Ringraziamo Beninio Jomok per il suo aiuto in zebrafish allevamento. Questo lavoro è finanziato dal NIH HL81753.

1. Preparazione di diapositive e da camera umidificata <ol><li> La camera umidificata può essere fatto da una scatola, come una scatola di punta svuotato. Avvolgere sia la camera e la copertura con fogli di alluminio per proteggere i campioni dalla luce. </li><li> All&#39;interno della camera, mettere un mucchio di tovaglioli di carta bagnata per mantenere l&#39;umidità all&#39;interno della camera, che impedirà i campioni cuore si asciughi. Impostare un micropiastre piccolo sulla sommità del tovagliolo di carta come una rastrelliera per le diapositive. </li><li> Disegnare sia le linee o cerchi sulla superficie di un vetrino da microscopio utilizzando la penna immEdge per fare i pozzi per i campioni del cuore con una dimensione di circa 5x5 mm. Lasciate asciugare le diapositive. </li><li> Mettere i vetrini in camera umidificata e aggiungere 50ul formaldeide fresca 4% in ogni pozzetto. </li></ol> 2. La dissezione del cuore embrionale <ol><li> Anestetizzare l&#39;embrione di pesce in acqua uovo E3 7 contenente 0,4% tricaine per circa 1 minuto fino a quando il movimento del corpo smettere di pesce ma hanno ancora cuore normalebattere. </li><li> Mettere un vetrino da microscopio concavo sul palco di un microscopio di dissezione. Trasferire gli embrioni al pozzo concavo. </li><li> Regolare la combinazione luce del microscopio da dissezione per rivelare chiaramente il cuore. A seconda delle preferenze personali, potrebbe essere uno dei due campi scuri o DIC-come condizione di luce polarizzata. </li><li> Pulire due siringhe da insulina con il 75% di etanolo, quindi asciugare. </li><li> Regolare il microscopio a dissezione maggiore ingrandimento e mettere a fuoco sul cuore. Fisicamente fissare un embrione con l&#39;inserimento di una punta ago nel tuorlo-somite confine vicino alla testa. Inserire un altro primo ago per la regione del cuore e tirare fuori il cuore in fretta. Se il cuore non è totalmente separata dalla embrione, tagliare il tessuto rimanente collegato tra il cuore e il tuorlo. </li><li> Regolare il microscopio a basso ingrandimento e mettere a fuoco sul cuore separati. Utilizzare un pipetteman 10μl e impostarlo al volume 1ml. Applicare la punta del pipetteman per disegnare una o toccare ilcuore del campione, in modo che il cuore isolato sarà all&#39;interno o attaccato alla superficie della punta. </li><li> Immergere la punta della pipetta nella soluzione di formaldeide al 4% sui vetrini preparati e premere il pistone pipetta per rilasciare il cuore nella soluzione. La tensione di superficie dell&#39;acqua aiuta anche a liberare il cuore dalla punta della pipetta alla soluzione. Mettere meno di 3 cuori in ogni pozzetto. </li></ol> 3. Immunocolorazione <ol><li> Chiudere la camera e metterlo su un agitatore con un movimento dolce dondolio. Fissare il campione di cuore per 20 minuti a temperatura ambiente. </li><li> Asciugare l&#39;acqua sulla parte posteriore delle diapositive e mettere le diapositive sul palco di un microscopio per dissezione tampone scambio. </li><li> Intingere nell&#39;acqua la punta di un pipetteman 10μl in una zona vuota di ogni bene e tenere la punta per quanto possibile dai campioni del cuore. Prendere precauzioni per evitare il cuore allegando alla punta, dato che il cuore può muovere con il flusso dell&#39;acqua quando la soluzione viene disegnato in punta. Cambiarela localizzazione punta, se necessario. </li><li> Smaltire la soluzione su un tessuto ed evitare la produzione di eventuali bolle nella punta perché il campione cuore si perde facilmente in presenza di bolle d&#39;aria. Mantenere i campioni cuore parzialmente a secco per un breve periodo può aiutare i cuori rimanere attaccati alle diapositive. </li><li> Aggiungere 50μl PBST per coprire il cuore del campione e incubare nella camera umidificata in un movimento a dondolo lento per 5 minuti. Ripetere il risciacquo una volta. Dopo questo passo, il cuore dei campioni possono essere conservati nella camera umidificata fino a 1 settimana a 4 ° C. </li><li> Blocca il campione di cuore con siero di pecora 10% in PBST per 1 ora a temperatura ambiente. </li><li> Incubare il campione cuore con anticorpi primari, come la diluizione 1:200 di β-catenina anticorpo e la diluizione di 1:200 MEF2 anticorpo specifico, nel 10% di siero ovino / PBST per 1 ora a temperatura ambiente. </li><li> Lavare il campione cuore con PBST tre volte (5 min.) A temperatura ambiente. </li><li> Incubare il campione con secondarioanticorpi, come ad esempio 1:1000 diluizione di Alexa-tagged anti-mouse e / o anticorpi anti-coniglio, in PBST per 0,5 ore. </li><li> Lavare i campioni cuore con PBST tre volte (5 min.) A temperatura ambiente. </li></ol> 4. Imaging <ol><li> Rimuovere PBST e aggiungere 10 microlitri di montaggio media in ciascun pozzetto. Rock the camera per diversi minuti fino alla soluzione diviene limpida. </li><li> Rimuovere la maggior parte del mezzo di montaggio e coprire tutti i pozzi in una diapositiva con un bicchiere di copertura microscopio (24x50). </li><li> Pesce cuore immagine utilizzando Zeiss Axioplan 2 microscopio dotato di ApoTome (Carl Zeiss). </li></ol> 5. Rappresentante Risultati Un esempio di un&#39;immagine che rivela membrana e nuclei di tutti i cardiomiociti zebrafish è mostrato in fig. 1. anticorpi mef2c e β-catenina sono stati utilizzati insieme per colorare i campioni cuore sezionato da 52 embrioni di pesce zebra HPF. Mentre le macchie di anticorpi mef2c i nuclei di cardiomiociti, β-cateninaanticorpi rivela il confine di ogni 8-10 cellule. Regolando la fase del microscopio composto, le immagini dei campioni del cuore può essere regolata per lo stesso orientamento, che permetterà l&#39;osservazione di curvatura esterno e curvatura interno nel cuore 11. Il numero totale dei cardiomiociti, le dimensioni delle cellule cardiomiociti, e la circolarità può essere misurata. Un altro esempio che rivela la struttura del sarcomero di un cuore embrionale è mostrato in fig. 2. Abbiamo eseguito immunostaining con F59, un anticorpo miosina, in un cuore sezionato embrionale. La rete miofibrilla in un cuore tutto embrionale può essere chiaramente rivelato a basso ingrandimento, mentre la band striato di filamenti spessi può essere rivelato a più alto ingrandimento (Fig. 2) 6. <img alt="Figura 1" src="/files/ftp_upload/3510/3510fig1.jpg" /> Figura 1. Immunocolorazione di MEF2 (rosso) e β-catenina (verde) per mostrare i nuclei e outlines di cardiomiociti in un ventricolo zebrafish. Barra di scala = 10μm <img alt="Figura 2" src="/files/ftp_upload/3510/3510fig2.jpg" /> Figura 2. Immnostaining della miosina per mostrare il filamento spesso in un ventricolo zebrafish sia a basso ingrandimento (A) o ad alto ingrandimento (B). Barra di scala = 10μm

