li><li> b) Una volta che il cuore è stato rimosso dalla cavità, utilizzare le due pinze per tenere il cuore e rimuovere il tessuto in più, o un paio di pinze per tenere il cuore e un micro-ago per rimuovere extra, non tessuto cardiaco. Il rivestimento epicardici, evidenti dalla sua pigmentazione nera, può essere rimossa dall&#39;esterno del cuore, se desiderato con delicatezza raschiando il tessuto. </li></ol> 3. Fotografia di cuori <ol><li> Preparare un 23g / l di agar nutriente piatto di Petri e coprire agar solidificato con PBS. </li><li> Utilizzando le pinze, fare un bene in agar che permette al cuore di riposare l&#39;orientamento desiderato per fotografare. </li><li> REMOVe il cuore dal tubo di PCR tenendo la punta del bulbo con la pinza con decisione. Mettere il cuore nel piatto agar. </li><li> Orientare il cuore in agar da fotografare. Il campione può essere ruotato dopo ogni immagine di tutti gli orientamenti possibili (Figura 6). </li><li> Utilizzare il tempo di esposizione in testa leggera e breve per fotografare il cuore. Effettuare le regolazioni per la qualità della luce utilizzando l&#39;esposizione necessaria per ottenere la migliore fotografia. </li></ol> 4. Rappresentante dei risultati: Un esempio di ben sezionato pulito cuore è mostrato in più orientamenti in Figura 6. Ogni centro avrà alcune differenze nella morfologia generale, ma il cuore deve essere completa, contenente un ventricolo integro, atrio e bulbo arterioso. Alcuni esempi di questa variazione nella morfologia generale sono mostrati in figura 7. Il nero macchiato tessuto epicardico può anche strappare dal cuore. <img src="/files/ftp_upload/3165/3165fig1.jpg" alt="Figura 1"/> Figura 1. Misurare il pesce dal muso al peduncolo caudale utilizzando una pinza digitale millimetri. <img src="/files/ftp_upload/3165/3165fig2.jpg" alt="Figura 2"/> Figura 2. Tenere il pesce con una pinza tra gli occhi e le branchie. <img src="/files/ftp_upload/3165/3165fig3.jpg" alt="Figura 3"/> Figura 3 A cardiaco specifico marcatore fluorescente transgenici come il Tg (myl7: GFP). Twu34, qui, può aiutare a visualizzare cuore sezionato da pesci più piccoli. <img src="/files/ftp_upload/3165/3165fig4.jpg" alt="Figura 4"/> Figura 4 Tre incisioni per rimuovere i muscoli pettorali e la pelle: 1 - trasversalmente attraverso le branchie 2 - taglio trasversale al ventre anteriore 3 - taglio sagittale sul lato ventrale che collega entrambi i tagli.. <img src="/files/ftp_upload/3165/3165fig5.jpg" alt="Figura 5"/> . Lettere Figura 5 Black Label le camere del cuore: bulbo arterioso (ba), ventricolo (v), e l&#39;atrio (a). A-La cavità superiore del corpo è aperto per mostrare il tessuto argenteo del pericardio. B-Dopo aver rimosso il pericardio, il cuore è visibile e facilmente rimovibile. <img src="/files/ftp_upload/3165/3165fig6.jpg" alt="Figura 6"/> Orientamenti Figura 6. Differenti del cuore sono mostrati. Lettere nere etichetta le camere del cuore: bulbo arterioso (ba), ventricolo (v), e l&#39;atrio (a). Grigio frecce indicano la posizione e l&#39;orientamento del cuore in riferimento alla sua posizione normale del corpo: anteriore (A), posteriore (P), dorsale (D), ventrale (V), sinistro (L) e destro (R). Bar scala indica 400μm. A-ventrale vista del cuore B-dorsale vista del cuore C-vista laterale dal lato sinistro D-laterale vista dal lato destro <img src="/files/ftp_upload/3165/3165fig7.jpg" alt="Figura 7"/> Figura 7. Morfologia dei cuori di diverse dimensioni è indicato. Lettere nere etichetta le camere del cuore: bulbo arterioso (ba), ventricolo (v), e l&#39;atrio (a). Barra di scala indicato 400 micron. AC-Hearts forma di pesce con meno di 12 millimetri SL DF-Hearts forma di pesce con SL più grandi di 12mm

