Wir danken Beninio Jomok für seine Hilfe beim Zebrafisch Tierhaltung. Diese Arbeit wird durch NIH HL81753 finanziert.

1. Vorbereitung von Dias und Feuchtekammer <ol><li> Die befeuchteten Kammer kann aus einer Kiste wie ein geleert tip box erfolgen. Wrap sowohl der Kammer und der mit Alufolie abdecken, um die Proben vor Licht zu schützen. </li><li> Im Inneren der Kammer, legte einen Haufen nasse Papiertücher, um die Feuchtigkeit in der Kammer, die das Herz Proben vor dem Austrocknen wird verhindert, aufrecht zu erhalten. Setzen Sie einen kleinen Microplatte auf der Oberseite des Papiertuch als Ablage für die Folien. </li><li> Zeichnen Sie Linien oder Kreise auf der Oberfläche eines Objektträgers mit dem immEdge Stift Brunnen für Herz-Proben mit einer Größe ca. 5x5 mm zu machen. Lassen Sie die Folien trocken. </li><li> Legen Sie die Folien in der feuchten Kammer und fügen 50ul frisch 4% Formaldehyd in jede Vertiefung. </li></ol> 2. Dissection von embryonalen Herzen <ol><li> Anesthetize sich der Embryo in E3 Ei Wasser 7 mit 0,4% Tricaine für ca. 1 Minute bis der Fisch zu stoppen Bewegung des Körpers, aber immer noch normales Herzzu schlagen. </li><li> Legen Sie eine konkave Objektträger auf der Bühne eines Dissektionsmikroskop. Übertragen Sie die Embryonen in die konkave gut. </li><li> Passen Sie das Licht Kombination der Präpariermikroskop zu zeigen deutlich das Herz. Je nach persönlicher Vorliebe, könnte es entweder Dunkelfeld oder DIC-like polarisiertem Licht Zustand sein. </li><li> Saubere zwei Insulin-Spritzen mit 75% Ethanol, dann trocknen lassen. </li><li> Passen Sie die Dissektionsmikroskop höhere Vergrößerung und Fokussierung auf das Herz. Körperlich fix ein Embryo durch Einstecken mit einem Nadelspitze in den Dotter-Somiten Grenze in der Nähe des Kopfes. Legen Sie eine andere Nadel in der Nähe der Herzgegend und ziehen das Herz schnell. Wenn das Herz nicht völlig aus dem Embryo getrennt, schneiden Sie die restlichen angeschlossenen Gewebe zwischen dem Herzen und dem Eigelb. </li><li> Passen Sie das Mikroskop geringer Vergrößerung und Fokussierung auf die getrennte Herzen. Verwenden Sie eine 10 &amp;mgr; l pipetteman und setzen Sie ihn auf 1μl Volumen. Übernehmen Sie die Spitze des pipetteman entweder ziehen oder berühren Sie dieHerz Probe, so dass das isolierte Herz wird entweder innerhalb oder an der Oberfläche der Spitze sein. </li><li> Tauchen Sie die Pipettenspitze in 4% Formaldehyd-Lösung auf vorbereitete Objektträger und drücken Sie die Pipette Kolben an das Herz in die Lösung veröffentlichen. Die Spannung der Wasseroberfläche hilft auch das Herz von Pipettenspitze, um die Lösung veröffentlichen. Legen Sie weniger als 3 Herzen in jede Vertiefung. </li></ol> 3. Immunfärbung <ol><li> Schließen Sie die Kammer und legte es auf einem Schüttler mit einer sanften Schaukelbewegung. Fix Herzen Probe für 20 Minuten bei Raumtemperatur. </li><li> Wischen Sie das Wasser auf der Rückseite der Folien und legen die Folien auf der Bühne eines Dissektionsmikroskop für Puffer auszutauschen. </li><li> Tauchen Sie die Spitze von einem 10 &amp;mgr; l pipetteman in einen leeren Bereich in jede Vertiefung und halten Sie die Spitze so weit wie möglich von Herz-Proben. Nehmen Sie Vorsichtsmaßnahmen, um das Herz Befestigung an der Spitze zu vermeiden, da Herzen mit dem Wasserstrom zu bewegen könnte, wenn