<ol>
	<li>Shin, J. T., Fishman, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+Zebrafish+to+human:+modular+medical+models.">From Zebrafish to human: modular medical models.</a> Annu. Rev. Genomics Hum. Genet. 3, 311-340 (2002).</li><li>Beis, D., Stainier, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+cell+biology:+following+the+zebrafish+trend.">In vivo cell biology: following the zebrafish trend.</a> Trends. Cell. Biol. 16, 105-112 (2006).</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>Nasevicius, A., Ekker, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+targeted+gene+'knockdown'+in+zebrafish.">Effective targeted gene 'knockdown' in zebrafish.</a> Nat. Genet. 26, 216-220 (2000).</li><li>Dahme, T., Katus, H. A., Rottbauer, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fishing+for+the+genetic+basis+of+cardiovascular+disease.">Fishing for the genetic basis of cardiovascular disease.</a> Dis. Model Mech. 2, 18-22 (2009).</li><li>Huang, W., Zhang, R., Xu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myofibrillogenesis+in+the+developing+zebrafish+heart:+A+functional+study+of+tnnt2.">Myofibrillogenesis in the developing zebrafish heart: A functional study of tnnt2.</a> Dev. Biol. 331, 237-249 (2009).</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=.">.</a> The Zebrafish Book. , (2007).</li><li>Buckingham, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle:+the+regulation+of+myogenesis.">Muscle: the regulation of myogenesis.</a> Curr. Opin. Genet. Dev. 4, 745-751 (1994).</li><li>Wheelock, M. J., Knudsen, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cadherins+and+associated+proteins.">Cadherins and associated proteins.</a> Vivo. 5, 505-513 (1991).</li><li>Sun, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+hypertrophy+involves+both+myocyte+hypertrophy+and+hyperplasia+in+anemic+zebrafish.">Cardiac hypertrophy involves both myocyte hypertrophy and hyperplasia in anemic zebrafish.</a> PLoS One. 4, e6596-e6596 (2009).</li><li>Auman, 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=Functional+modulation+of+cardiac+form+through+regionally+confined+cell+shape+changes.">Functional modulation of cardiac form through regionally confined cell shape changes.</a> PLoS Biol. 5, e53-e53 (2007).</li><li>Chen, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depletion+of+zebrafish+essential+and+regulatory+myosin+light+chains+reduces+cardiac+function+through+distinct+mechanisms.">Depletion of zebrafish essential and regulatory myosin light chains reduces cardiac function through distinct mechanisms.</a> Cardiovasc. Res. 79, 97-108 (2008).</li></ol>

