Damos las gracias a Beninio Jomok por su ayuda en la cría de pez cebra. Este trabajo está financiado por el NIH HL81753.

1. Preparación de las placas y cámara húmeda <ol><li> La cámara húmeda se puede hacer de una caja como una caja vacía punta. Envuelva tanto la cámara y la tapa con papel de aluminio para proteger las muestras de la luz. </li><li> Dentro de la cámara, poner un montón de toallas de papel húmedo para mantener la humedad dentro de la cámara, lo que evitará que las muestras de corazón de secado. Establecer una microplaca pequeña en la parte superior de la toalla de papel en un estante para las diapositivas. </li><li> Dibujar líneas o círculos en la superficie de un portaobjetos de microscopio utilizando el lápiz immEdge hacer pozos para las muestras de corazón con una dimensión de 5x5 mm. Deje que los portaobjetos secos. </li><li> Poner los portaobjetos en la cámara húmeda y añadir 50 ul formaldehído fresco 4% en cada pocillo. </li></ol> 2. La disección del corazón embrionario <ol><li> Anestesiar a los embriones de pez en el agua de huevo E3 7, que contiene 0,4% tricaína de 1 minuto hasta que el movimiento del cuerpo de peces, pero todavía tienen parada normal del corazónpaliza. </li><li> Ponga un portaobjetos cóncavo en el escenario de un microscopio de disección. Transferir los embriones al pozo cóncavo. </li><li> Ajustar la combinación de la luz del microscopio de disección para revelar claramente el corazón. Dependiendo de la preferencia personal, podría ser cualquiera de campo oscuro o CID-como condición de la luz polarizada. </li><li> Limpiar dos jeringas de insulina con un 75% de etanol, luego se seca. </li><li> Ajustar el microscopio de disección a un aumento mayor y se centran en el corazón. Para fijar físicamente un embrión mediante la inserción de una aguja en el límite con la yema somite cerca de la cabeza. Insertar otra aguja cerca de la región del corazón y tirar el centro hacia fuera rápidamente. Si el corazón no está totalmente separada del embrión, cortar el tejido que ha quedado conectado entre el corazón y la yema. </li><li> Ajustar el microscopio para reducir la ampliación y se centran en el corazón separado. Use un pipetteman 10μl y configurarlo para que el volumen 1μl. Aplicar la punta de la pipetteman Para dibujar o tocar elcorazón de la muestra, de manera que el corazón aislado será dentro o adjunto a la superficie de la punta. </li><li> Sumerja la punta de la pipeta en la solución de formaldehído al 4% en portaobjetos preparados y bajar el pistón de la pipeta para liberar el corazón en la solución. La tensión de la superficie del agua también ayuda a liberar el corazón de la punta de la pipeta a la solución. Con menos del 3 corazones en cada pocillo. </li></ol> 3. Inmunotinción <ol><li> Cerrar la cubeta y lo puso en la coctelera con un movimiento de vaivén. Fijar la muestra del corazón durante 20 minutos a temperatura ambiente. </li><li> Limpie el agua en la parte posterior de las diapositivas y poner las diapositivas en el escenario de un microscopio de disección para el intercambio de buffer. </li><li> Que moje la punta de un pipetteman 10μl en una región vacía en cada pozo y mantener la punta de la medida de lo posible a partir de muestras de corazón. Tomar precauciones para evitar que el corazón inherentes a la punta, ya que los corazones puedan moverse con el flujo de agua cuando la solución se introduce en la punta. Cambiola localización de la punta, si es necesario. </li><li> Deseche la solución en un pañuelo y evitar la producción de las burbujas en la punta ya que la muestra del corazón se pierde fácilmente en la presencia de burbujas de aire. Mantener las muestras de corazón parcialmente seco por un corto tiempo puede ayudar a los corazones de permanecer conectados a las diapositivas. </li><li> Añadir 50μl PBST para cubrir la muestra y se incuba en el corazón de la cámara húmeda en un movimiento de balanceo lento durante 5 minutos. Repita el enjuague una vez más. Después de este paso, las muestras del corazón pueden ser almacenados en la cámara húmeda hasta 1 semana a 4 ° C. </li><li> Bloque de la muestra de corazón con suero de oveja 10% en PBST durante 1 hora a temperatura ambiente. </li><li> Incubar la muestra de corazón con los anticuerpos primarios, tales como dilución 1:200 de la β-catenina de anticuerpos y la dilución 1:200 de MEF2 anticuerpo específico, en el 10% suero de oveja / PBST durante 1 hora a temperatura ambiente. </li><li> Lavar la muestra con el corazón PBST tres veces (5 min. Cada uno) a temperatura ambiente. </li><li> Incubar la muestra con la secundariaanticuerpos, tales como dilución 1:1000 de Alexa-etiquetados anti-ratón y / o anticuerpos anti-conejo, en PBST durante 0,5 horas. </li><li> Lavado de las muestras de corazón con PBST tres veces (5 min. Cada uno) a temperatura ambiente. </li></ol> 4. Imágenes <ol><li> Quitar PBST y se añaden 10 l medio de montaje en cada pocillo. Rocas de la cámara durante varios minutos hasta que la solución se aclare. </li><li> Eliminar la mayor parte del medio de montaje y cubrir todos los pozos en una diapositiva con una cubierta de cristal de microscopio (24x50). </li><li> Corazón la imagen de peces utilizando Zeiss Axioplan 2 microscopio equipado con ApoTome (Carl Zeiss). </li></ol> 5. Resultados representante Un ejemplo de una imagen que revela la membrana y el núcleo de todos los cardiomiocitos del pez cebra se muestra en la fig. 1. anticuerpos MEF2C y β-catenina se utiliza junto a manchar las muestras de corazón disecado de 52 HPF embrión de pez cebra. Mientras que las manchas de MEF2C anticuerpos de los núcleos de cardiomiocitos, β-cateninaanticuerpos revela el borde de cada celda de 8.10. Mediante el ajuste de la platina del microscopio compuesto, las imágenes de las muestras de corazón se puede ajustar a la misma orientación, lo que permitirá la observación de la curvatura exterior y la curvatura interna en el corazón 11. El número de cardiomiocitos total, tamaño de la celda de cardiomiocitos, y la circularidad se puede medir. Otro ejemplo que revela la estructura del sarcómero de un corazón embrionario se muestra en la fig. 2. Se realizó inmunotinción con F59, un anticuerpo miosina, en un corazón disecado embrionarias. La red de miofibrillas en el corazón embrionario completo puede ser revelado claramente a un menor aumento, mientras que la banda estriada del filamento grueso se puede revelar a mayor aumento (Fig. 2) 6. <img alt="Figura 1" src="/files/ftp_upload/3510/3510fig1.jpg" /> Figura 1. Inmunotinción de MEF2 (rojo) y β-catenina (verde) para mostrar los núcleos y outlines de los cardiomiocitos en un ventrículo del pez cebra. Barra de escala = 10μm <img alt="Figura 2" src="/files/ftp_upload/3510/3510fig2.jpg" /> Figura 2. Immnostaining de la miosina para mostrar el filamento grueso en un ventrículo del pez cebra en cualquiera de bajo aumento (A) o aumento (B). Barra de escala = 10μm

