Nous remercions Beninio Jomok pour son aide dans le poisson-zèbre élevage. Ce travail est financé par le NIH HL81753.

1. Préparation des lames et la chambre humidifié <ol><li> La chambre humide peut être faite d&#39;une boîte comme une boîte de pointe vidé. Enveloppez la fois la chambre et le couvrir de papier aluminium pour protéger les échantillons de la lumière. </li><li> A l&#39;intérieur de la chambre, mettre une pile de serviettes en papier humide pour maintenir l&#39;humidité dans la chambre, ce qui empêchera les échantillons coeur de sécher. Régler une microplaque petits sur le dessus de serviette en papier comme un support pour les diapos. </li><li> Dessinez des lignes ou des cercles, soit sur la surface d&#39;une lame de microscope à l&#39;aide du stylet pour faire immEdge puits pour les échantillons de cœur avec une dimension environ 5x5 mm. Laissez les lames sèches. </li><li> Mettez les diapositives dans la chambre humide et ajouter 50ul de formaldéhyde frais 4% dans chaque puits. </li></ol> 2. La dissection de coeur embryonnaire <ol><li> Anesthetize l&#39;embryon de poisson dans l&#39;eau d&#39;œuf E3 7 contenant 0,4% tricaïne pendant environ 1 minute jusqu&#39;à ce que le mouvement du corps du poisson arrêter, mais ont encore cardiaque normalbattre. </li><li> Mettez une lame de microscope concave sur la platine d&#39;un microscope de dissection. Transfert des embryons pour le bien concave. </li><li> Ajustez la combinaison de la lumière du microscope disséquant révéler clairement le cœur. Selon les préférences personnelles, il peut être soit en champ sombre ou DIC-comme condition de la lumière polarisée. </li><li> Nettoyer deux seringues d&#39;insuline avec de l&#39;éthanol 75%, puis séchez. </li><li> Régler le microscope à dissection au plus fort grossissement et de se concentrer sur le cœur. Fixer physiquement un embryon en insérant une aiguille dans la limite de jaune-somite près de la tête. Insérez un autre proche aiguille pour la région du coeur et tirez le cœur rapidement. Si le cœur n&#39;est pas totalement séparé de l&#39;embryon, couper le tissu restant connecté entre le cœur et le jaune. </li><li> Régler le microscope faible grossissement et de se concentrer sur le cœur séparé. Utilisez un pipetteman 10 ul et réglez le volume 1 microlitre. Appliquer la pointe de l&#39;pipetteman à dessiner ou toucher lescoeur de l&#39;échantillon, de sorte que le cœur isolé sera à l&#39;intérieur ou attachés à la surface de la pointe. </li><li> Trempez la pointe de la pipette dans la solution de formaldéhyde à 4% sur les lames préparées et appuyer sur le piston pipette pour libérer le cœur dans la solution. La tension de surface de l&#39;eau aide aussi à libérer le cœur de pointe de la pipette à la solution. Mettez moins de 3 coeurs dans chaque puits. </li></ol> 3. Immunocoloration <ol><li> Fermer la chambre et le mettre sur un shaker avec un mouvement doux balancement. Fixer l&#39;échantillon coeur pendant 20 minutes à température ambiante. </li><li> Essuyez l&#39;eau sur le dos de la glisse et de mettre la glisse sur la platine d&#39;un microscope de dissection pour le tampon de l&#39;échange. </li><li> Tremper la pointe d&#39;un pipetteman 10 ul dans une région vide dans chaque puits et de garder la pointe autant que possible à partir d&#39;échantillons du cœur. Prenez garde à éviter le cœur attachés à l&#39;extrémité, car le cœur peut se déplacer avec le débit d&#39;eau lorsque la solution est aspiré dans l&#39;embout. Changerla localisation pointe, si nécessaire. </li><li> Eliminer la solution sur un mouchoir et éviter de produire des bulles dans la pointe du cœur, puisque l&#39;échantillon est facilement perdu dans la présence de bulles d&#39;air. Garder les échantillons coeur sécher partiellement pour une courte durée peut aider les cœurs restent attachés à la glisse. </li><li> Ajouter 50 pl PBST pour couvrir l&#39;échantillon coeur et incuber dans la chambre humide dans un mouvement de bascule lente pendant 5 min. Répéter le rinçage une fois de plus. Après cette étape, les échantillons du cœur peuvent être stockés dans la chambre humidifié jusqu&#39;à 1 semaine à 4 ° C. </li><li> Bloc de l&#39;échantillon coeur avec du sérum de mouton de 10% en PBST pendant 1 heure à température ambiante. </li><li> Incuber l&#39;échantillon coeur avec des anticorps primaires, tels que la dilution de 1:200 β-caténine anticorps et la dilution de 1:200 MEF2 anticorps spécifique, chez les ovins 10% de sérum / PBST pendant 1 heure à température ambiante. </li><li> Laver l&#39;échantillon avec PBST coeur à trois reprises (5 min. Chacun) à température ambiante. </li><li> Incuber l&#39;échantillon avec du secondaireanticorps, comme dilution de 1:1000 d&#39;Alexa-marqués anti-souris et / ou anticorps anti-lapin, dans le PBST pendant 0,5 heure. </li><li> Laver les échantillons coeur avec PBST trois reprises (5 min. Chacun) à température ambiante. </li></ol> 4. Imagerie <ol><li> Retirer PBST et ajouter 10 pl de milieu de montage à chaque puits. Rocher de la chambre pendant quelques minutes jusqu&#39;à ce que la solution devient limpide. </li><li> Retirer la plupart des moyennes de montage et couvrent l&#39;ensemble des puits dans une diapositive avec un couvercle en verre de microscope (24x50). </li><li> Coeur de poissons à l&#39;aide d&#39;images Zeiss Axioplan 2 microscope équipé d&#39;ApoTome (Carl Zeiss). </li></ol> 5. Les résultats représentatifs Un exemple d&#39;une image qui révèle la membrane et des noyaux de tous les cardiomyocytes poisson zèbre est montré dans la Fig. 1. anticorps mef2c et β-caténine ont été utilisés ensemble pour colorer les échantillons coeur disséqué à partir de 52 embryons de poissons zèbres HPF. Alors que les taches d&#39;anticorps mef2c les noyaux de cardiomyocytes, β-caténined&#39;anticorps révèle la frontière de chaque cellule de 80-10. En ajustant la platine du microscope composé, des images des échantillons cœur peut être ajusté à la même orientation, ce qui permettra l&#39;observation de la courbure extérieure et courbure interne dans le coeur 11. Le nombre total des cardiomyocytes, la taille des cellules cardiomyocytes, et la circularité peut alors être mesuré. Un autre exemple qui révèle la structure du sarcomère d&#39;un cœur embryonnaire est montré dans la Fig. 2. Nous avons effectué immunomarquage utilisant F59, un anticorps myosine, dans un cœur découpé embryonnaire. Le réseau myofibrille dans un coeur entier embryonnaires peuvent être clairement révélé à faible grossissement, tandis que la bande striée de filaments d&#39;épaisseur peut être révélé à un plus fort grossissement (Fig. 2) 6. <img alt="Figure 1" src="/files/ftp_upload/3510/3510fig1.jpg" /> Figure 1. Immunocoloration des MEF2 (rouge) et β-caténine (vert) pour montrer les noyaux et outlines de cardiomyocytes dans un ventricule du poisson zèbre. Barre d&#39;échelle = 10 microns <img alt="Figure 2" src="/files/ftp_upload/3510/3510fig2.jpg" /> Figure 2. Immnostaining de la myosine de montrer le filament épais dans un ventricule zebrafish soit à un faible grossissement (A) ou à fort grossissement (B). Barre d&#39;échelle = 10 microns