<ol>
	<li>Ahmad, N., Long, S., Rebagliati, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+southpaw+joins+the+roster:+the+role+of+the+zebrafish+nodal-related+gene+southpaw+in+cardiac+LR+asymmetry.">A southpaw joins the roster: the role of the zebrafish nodal-related gene southpaw in cardiac LR asymmetry.</a> Trends Cardiovasc Med. 14, 43-49 (2004).</li><li>Armstrong, E. J., Bischoff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+valve+development:+endothelial+cell+signaling+and+differentiation.">Heart valve development: endothelial cell signaling and differentiation.</a> Circ Res. 95, 459-470 (2004).</li><li>Bakkers, J., Verhoeven, M. C., Abdelilah-Seyfried, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shaping+the+zebrafish+heart:+from+left-right+axis+specification+to+epithelial+tissue+morphogenesis.">Shaping the zebrafish heart: from left-right axis specification to epithelial tissue morphogenesis.</a> Dev Biol. 330, 213-220 (2009).</li><li>Glickman, N. S., Yelon, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+development+in+zebrafish:+coordination+of+form+and+function.">Cardiac development in zebrafish: coordination of form and function.</a> Semin Cell Dev Biol. 13, 507-513 (2002).</li><li>Heicklen-Klein, A., McReynolds, L. J., Evans, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+the+zebrafish+model+to+study+GATA+transcription+factors.">Using the zebrafish model to study GATA transcription factors.</a> Semin Cell Dev Biol. 16, 95-106 (2005).</li><li>Schoenebeck, J. J., Yelon, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Illuminating+cardiac+development:+Advances+in+imaging+add+new+dimensions+to+the+utility+of+zebrafish+genetics.">Illuminating cardiac development: Advances in imaging add new dimensions to the utility of zebrafish genetics.</a> Semin Cell Dev Biol. 18, 27-35 (2007).</li><li>Beis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+and+cellular+analyses+of+zebrafish+atrioventricular+cushion+and+valve+development.">Genetic and cellular analyses of zebrafish atrioventricular cushion and valve development.</a> Development. 132, 4193-4204 (2005).</li><li>Lepilina, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dynamic+epicardial+injury+response+supports+progenitor+cell+activity+during+zebrafish+heart+regeneration.">A dynamic epicardial injury response supports progenitor cell activity during zebrafish heart regeneration.</a> Cell. 127, 607-619 (2006).</li><li>Wills, A. A., Holdway, J. E., Major, R. J., Poss, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulated+addition+of+new+myocardial+and+epicardial+cells+fosters+homeostatic+cardiac+growth+and+maintenance+in+adult+zebrafish.">Regulated addition of new myocardial and epicardial cells fosters homeostatic cardiac growth and maintenance in adult zebrafish.</a> Development. 135, 183-192 (2008).</li><li>Parichy, D. M., Elizondo, M. R., Mills, M. G., Gordon, T. N., Engeszer, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+table+of+postembryonic+zebrafish+development:+staging+by+externally+visible+anatomy+of+the+living+fish.">Normal table of postembryonic zebrafish development: staging by externally visible anatomy of the living fish.</a> Dev Dyn. 238, 2975-3015 (2009).</li><li>Huang, C. J., Tu, C. T., Hsiao, C. D., Hsieh, F. J., Tsai, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germ-line+transmission+of+a+myocardium-specific+GFP+transgene+reveals+critical+regulatory+elements+in+the+cardiac+myosin+light+chain+2+promoter+of+zebrafish.">Germ-line transmission of a myocardium-specific GFP transgene reveals critical regulatory elements in the cardiac myosin light chain 2 promoter of zebrafish.</a> Dev Dyn. 228, 30-40 (2003).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Zebrafish+Book,+A+guide+for+the+laboratory+use+of+zebrafish+(Danio+rerio).">The Zebrafish Book, A guide for the laboratory use of zebrafish (Danio rerio).</a> , (1995).</li></ol>

Nessun conflitto di interessi dichiarati.