Nessun conflitto di interessi dichiarati.

Rispetto ai metodi classici montare tutto immunostaining, il nostro metodo ha i seguenti vantaggi. In primo luogo, molto più forte dei segnali fluorescenti possono essere congruenti a causa di una migliore penetrazione. Nel metodo tutto immunostaining monte, il tessuto cutaneo denso che circonda il cuore ha ridotto significativamente la penetrazione di molti anticorpi, nel fondo di elevata in tutto il corpo. Questo problema è particolarmente grave per gli embrioni di età superiore ai 3 giorni di post-fertilizzazione ...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> tricaine </td><td> Organici di ricerca </td><td> 3007T </td></tr><tr><td> Formaldeide </td><td> Polysciences </td><td> 04018 </td></tr><tr><td> Siringa di insulina </td><td> Becton Dickinson </td><td> 329461 </td></tr><tr><td> Triton X-100 </td><td> Sigma </td><td> T8532 </td></tr><tr><td> Anti-β-catenina anticorpi </td><td> Sigma </td><td> C7207 </td></tr><tr><td> Anticorpi anti-Mef2c </td><td> Santa Cruz Biotech </td><td> SC313 </td></tr><tr><td> F59 </td><td> Zfin </td><td></td></tr><tr><td> Anti-α-actinina anticorpi </td><td> Sigma </td><td> A7811 </td></tr><tr><td> Anticorpi anti-Ilk </td><td> Segnalazione cellulare </td><td> # 3862 </td></tr><tr><td> Alexa fluor 568 capra anti IgG di coniglio </<html> td&gt; <td> Invitrogen </td><td> A11011 </td></tr><tr><td> Alexa fluor 488 capra anti IgG1 di topo </td><td> Invitrogen </td><td> A21121 </td></tr><tr><td> Mezzo di montaggio per fluorescenza </td><td> Vettore </td><td> H-1200 </td></tr><tr><td> ImmEdge penna </td><td> Vettore </td><td> H-4000 </td></tr><tr><td> Poli-L-lisina vetrini rivestiti </td><td> Microscopia elettronica a scienze </td><td> 63410-01 </td></tr><tr><td> Microscopio copertura in vetro </td><td> Pescatore </td><td> 12-543-D </td></tr><tr><td> Vetrini da microscopio concavo </td><td> Pescatore </td><td> 7-1305-8 </td></tr><tr><td> Dissezione microscopio </td><td> Leica MZ95 </td><td></td></tr><tr><td> Microscopio composto </td><td> Zeiss </td><td> Axioplan2 </td></tr><tr><td> ApoTome </td><td> Zeiss </td><td></td></tr></tbody></table> PBST: 1 x PBS 0,5% Triton-X 100 ENT &quot;&gt; 25X Tricaine: 400 mg Tricaine 97,9 mL DDH 2 O 2,1 ml (1M Tris pH9) Regolare il pH a 7,0