die Lösung in die Spitze gezogen wird. Änderungdie Spitze Lokalisierung, falls nötig. </li><li> Entsorgen Sie die Lösung auf ein Gewebe und vermeiden jegliche Blasen in die Spitze, da das Herz Probe wird leicht in Gegenwart von Luftblasen verloren. Keeping das Herz Proben teilweise trocken für eine kurze Zeit kann helfen, die Herzen zu bleiben an den Folien. </li><li> Add 50 ul PBST das Herz Probe abdecken und inkubieren in der feuchten Kammer in eine langsame Schaukelbewegung für 5 min. Wiederholen Sie die Spülung noch einmal. Nach diesem Schritt kann das Herz Proben in der feuchten Kammer bis zu 1 Woche bei 4 ° C gelagert werden </li><li> Blockieren Sie die Herzen Probe mit 10% Schafserum in PBST für 1 Stunde bei Raumtemperatur. </li><li> Inkubieren Sie die Herzen Probe mit primärer Antikörper, wie zB 1:200 Verdünnung von β-Catenin-Antikörper und 1:200 Verdünnung MEF2 spezifischen Antikörper, in 10% Schafserum / PBST für 1 Stunde bei Raumtemperatur. </li><li> Waschen Sie die Herzen Probe mit PBST dreimal (5 min.) Bei Raumtemperatur. </li><li> Inkubieren Sie die Probe mit sekundärenAntikörper, wie 1:1000 Verdünnung von Alexa-markierten anti-Maus und / oder anti-Kaninchen-Antikörper, in PBST 0,5 Stunden. </li><li> Waschen Sie die Herzen Proben mit PBST dreimal (5 min.) Bei Raumtemperatur. </li></ol> 4. Imaging <ol><li> Entfernen PBST und mit 10 ul Eindeckmedium in jede Vertiefung. Rock the Kammer für einige Minuten, bis die Lösung klar wird. </li><li> Entfernen Sie die meisten der Eindeckmedium und decken alle der Brunnen in eine Folie mit einem Mikroskop Deckglas (24x50). </li><li> Bild Fisch Herz mit Zeiss Axioplan 2 Mikroskop mit ApoTome (Carl Zeiss) ausgestattet. </li></ol> 5. Repräsentative Ergebnisse Ein Beispiel für ein Bild, das Membran und Kerne aller Zebrafisch Kardiomyozyten offenbart, ist in Abb. dargestellt. 1. mef2c und β-Catenin-Antikörper wurden zusammen, um Herz-Proben von 52 hpf Zebrafischembryo seziert Flecken verwendet. Während mef2c Antikörper färbt die Kerne von Herzmuskelzellen, β-CateninAntikörper zeigt die Grenze der einzelnen Zellen 8-10. Durch die Anpassung der Phase des zusammengesetzten Mikroskop können die Bilder des Herzens Proben der gleichen Orientierung, die die Beobachtung der äußeren Krümmung und der inneren Krümmung im Herzen 11 ermöglichen wird eingestellt werden. Die gesamte Anzahl Kardiomyozyten, Kardiomyozyten Zellgröße und Zirkularität kann dann gemessen werden. Ein weiteres Beispiel, das Sarkomer Struktur eines embryonalen Herzens offenbart, ist in Abb. dargestellt. 2. Wir führten Immunfärbung mit F59, ein Myosin-Antikörper, in einem aufgeschnittenen embryonalen Herzen. Die Myofibrillen Netzwerk in eine ganze embryonale Herz kann deutlich niedrigeren Vergrößerung offenbart werden, während die gestreiften Band aus dickem Faden bei höherer Vergrößerung (Abb. 2) 6 enthüllt werden kann. <img alt="Abbildung 1" src="/files/ftp_upload/3510/3510fig1.jpg" /> Abbildung 1. Immunfärbung von MEF2 (rot) und β-Catenin (grün), die Kerne und outl zeigenines der Kardiomyozyten in einem Zebrafisch Ventrikels. Balken = 10 um <img alt="Abbildung 2" src="/files/ftp_upload/3510/3510fig2.jpg" /> Abbildung 2. Immnostaining von Myosin der dicken Filamente in einem Zebrafisch Ventrikel entweder geringer Vergrößerung (A) oder mit hoher Vergrößerung (B) zu zeigen. Balken = 10 um