Questi metodi per la dissezione del cuore zebrafish di post-embrionale di pesce permette la rimozione completa di un cuore integro. Mentre questa tecnica è semplice e fornisce guide per tagli esterni che evitare di danneggiare il cuore, una mano ferma è essenziale per i più piccoli pesci. Un passo fondamentale durante la dissezione è quello di identificare il pericardio argenteo come il cuore è direttamente all&#39;interno di questa uscita. Visualizzando il pericardio fornisce un indica...

<table border="1"><tbody><tr><th> Materiale </th><th> Azienda </th><th> Numero di catalogo </th><th> Commenti (opzionale) </th></tr><tr><td> Paraformaldeide </td><td> EMD </td><td> PX0055 </td><td> .04g/mL In PBS </td></tr><tr><td> Pinza # 55 </td><td> FST </td><td> 11295-51 </td><td></td></tr><tr><td> Micro-forbici </td><td> FST </td><td> 15005-08 </td><td></td></tr><tr><td> Pin titolare </td><td> FST </td><td> 26016-12 </td><td></td></tr><tr><td> Micro-aghi </td><td> FST </td><td> 10130-10 </td><td></td></tr><tr><td> Nutrient Agar </td><td> BBL </td><td> 11472 </td><td> 23g / L in H 2 O distillata </td></tr><tr><td> Petri </td><td> BD Falcon </td><td> 351029 </td><td></td></tr><tr><td> PCR 8-tubo strisce </td><td> USA scientifico </td><td> 1402-2500 </td><td></td></tr><tr><td> Calibro digitale </td><td> Fisher Scientific </td><td> 14-648-17 </td><td></td></tr></tbody></table>

heart dissection in larval, juvenile and adult zebrafish, danio rerio

Zebrafish sono diventati un organismo modello utile e pratico per lo studio dello sviluppo embrionale cuore (vedi recensioni più recenti 1-6), tuttavia, il lavoro esaminando post-embrionale attraverso lo sviluppo cardiaco adulto è stato limitato 7-10. Esaminando la morfologia cambia del cuore maturazione e l&#39;invecchiamento sono limitate dalla mancanza di tecniche disponibili per la stadiazione e isolare i cuori giovani che adulti. Al fine di analizzare lo sviluppo cuore oltre la durata della vita del pesce, si sezionano i cuori pesce zebra in numerose fasi e fotografarli per ulteriori analisi 11. Le caratteristiche morfologiche del cuore può essere facilmente quantificata e cuori individuali possono essere ulteriormente analizzati da una serie di metodi standard. Zebrafish crescere a tassi variabili e la maturazione si correla meglio con dimensione dei pesci di età, quindi, post-fissazione, abbiamo lunghezza pesce fotografare e misurare come un indicatore di maturazione di pesce. Questo protocollo spiega due distinte tecniche di dissezione dimensioni dipendenti per zebrafish, che vanno dalla lunghezza standard di larve da 3,5 mm (SL) con i cuori di lunghezza ventricolo 100μm (VL), agli adulti, con SL di 30mm e VL 1mm o più grandi. Larve e adulti hanno il corpo ben distinte e la morfologia degli organi. Le larve non sono solo molto più piccoli, hanno meno pigmento e ogni organo è visivamente molto difficile da identificare. Per questa ragione, usiamo tecniche di dissezione distinte. Abbiamo usato pre-dissezione procedure di fissaggio, come abbiamo scoperto che il cuore sezionato direttamente dopo euthanization hanno una morfologia più variabile, con palloncino molto sciolto e, come atri rispetto al cuore rimosso fissazione seguente. Il pesce fisso prima di dissezione, trattenere in vivo la morfologia e la posizione della camera (dati non riportati). In aggiunta, a scopo dimostrativo, approfittiamo del cuore (infarto) specifiche GFP transgenici Tg (myl7: GFP) twu34 (12), che ci permette di visualizzare il cuore e il servizio è particolarmente utile nelle fasi iniziali di sviluppo quando la cardiaco morfologia è meno distinto dai tessuti circostanti. Dissezione del cuore fa un&#39;ulteriore analisi della biologia cellulare e molecolare alla base dello sviluppo del cuore e con la maturazione di ibridazione in situ, immunoistochimica, estrazione di RNA o altri metodi analitici più facile in post-embrionale di zebrafish. Questo protocollo fornisce una valida tecnica per lo studio della maturazione dello sviluppo cardiaco e l&#39;invecchiamento.