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	<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>

No hay conflictos de interés declarado.

En comparación con los métodos clásicos de todo el montaje inmunoticción, nuestro método tiene las siguientes ventajas. En primer lugar, mucho más fuertes señales fluorescentes pueden ser obtenidos consistentemente, debido a una mejor penetración. En el método de tinción de montaje conjunto, el denso tejido de la piel que rodea el corazón reduce significativamente la penetración de muchos anticuerpos, lo que resulta en el fondo de alto en todo el cuerpo. Este problema es especialmente grave para los embrione...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td></tr><tr><td> tricaína </td><td> Orgánicos de investigación </td><td> 3007T </td></tr><tr><td> Formaldehído </td><td> Polysciences </td><td> 04018 </td></tr><tr><td> La insulina jeringa </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 de anticuerpos </td><td> Sigma </td><td> C7207 </td></tr><tr><td> Anticuerpos 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 anticuerpos </td><td> Sigma </td><td> A7811 </td></tr><tr><td> Anticuerpo anti-calaña </td><td> Señalización celular </td><td> # 3862 </td></tr><tr><td> Alexa Fluor 568 cabra anti IgG de conejo </<html> td&gt; <td> Invitrogen </td><td> A11011 </td></tr><tr><td> Alexa Fluor 488 cabra anti IgG1 de ratón </td><td> Invitrogen </td><td> A21121 </td></tr><tr><td> Medio de montaje para fluorescencia </td><td> Vector </td><td> H-1200 </td></tr><tr><td> ImmEdge pluma </td><td> Vector </td><td> H-4000 </td></tr><tr><td> Poli-L-lisina portaobjetos recubiertos </td><td> Electrónica de ciencias de la microscopía </td><td> 63410-01 </td></tr><tr><td> Microscopio cubierta de vidrio </td><td> Pescador </td><td> 12 a 543-D </td></tr><tr><td> Diapositivas cóncavo microscopio </td><td> Pescador </td><td> 7-1305-8 </td></tr><tr><td> Disección microscopio </td><td> Leica MZ95 </td><td></td></tr><tr><td> Compuesto microscopio </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 tricaína: 400 mg tricaína 97.9 mL ddH2O 2,1 ml (1M Tris pH 9) Ajustar el pH a 7.0