<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>

Pas de conflits d&#39;intérêt déclarés.

Comparé à l&#39;ensemble des méthodes classiques immunomarquage monter, notre méthode présente les avantages suivants. D&#39;abord, beaucoup plus fort des signaux fluorescents peuvent être systématiquement obtenu grâce à une meilleure pénétration. Dans la méthode immunologique monter ensemble, les tissus de la peau épaisse qui entoure le cœur significativement réduit la pénétration de nombreux anticorps, résultant en bruit de fond élevé dans le corps entier. Ce problème est particulièrement grave p...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Société </td><td> Numéro de catalogue </td></tr><tr><td> tricaïne </td><td> Organiques de recherche </td><td> 3007T </td></tr><tr><td> Formaldéhyde </td><td> Polysciences </td><td> 04018 </td></tr><tr><td> Seringue à insuline </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-caténine d&#39;anticorps </td><td> Sigma </td><td> C7207 </td></tr><tr><td> Anti-Mef2c d&#39;anticorps </td><td> Santa Cruz Biotech </td><td> SC313 </td></tr><tr><td> F59 </td><td> Zfin </td><td></td></tr><tr><td> Anti-α-actinine d&#39;anticorps </td><td> Sigma </td><td> A7811 </td></tr><tr><td> Anti-Ilk d&#39;anticorps </td><td> La signalisation cellulaire </td><td> # 3862 </td></tr><tr><td> Alexa Fluor 568 de chèvre anti IgG de lapin </<html> td&gt; <td> Invitrogen </td><td> A11011 </td></tr><tr><td> Alexa Fluor 488 chèvre anti-souris IgG1 </td><td> Invitrogen </td><td> A21121 </td></tr><tr><td> Milieu de montage pour la fluorescence </td><td> Vecteur </td><td> H-1200 </td></tr><tr><td> ImmEdge stylo </td><td> Vecteur </td><td> H-4000 </td></tr><tr><td> Poly-L-lysine lames revêtues </td><td> La microscopie électronique en sciences </td><td> 63410-01 </td></tr><tr><td> Couvercle en verre de microscope </td><td> Fisher </td><td> 12 à 543-D </td></tr><tr><td> Lames de microscope concave </td><td> Fisher </td><td> 7-1305-8 </td></tr><tr><td> Microscope à dissection </td><td> Leica MZ95 </td><td></td></tr><tr><td> Microscope composé </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% de Triton X-100 ent &quot;&gt; 25X tricaïne: 400 mg de tricaïne 97,9 ml ddH 2 O 2,1 ml (1M Tris pH9) Ajuster le pH à 7,0