Watch this Scientific Journal Video about Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio at JoVE.com

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

Un chiaro, metodo standardizzato per la dissezione e l&#39;isolamento del cuore zebrafish a più stadi di sviluppo sono descritti. Tecniche di annotazione e la quantificazione vengono anche discussi.

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 ...

heart-dissection-in-larval-juvenile-and-adult-zebrafish-danio-rerio

Research

JoVE Journal

Biology

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

21.3K Views.  Queens College, City University of New York. Un chiaro, metodo standardizzato per la dissezione e l'isolamento del cuore zebrafish a più stadi di sviluppo sono descritti. Tecniche di annotazione e la quantificazione vengono anche discussi.

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

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 ...

Ricerca

Biologia

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 ...

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish (Danio rerio)

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the cellular and molecular features of the epithelia, which are mainly composed of two types of cells: hair cells (HCs) and supporting cells (SCs). Here we choose to study the inner ear sensory epithelia in adult zebrafish not only because the epithelial structures are highly conserved in all vertebrates studied, but also because the adult zebrafish is able to regenerate HCs, an ability that mammals lose shortly after birth. We use the inner ear of adult zebrafish as a model system to study the mechanisms of inner ear HC regeneration in adult vertebrates that could be helpful for clinical therapy of hearing/balance deficits in human as a result of HC loss.
Here we demonstrate how to do gross and fine dissections of inner ear sensory epithelia in adult zebrafish. The gross dissection removes the tissues surrounding the inner ear and is helpful for preparing tissue sections, which allows us to examine the detailed structure of the sensory epithelia. The fine dissection cleans up the non-sensory-epithelial tissues of each individual epithelium and enables us to examine the heterogeneity of the whole epithelium easily in whole-mount epithelial samples.

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish Danio rerio

The inner ear sensory epithelium of adult zebrafish is a good model system for understanding the mechanisms of hair cell regeneration in adult vertebrates. This protocol demonstrates the fine dissection of the epithelia, through which we can get tissue samples for studying the regenerative events at cellular and subcellular levels.

Dissezione lordo e Arti di epiteli sensoriali dell&#39;orecchio interno in adulti Zebrafish ( Danio rerio)

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the ...

Dissection of Organs from the Adult Zebrafish

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental analysis of the embryo and larva is extensive and the morphology has been well documented, descriptions of adult zebrafish anatomy and studies of development of the adult structures and organs, together with techniques for working with adults are lacking. The organs of the larva undergo significant changes in their overall structure, morphology, and anatomical location during the larval to adult transition.  Externally, the transparent larva develops its characteristic adult striped pigment pattern and paired pelvic fins, while internally, the organs undergo massive growth and remodeling.  In addition, the bipotential gonad primordium develops into either testis or ovary.  This protocol identifies many of the organs of the adult and demonstrates methods for dissection of the brain, gonads, gastrointestinal system, heart, and kidney of the adult zebrafish. The dissected organs can be used for in situ hybridization, immunohistochemistry, histology, RNA extraction, protein analysis, and other molecular techniques.  This protocol will assist in the broadening of studies in the zebrafish to include the remodeling of larval organs, the morphogenesis of organs specific to the adult and other investigations of the adult organ systems.

This protocol describes a procedure for identifying and dissecting organs from the adult zebrafish.