<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>

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Im Vergleich zu klassischen whole mount Immunfärbung Methoden, hat unsere Methode folgende Vorteile. Erstens kann viel stärkere Fluoreszenzsignale konsequent erreicht werden durch die verbesserte Penetration. In der ganze Berg Immunfärbung Methode, die dichten Haut umgebende Gewebe des Herzens deutlich reduziert das Eindringen von vielen Antikörper, was zu hohen Hintergrund in den ganzen Körper. Dieses Problem ist besonders gravierend für Embryonen älter als 3-Tages-post-Fertilisation (DPF). Im Gegensatz dazu sez...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td></tr><tr><td> Tricaine </td><td> Research Organics </td><td> 3007T </td></tr><tr><td> Formaldehyd </td><td> Polysciences </td><td> 04018 </td></tr><tr><td> Insulin Spritze </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-Catenin-Antikörper </td><td> Sigma </td><td> C7207 </td></tr><tr><td> Anti-Mef2c Antikörper </td><td> Santa Cruz Biotech </td><td> SC313 </td></tr><tr><td> F59 </td><td> Zfin </td><td></td></tr><tr><td> Anti-α-Antikörper actinin </td><td> Sigma </td><td> A7811 </td></tr><tr><td> Anti-Ilk Antikörper </td><td> Zelluläre Signale </td><td> # 3862 </td></tr><tr><td> Alexa Fluor 568 Ziege anti-Kaninchen IgG </<html> td&gt; <td> Invitrogen </td><td> A11011 </td></tr><tr><td> Alexa Fluor 488 Ziege anti-Maus IgG1 </td><td> Invitrogen </td><td> A21121 </td></tr><tr><td> Eindeckmedium für die Fluoreszenz- </td><td> Vektor </td><td> H-1200 </td></tr><tr><td> ImmEdge Stift </td><td> Vektor </td><td> H-4000 </td></tr><tr><td> Poly-L-Lysin beschichteten Objektträger </td><td> Electron Microscopy Sciences </td><td> 63410-01 </td></tr><tr><td> Mikroskop Deckglas </td><td> Fischer </td><td> 12 bis 543-D </td></tr><tr><td> Konkave Objektträger </td><td> Fischer </td><td> 7-1305-8 </td></tr><tr><td> Dissektionsmikroskop </td><td> Leica MZ95 </td><td></td></tr><tr><td> Zusammengesetztes Mikroskop </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 pH 9) Der pH-Wert auf 7,0