immunostaining of dissected zebrafish embryonic heart

L&#39;embryon de poisson zèbre devient un populaire dans le modèle in vivo pour l&#39;étude des vertébrés développement cardiaque et les maladies cardiaques humaines en raison de sa embryologie et la génétique avantageuse 1,2. Environ 100-200 embryons sont disponibles chaque semaine d&#39;un seul couple de poissons adultes. Les embryons transparents qui se développent ex utero les rendent idéales pour évaluer les défauts cardiaques 3. L&#39;expression de tout gène peut être manipulé par l&#39;intermédiaire morpholino la technologie d&#39;injection ou de l&#39;ARN 4. Par ailleurs, avant de cribles génétiques ont déjà généré une liste de mutants qui affectent différentes perspectives de cardiogenèse 5. Whole immunomarquage de montage est une technique importante dans ce modèle animal de révéler le profil d&#39;expression de la protéine ciblée à un tissu particulier 6. Toutefois, les images à haute résolution qui peut révéler des structures cellulaires ou subcellulaires ont été difficiles, principalement en raison de la locat physiquesion du cœur et la faible pénétration de l&#39;anticorps. Ici, nous présentons une méthode pour répondre à ces goulots d&#39;étranglement en disséquant le cœur d&#39;abord et ensuite la conduite du processus de taches sur la surface d&#39;une lame de microscope. Pour éviter la perte des échantillons de petit coeur et pour faciliter la manipulation solution, nous avons limité les échantillons coeur dans un cercle à la surface du microscope diapositives tirées par un stylo immEdge. Après la coloration, les signaux de fluorescence peut être directement observé par un microscope composé. Notre nouvelle méthode améliore significativement la pénétration des anticorps, car un cœur à partir d&#39;un embryon de poisson ne se compose que de quelques couches de cellules. Des images de haute qualité de coeurs intacts peuvent être obtenus dans un délai très réduit pour le défilé des embryons de poisson zèbre âgés de jour 2 au jour 6. Notre méthode peut être potentiellement étendue à d&#39;autres organes disséqués tache soit de poisson zèbre ou autres petits animaux.

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

Immunostaining of Dissected Zebrafish Embryonic Heart

Un moyen rapide pour effectuer immunocoloration des embryons de poisson zèbre coeur est décrite. Comparativement à l&#39;approche immunologique monter ensemble, cette méthode augmente considérablement la pénétration de l&#39;anticorps, ce qui permet d&#39;obtenir des images haute résolution qui révèlent des structures cellulaires / subcellulaires dans le coeur dans un temps de traitement beaucoup plus réduite.

Immunocoloration des Disséqué coeur embryonnaire Zebrafish

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 vues.  Mayo Clinic College of Medicine. Un moyen rapide pour effectuer immunocoloration des embryons de poisson zèbre coeur est décrite. Comparativement à l'approche immunologique monter ensemble, cette méthode augmente considérablement la pénétration de l'anticorps, ce qui permet d'obtenir des images haute résolution qui révèlent des structures cellulaires / subcellulaires dans le coeur dans un temps de traitement beaucoup plus réduite.

Vidéo: 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.

En direct l&#39;imagerie du cerveau de poisson zèbre embryonnaires par microscopie 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 ...

Recherche

Biologie

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.

Tissu ciblé chimères embryonnaires: Zebrafish cellulaire Gastrula 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 ...

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.

Préparation d&#39;embryons pour la microscopie électronique des Drosophile Tube cardiaque embryonnaire

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.

Microdissection de tissus embryonnaires Zebrafish Eye

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.

Whole-Mont analyse immunohistochimique à la vascularisation cutanée Limb embryonnaires: un modèle pour l&#39;étude vasculaire morphogenèse de branchement de l&#39;embryon

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.

Dissection du cœur chez les larves, le poisson zèbre juvéniles et adultes, Danio rerio</em

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

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 système d&#39;<em&gt; Ex vivo</emLa culture&gt; de pancréas embryonnaire

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-infligé des blessures de poisson zèbre muscle squelettique embryonnaire

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.

Analyse des embryonnaire et larvaire de poisson-zèbre squelettiques fibres musculaires de préparations dissociées

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; Méthode de la culture pour le embryonnaire Souris Coeur

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