Dissezione di organi dal Zebrafish adulti

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental ...

Dissection and Staining of Drosophila Larval Ovaries

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells form, and how they interact with their environment is therefore crucial for understanding development, homeostasis and disease. The ovary of the fruit fly Drosophila melanogaster has served as an influential model for the interaction of germ line stem cells (GSCs) with their somatic support cells (niche) 1, 2. The known location of the niche and the GSCs, coupled to the ability to genetically manipulate them, has allowed researchers to elucidate a variety of interactions between stem cells and their niches 3-12.
Despite the wealth of information about mechanisms controlling GSC maintenance and differentiation, relatively little is known about how GSCs and their somatic niches form during development. About 18 somatic niches, whose cellular components include terminal filament and cap cells (Figure 1), form during the third larval instar 13-17. GSCs originate from primordial germ cells (PGCs). PGCs proliferate at early larval stages, but following the formation of the niche a subgroup of PGCs becomes GSCs 7, 16, 18, 19. Together, the somatic niche cells and the GSCs make a functional unit that produces eggs throughout the lifetime of the organism.
Many questions regarding the formation of the GSC unit remain unanswered. Processes such as coordination between precursor cells for niches and stem cell precursors, or the generation of asymmetry within PGCs as they become GSCs, can best be studied in the larva. However, a methodical study of larval ovary development is physically challenging. First, larval ovaries are small. Even at late larval stages they are only 100&mu;m across. In addition, the ovaries are transparent and are embedded in a white fat body. Here we describe a step-by-step protocol for isolating ovaries from late third instar (LL3) Drosophila larvae, followed by staining with fluorescent antibodies. We offer some technical solutions to problems such as locating the ovaries, staining and washing tissues that do not sink, and making sure that antibodies penetrate into the tissue. This protocol can be applied to earlier larval stages and to larval testes as well.

Dissection and Staining of Drosophila Larval Ovaries

How niches and stem cells form during development is an important question with practical implications. In the Drosophila ovary, germ line stem cells and their somatic niches form during larval development. This video demonstrates how to dissect, stain and mount female gonads from late third instar (LL3) Drosophila larvae.

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 ...

Dissection of the Adult Zebrafish Kidney

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem cells during normal homeostasis and disease states. The understanding of adult stem cells has broad reaching implications for the future of regenerative medicine1. For example, better knowledge about adult stem cell biology can facilitate the design of therapeutic strategies in which organs are triggered to heal themselves or even the creation of methods for growing organs in vitro that can be transplanted into humans1. The zebrafish has become a powerful animal model for the study of vertebrate cell biology2. There has been extensive documentation and analysis of embryonic development in the zebrafish3. Only recently have scientists sought to document adult anatomy and surgical dissection techniques4, as there has been a progressive movement within the zebrafish community to broaden the applications of this research organism to adult studies. For example, there are expanding interests in using zebrafish to investigate the biology of adult stem cell populations and make sophisticated adult models of diseases such as cancer5. Historically, isolation of the zebrafish adult kidney has been instrumental for studying hematopoiesis, as the kidney is the anatomical location of blood cell production in fish6,7. The kidney is composed of nephron functional units found in arborized arrangements, surrounded by hematopoietic tissue that is dispersed throughout the intervening spaces. The hematopoietic component consists of hematopoietic stem cells (HSCs) and their progeny that inhabit the kidney until they terminally differentiate8. In addition, it is now appreciated that a group of renal stem/progenitor cells (RPCs) also inhabit the zebrafish kidney organ and enable both kidney regeneration and growth, as observed in other fish species9-11. In light of this new discovery, the zebrafish kidney is one organ that houses the location of two exciting opportunities for adult stem cell biology studies. It is clear that many outstanding questions could be well served with this experimental system. To encourage expansion of this field, it is beneficial to document detailed methods of visualizing and then isolating the adult zebrafish kidney organ. This protocol details our procedure for dissection of the adult kidney from both unfixed and fixed animals.
Dissection of the kidney organ can be used to isolate and characterize hematopoietic and renal stem cells and their offspring using established techniques such as histology, fluorescence activated cell sorting (FACS)11,12, expression profiling13,14, and transplantation11,15. 
We hope that dissemination of this protocol will provide researchers with the knowledge to implement broader use of zebrafish studies that ultimately can be translated for human application.