immunostaining of dissected zebrafish embryonic heart

Zebrafish embrione diventa un popolare modello vertebrato vivo per lo studio dello sviluppo delle malattie cardiache e cuore umano grazie alla sua vantaggiosa embriologia e genetica 1,2. Circa 100-200 embrioni sono disponibili ogni settimana da una singola coppia di pesci adulti. Gli embrioni che si sviluppano in utero trasparente ex li rendono ideali per la valutazione dei difetti cardiaci 3. L&#39;espressione di un gene può essere manipolato tramite morfolino tecnologia o RNA iniezione 4. Inoltre, in avanti schermi genetici hanno già generato una lista di mutanti che interessano diverse prospettive di cardiogenesis 5. Tutto immunocolorazione mount è una tecnica importante in questo modello animale per rivelare il pattern di espressione della proteina bersaglio di un particolare tessuto 6. Tuttavia, le immagini ad alta risoluzione che possono rivelare le strutture cellulari o subcellulari sono stati difficili, principalmente a causa della Locat fisicoione del cuore e la scarsa penetrazione degli anticorpi. Qui vi presentiamo un metodo per affrontare questi colli di bottiglia da dissezionare il cuore e poi condurre il processo di colorazione sulla superficie di un vetrino da microscopio. Per prevenire la perdita dei campioni piccolo cuore e da facilitare la manipolazione soluzione, abbiamo ristretto il cuore dei campioni all&#39;interno di un cerchio sulla superficie del microscopio diapositive tratte da una penna immEdge. Dopo la colorazione, i segnali di fluorescenza può essere osservata direttamente da un microscopio composto. Il nostro nuovo metodo migliora significativamente la penetrazione per gli anticorpi, perché un cuore da un pesce embrionale costituito solo da pochi strati di cellule. Immagini di alta qualità dal cuore intatto può essere ottenuto in tempi molto ridotti per la processione embrioni di zebrafish anni dal giorno 2 al giorno 6. Il nostro metodo può essere potenzialmente esteso a macchia di altri organi sezionati sia da zebrafish o altri piccoli animali.

Watch this Scientific Journal Video about Immunostaining of Dissected Zebrafish Embryonic Heart at JoVE.com

Immunostaining of Dissected Zebrafish Embryonic Heart

Un modo rapido per condurre immunocolorazione cuore embrionale di zebrafish è descritto. Rispetto alla intero approccio immunostaining monte, questo metodo aumenta notevolmente la penetrazione degli anticorpi, che permette di ottenere immagini ad alta risoluzione che mostrano strutture cellulari / subcellulari nel cuore entro un tempo di elaborazione molto più ridotto.

Immunocolorazione di Dissected Cuore Zebrafish embrionali

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology ...

immunostaining-of-dissected-zebrafish-embryonic-heart

Research

JoVE Journal

Biology

17.6K Views.  Mayo Clinic College of Medicine. Un modo rapido per condurre immunocolorazione cuore embrionale di zebrafish è descritto. Rispetto alla intero approccio immunostaining monte, questo metodo aumenta notevolmente la penetrazione degli anticorpi, che permette di ottenere immagini ad alta risoluzione che mostrano strutture cellulari / subcellulari nel cuore entro un tempo di elaborazione molto più ridotto.

Video: Immunostaining of Dissected Zebrafish Embryonic Heart

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

Vivere Imaging of the Brain Zebrafish embrionali da microscopia confocale

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

Ricerca

Biologia

Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups of cells in a larger context are altered.  Watching how one cell interacts with and behaves in a unique environment is essential to characterizing cell functions.  Determining how the localized misexpression of a specific protein influences surrounding cells provides insightful information on the roles that protein plays in a variety of developmental processes.  Our lab uses the zebrafish model system to uniquely combine genetic approaches with classical transplantation techniques to generate genotypic or phenotypic chimeras.  We study neuron-glial cell interactions during the formation of forebrain commissures in zebrafish. This video describes a method that allows our lab to investigate the role of astroglial populations in the diencephalon and the roles of specific guidance cues that influence projecting axons as they cross the midline. Due to their transparency zebrafish embryos are ideal models for this type of ectopic cell placement or localized gene misexpression. Tracking transplanted cells can be accomplished using a vital dye or a transgenic fish line expressing a fluorescent protein. We demonstrate here how to prepare donor embryos with a vital dye tracer for transplantation, as well as how to extract and transplant cells from one gastrula staged embryo to another.  We present data showing ectopic GFP+ transgenic cells within the forebrain of zebrafish embryos and characterize the location of these cells with respect to forebrain commissures.  In addition, we show laser scanning confocal timelapse microscopy of Alexa 594 labeled cells transplanted into a GFP+ transgenic host embryo.  These data provide evidence that gastrula staged transplantation enables the targeted positioning of ectopic cells to address a variety of questions in Developmental Biology.