immunostaining of dissected zebrafish embryonic heart

Embrión de pez cebra se convierte en un popular modelo de vertebrados vivo para el estudio de desarrollo cardiaco y las enfermedades del corazón humano, debido a su ventajosa embriología y la genética 1,2. Unos 100-200 embriones están disponibles todas las semanas de un par de peces adultos. Los embriones transparentes que se desarrollan fuera del útero lo hacen ideal para la evaluación de defectos cardíacos 3. La expresión de cualquier gen pueden ser manipulados a través de la tecnología morpholino o ARN de inyección 4. Por otra parte, hacia las pantallas de genética ya han generado una lista de mutantes que afectan a las diferentes perspectivas de la cardiogénesis 5. Toda la inmunotinción de montaje es una técnica importante en este modelo animal para revelar el patrón de expresión de la proteína dirigida a un determinado tejido 6. Sin embargo, las imágenes de alta resolución que pueden revelar las estructuras celulares o subcelulares han sido difíciles, debido principalmente a la física location del corazón y de la escasa penetración de los anticuerpos. A continuación, presentamos un método para hacer frente a estos cuellos de botella mediante la disección de corazón y luego llevar a cabo el proceso de tinción de la superficie de un portaobjetos de microscopio. Para evitar la pérdida de las muestras de corazón pequeño y para facilitar el manejo de la solución, que limita las muestras de corazón dentro de un círculo en la superficie del portaobjetos dibujado por una pluma immEdge. Después de la tinción, las señales de fluorescencia puede ser observada directamente por un microscopio compuesto. Nuestro nuevo método mejora significativamente la penetración de los anticuerpos, ya que un corazón de un pez de embriones sólo se compone de capas de células de unos pocos. Imágenes de alta calidad de los corazones intactos se pueden obtener en un plazo muy reducido para la procesión de embriones de pez cebra edades comprendidas entre los día 2 al día 6. Nuestro método puede ser potencialmente extenderse a otros órganos disecados mancha de cualquiera de pez cebra y otros animales pequeños.

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

Immunostaining of Dissected Zebrafish Embryonic Heart

Una forma rápida para llevar a cabo la inmunotinción del corazón embrionario del pez cebra se describe. En comparación con el enfoque de montaje inmunotinción todo, este método aumenta considerablemente la penetración de los anticuerpos, lo que permite obtener imágenes de alta resolución que revelan la estructura celular / subcelular en el corazón dentro de un tiempo de procesamiento muy reducida.

Inmunotinción de Disección del corazón embrionario de pez cebra

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 vistas.  Mayo Clinic College of Medicine. Una forma rápida para llevar a cabo la inmunotinción del corazón embrionario del pez cebra se describe. En comparación con el enfoque de montaje inmunotinción todo, este método aumenta considerablemente la penetración de los anticuerpos, lo que permite obtener imágenes de alta resolución que revelan la estructura celular / subcelular en el corazón dentro de un tiempo de procesamiento muy reducida.

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.

Imágenes en vivo del cerebro del embrión de pez cebra mediante microscopía confocal

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 ...

Investigación

Biología

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.

Tejido diana quimeras embrionarias: pez cebra Gástrula Trasplante de células

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.

Preparación de embriones para Microscopía Electrónica de la Drosophila Tubo cardíaco embrionario

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.

Microdisección de los tejidos del ojo de pez cebra embrionarias

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.

Todo el montaje El análisis inmunohistoquímico de la piel del miembro Vasculatura embrionarias: un sistema modelo para estudiar la morfogénesis vascular ramificación en embriones

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.

La disección del corazón en las larvas, juveniles y adultos de pez cebra, 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 para la<em&gt; Ex vivo</em&gt; Cultivo de páncreas embrionario

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-infligido lesiones de músculo esquelético embrionario de pez cebra

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.

Análisis de embriones y larvas de pez cebra esqueléticas miofibras de Preparativos disociadas

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; Método de la cultura para el corazón de embriones de ratón

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