The zebrafish kidney is home to both renal and hematopoietic adult stem/progenitor cells, and represents an outstanding opportunity to study these cell types and their progeny in a vertebrate model organism. Here, we demonstrate a detailed dissection procedure that enables the researcher to identify and surgically remove the adult zebrafish kidney, which can be used for applications such as cell isolation, transplantation, and expression studies of kidney and/or blood cell populations.

Dissezione del rene Zebrafish adulti

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

In elettroporazione in vivo dei Morpholinos nella retina Zebrafish per adulti

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

Regular Care and Maintenance of a Zebrafish (Danio rerio) Laboratory: An Introduction

This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and behavioural research. As a vertebrate, zebrafish share considerable genetic sequence similarity with humans and are being used as an animal model for various human disease conditions. The advantages of zebrafish in comparison to other common vertebrate models include high fecundity, low maintenance cost, transparent embryos, and rapid development. Due to the spur of interest in zebrafish research, the need to establish and maintain a productive zebrafish housing facility is also increasing. Although literature is available for the maintenance of a zebrafish laboratory, a concise video protocol is lacking. This video illustrates the protocol for regular housing, feeding, breeding and raising of zebrafish larvae. This process will help researchers to understand the natural behaviour and optimal conditions of zebrafish husbandry and hence troubleshoot experimental issues that originate from the fish husbandry conditions. This protocol will be of immense help to researchers planning to establish a zebrafish laboratory, and also to graduate students who are intending to use zebrafish as an animal model.

Regular Care and Maintenance of a Zebrafish Danio rerio Laboratory: An Introduction

This protocol outlines regular maintenance and care to maintain optimal conditions for zebrafish husbandry. The video illustrates the protocol for system maintenance, regular housing, feeding, breeding, and raising of zebrafish larvae.

Cura e manutenzione periodiche di Zebrafish (<em&gt; Danio rerio</em&gt;) Laboratorio: Introduzione

This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and ...

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish. Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated.1 These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present a systematic approach for developing physiologically relevant, sensitive and specific in vivo assays for interpreting variation in human pathology. Transient genetic manipulation via microinjection of WT and mutant human mRNA and morpholino (MO) antisense oligonucleotides harness the tractability of the developing zebrafish embryo to rapidly assay pathogenic mutations, especially, but not exclusively, in the context of human developmental disorders.

In Vivo Modellazione del genoma umano Morbid utilizzando Danio rerio

Here,  we present methods for the development of assays to query potentially clinically  significant nonsynonymous changes using in  vivo complementation ...

Gavaging Adult Zebrafish

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or agents in solutions for scientific research. Current methods for administering compounds orally to adult zebrafish are inaccurate due to variability in voluntary consumption by the fish. A gavage procedure was developed to deliver precise quantities of infectious agents to zebrafish for study in biomedical research. Adult zebrafish over 6 months of age were anesthetized with 150 mg/L of buffered MS-222 and gavaged with 5 &mu;l of solution using flexible catheter implantation tubing attached to a cut 22-G needle tip. The flexible tubing was lowered into the oral cavity of the zebrafish until the tip of the tubing extended past the gills (approximately 1 cm). The solution was then injected slowly into the intestinal tract. This method was effective 88% of the time, with fish recovering uneventfully. This procedure is also efficient as one person can gavage 20-30 fish in one hour. This method can be used to precisely administer agents for infectious diseases studies, or studies of other compounds in adult zebrafish.

The increasing use of zebrafish as an animal model requires the development of effective methods for the delivery of known quantities of compounds and solutions. The gavage procedure described below allows for the oral delivery of precise volumes of solution reliably, safely and efficiently to adult zebrafish.

Gavaging Zebrafish adulti

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or ...