Zebrafish cell transplantation enables the combination of genetics and embryology to generate tissue specific chimeras. This video demonstrates gastrula staged cell transplantations that have allowed our lab to investigate the roles of astroglial populations and specific guidance cues during commissure formation in the forebrain.

Tessuto Chimere mirate embrionali: Zebrafish gastrula trapianto di cellule

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

Preparazione di embrioni per la microscopia elettronica del Drosophila Tubo cuore embrionale

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

Microdissection of Zebrafish Embryonic Eye Tissues

Zebrafish is a popular animal model for research on eye development because of its rapid ex utero development and good fecundity. By 3 days post fertilization (dpf), the larvae will show the first visual response. Many genes have been identified to control a proper eye development, but we are far from a complete understanding of the underlying genetic architecture. Whole genome gene expression profiling is a useful tool to elucidate genetic regulatory network for eye development. However, the small size of the embryonic eye in zebrafish makes it challenging to obtain intact and pure eye tissues for expression analysis. For example, the anterior-posterior length of the eye between day 2 and 3 is only approximately 200-300 &mu;m, while the diameter of the lens is less 100 &mu;m. Also, the retinal pigment epithelium (RPE) underlying the retina is just a single-layer epithelium. While gene expression profiles can be obtained from the whole embryo, they do not accurately represent the expression of these tissues. Therefore pure tissue must be obtained for a successful gene expression profiling of eye development. To address this issue, we have developed an approach to microdissect intact retina and retina with RPE attached from 1-3 dpf, which cover major stages of eye morphogenesis. All procedures can be done with fine forceps and general laboratory supplies under standard stereomicroscopes. For retinal dissection, the single-layer RPE is removed and peeled off by brushing action and the preferential adherence of the RPE remnants to the surface of the culture plate for dissection. For RPE-attached retinal dissection, the adherence of RPE to the dissection plate is removed before the dissection so that the RPE can be completely preserved with the retina. A careful lifting action of this tissue can efficiently separate the presumptive choroid and sclera. The lens can be removed in both cases by a chemically etched tungsten needle. In short, our approach can obtain intact eye tissues and has been successfully utilized to study tissue-specific expression profiles of zebrafish retina1, 2 and retinal pigment epithelium3.

This article describes an approach to microdissect zebrafish retinas with and without retinal pigment epithelium attached, from one to three days postfertilization embryos.

Microdissezione dei tessuti oculari Zebrafish embrionali

Zebrafish is a popular animal model for research on eye development because of its rapid ex utero development and good fecundity. By 3 days post ...

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching morphogenesis. We have developed the limb skin vasculature model to study vascular development in which a pre-existing primitive capillary plexus is reorganized into a hierarchically branched vascular network. Whole-mount confocal microscopy with multiple labelling allows for robust imaging of intact blood vessels as well as their cellular components including endothelial cells, pericytes and smooth muscle cells, using specific fluorescent markers. Advances in this limb skin vasculature model with genetic studies have improved understanding molecular mechanisms of vascular development and patterning. The limb skin vasculature model has been used to study how peripheral nerves provide a spatial template for the differentiation and patterning of arteries. This video article describes a simple and robust protocol to stain intact blood vessels with vascular specific antibodies and fluorescent secondary antibodies, which is applicable for vascularized embryonic organs where we are able to follow the process of vascular development.

We introduce a whole-mount immunohistochemistry and laser scanning confocal microscopy with multiple labelling for analyzing intricate vascular network formation in mouse embryonic limb skin.

Con tutto il supporto per l&#39;analisi immunoistochimica vasi embrionali pelle degli arti: un sistema modello per studiare morfogenesi vascolare diramazione in embrione

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching ...