immunostaining of dissected zebrafish embryonic heart

Zebrafisch-Embryo wird zu einem beliebten In-vivo-Modell zum Studium der Wirbeltiere kardiale Entwicklung und menschlichen Herzerkrankungen aufgrund seiner vorteilhaften Embryologie und Genetik 1,2. Etwa 100-200 Embryonen sind leicht zugänglich jede Woche von einem einzigen Paar von erwachsenen Fischen. Die transparente Embryonen, die ex utero entwickeln eignen sie sich ideal für die Beurteilung der Herzfehler 3. Der Ausdruck eines beliebigen Gens kann über Morpholino Technologie-oder RNA-Injektion 4 manipuliert werden. Darüber hinaus freuen genetische Screens haben bereits eine Liste von Mutanten, die unterschiedlichen Perspektiven der Kardiogenese 5 berühren generiert. Whole mount Immunfärbung ist eine wichtige Technik in diesem Tiermodell die Expressionsmuster des Zielproteins zu einem bestimmten Gewebe 6 offenbaren. Allerdings haben Bilder mit hoher Auflösung, dass zelluläre oder subzelluläre Strukturen offenbaren kann schwierig gewesen, vor allem aufgrund der physikalischen location des Herzens und der schlechten Penetration der Antikörper. Hier stellen wir eine Methode, um diese Engpässe durch Sezieren Herz zuerst und dann die Durchführung der Färbung auf der Oberfläche eines Objektträgers Adresse. Um den Verlust von kleinen Herzen Proben zu verhindern und die Lösung Handhabung zu erleichtern, haben wir eingeschränkt Herzen Proben innerhalb eines Kreises auf der Oberfläche der Objektträger durch eine immEdge Stift gezeichnet. Nach der Färbung kann die Fluoreszenz-Signale direkt von einem zusammengesetzten Mikroskop beobachtet werden. Unsere neue Methode verbessert das Eindringen von Antikörpern, da ein Herz aus einem embryonalen Fisch besteht nur aus wenigen Zellschichten. Qualitativ hochwertige Bilder von intakten Herzen kann innerhalb eines stark reduzierten Prozession Zeit für Zebrafischembryonen im Alter von Tag 2 bis Tag 6 gewonnen werden. Unsere Methode kann möglicherweise ausgeweitet werden auf andere Organe entweder von Zebrafisch oder andere kleine Tiere seziert Fleck.

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

Immunostaining of Dissected Zebrafish Embryonic Heart

Eine schnelle Weg zur Immunfärbung von Zebrafisch-Embryonen Herzen Verhalten beschrieben wird. Im Vergleich zu der ganze Berg Immunfärbung Ansatz, diese Methode drastisch erhöht die Penetration der Antikörper, die die Erzielung einer hohen Auflösung, dass zelluläre / subzelluläre Strukturen offenbaren sich im Herzen innerhalb von einer deutlich reduzierten Bearbeitungszeit ermöglicht.

Immunfärbung von Dissected Zebrafisch embryonale Herz

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 Aufrufe.  Mayo Clinic College of Medicine. Eine schnelle Weg zur Immunfärbung von Zebrafisch-Embryonen Herzen Verhalten beschrieben wird. Im Vergleich zu der ganze Berg Immunfärbung Ansatz, diese Methode drastisch erhöht die Penetration der Antikörper, die die Erzielung einer hohen Auflösung, dass zelluläre / subzelluläre Strukturen offenbaren sich im Herzen innerhalb von einer deutlich reduzierten Bearbeitungszeit ermöglicht.

Serie: 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.

Live-Imaging der Zebrafisch embryonalen Gehirn durch konfokale Mikroskopie

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 ...

Forschung

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.

Tissue Gezielte Embryonale Chimären: Zebrafisch 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 ...

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.

Vorbereitung der Embryonen für Elektronenmikroskopie der Drosophila Embryonale Herz Rohr

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.

Mikrodissektion von Zebrafisch Embryonale Augentücher

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-mount Immunhistochemische Analyse für Embryonale Limb Haut Gefäßsystem: ein Modellsystem zur Vascular Branching Morphogenese in Embryo-Studie

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.

Herz Dissection in Larven, juvenile und adulte Zebrafisch, 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.

Ein System für<em&gt; Ex vivo</em&gt; Kultivierung von embryonalen Pankreas

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-Schädigung der Zebrafisch Embryonale Skeletal Muscle

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 der embryonalen Zebrafisch-Larven-und Skelett Muskelfasern aus Dissoziierte Vorbereitungen

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; Kultur Verfahren zur embryonalen Maus Herz

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