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 ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

Un sistema per<em&gt; Ex vivo</emColtura&gt; del pancreas embrionale

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections (bupivacaine, cardiotoxin or notexin), muscle transplantations (denervation-devascularization induced regeneration), intensive exercise, but also murine muscular dystrophy models such as the mdx mouse (for a review of these approaches see 1). In zebrafish, genetic approaches include mutants that exhibit muscular dystrophy phenotypes (such as runzel2 or sapje3) and antisense oligonucleotide morpholinos that block the expression of dystrophy-associated genes4. Besides, chemical approaches are also possible, e.g. with Galanthamine, a chemical compound inhibiting acetylcholinesterase, thereby resulting in hypercontraction, which eventually leads to muscular dystrophy5. However, genetic and pharmacological approaches generally affect all muscles within an individual, whereas the extent of physically inflicted injuries are more easily controlled spatially and temporally1. Localized physical injury allows the assessment of contralateral muscle as an internal control. Indeed, we recently used laser-mediated cell ablation to study skeletal muscle regeneration in the zebrafish embryo6, while another group recently reported the use of a two-photon laser (822 nm) to damage very locally the plasma membrane of individual embryonic zebrafish muscle cells7.
Here, we report a method for using the micropoint laser (Andor Technology) for skeletal muscle cell injury in the zebrafish embryo. The micropoint laser is a high energy laser which is suitable for targeted cell ablation at a wavelength of 435 nm. The laser is connected to a microscope (in our setup, an optical microscope from Zeiss) in such a way that the microscope can be used at the same time for focusing the laser light onto the sample and for visualizing the effects of the wounding (brightfield or fluorescence). The parameters for controlling laser pulses include wavelength, intensity, and number of pulses.
Due to its transparency and external embryonic development, the zebrafish embryo is highly amenable for both laser-induced injury and for studying the subsequent recovery. Between 1 and 2 days post-fertilization, somitic skeletal muscle cells progressively undergo maturation from anterior to posterior due to the progression of somitogenesis from the trunk to the tail8, 9. At these stages, embryos spontaneously twitch and initiate swimming. The zebrafish has recently been recognized as an important vertebrate model organism for the study of tissue regeneration, as many types of tissues (cardiac, neuronal, vascular etc.) can be regenerated after injury in the adult zebrafish10, 11.

The method presented here comprises the precise injury of live zebrafish embryos with high-energy laser pulses and the subsequent analysis of these injuries and their recovery with time. We also show how genetically labeled single or groups of skeletal muscle cells can be tracked during and after laser light induced damage.

Laser-lesioni del muscolo scheletrico embrionale Zebrafish

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections ...

Analysis of Embryonic and Larval Zebrafish Skeletal Myofibers from Dissociated Preparations

The zebrafish has proven to be a valuable model system for exploring skeletal muscle function and for studying human muscle diseases. Despite the many advantages offered by in vivo analysis of skeletal muscle in the zebrafish, visualizing the complex and finely structured protein milieu responsible for muscle function, especially in whole embryos, can be problematic. This hindrance stems from the small size of zebrafish skeletal muscle (60 &mu;m) and the even smaller size of the sarcomere. Here we describe and demonstrate a simple and rapid method for isolating skeletal myofibers from zebrafish embryos and larvae. We also include protocols that illustrate post preparation techniques useful for analyzing muscle structure and function. Specifically, we detail the subsequent immunocytochemical localization of skeletal muscle proteins and the qualitative analysis of stimulated calcium release via live cell calcium imaging. Overall, this video article provides a straight-forward and efficient method for the isolation and characterization of zebrafish skeletal myofibers, a technique which provides a conduit for myriad subsequent studies of muscle structure and function.

Zebrafish are an emerging system for modeling human disorders of the skeletal muscle. We describe a fast and efficient method to isolate skeletal muscle myofibers from embryonic and larval zebrafish. This method yields a high-density myofiber preparation suitable for study of single skeletal muscle fiber morphology, protein subcellular localization, and muscle physiology.

Analisi delle embrionali e larvali Zebrafish scheletriche miofibre da Preparazioni dissociata

The zebrafish has  proven to be a valuable model system for exploring skeletal muscle function and  for studying human muscle diseases. Despite  the many ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

A Novel<em&gt; Ex vivo</em&gt; Metodo di Cultura per il mouse embrionale del cuore

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

Methods Article

Immunocolorazione di Dissected Cuore Zebrafish embrionali