Name: immunostaining phospho-epitopes in ciliated organs of whole mount zebrafish embryos
Uploaded: 2016-02-19T14:00:00.000+00:00
Duration: 8 min 42 s
Description: Watch this Scientific Journal Video about Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos at JoVE.com

Este trabajo fue apoyado por la beca de la Fundación Nacional de Ciencia IOS-0817658.

Los procedimientos de pez cebra en este protocolo han sido aprobados por el Comité Institucional de Cuidado y Uso de Animales (IACUC) de la Universidad de Virginia Commonwealth. 1. Preparación de los reactivos <ol><li> PFA al 4% / PBS. Pesar 8 g de paraformaldehído (PFA) en la campana de humos. Aunque todavía en la campana de humos, se disuelve el PFA seco en ~ 80 ml ​​de H 2 O destilada con agitación y calentamiento a 50 ° C. Mientras se agita, añadir 3 - 10 gotas de NaOH 1N fresco hasta PFA se haya disuelto completamente y la solución se aclara. Retirar del fuego y añadir 100 ml de solución salina tamponada 2x con fosfato (PBS). Se ajusta el volumen a 200 ml con H2O destilada Enfriar a ta y confirmar pH de 7,4 antes de su uso. Almacenar en la oscuridad a 4 ° C, pero el uso dentro de una semana. </li><li> Utilizar el 100% de metanol sin ningún tipo de suplementos. Utilizar etanol al 95% sin ningún tipo de suplementos. </li><li> Preparar tamponada con fosfato Tween (PBT). Suplemento solución salina tamponada con fosfato (PBS) con 0,1% de Tween-20. Añadir 0.5 ml de una solución de Tween-20 de la 20% a 100 ml de 1x PBS. Almacenar a temperatura ambiente. </li><li> Preparar tamponada con fosfato Triton-X (PbTx). Suplemento PBS con 0,1% de Triton X-100. Añadir 0,5 ml de un 20% de Triton X-100 en el almacén a 100 ml de PBS. Almacenar a temperatura ambiente. </li><li> Preparar 10% NGS / PbTx. suero de cabra (NGS) bloquea normales sitios de unión no específicos. NGS almacenar en alícuotas a -20 ° C. Para utilizar, añadir 1 ml a 9 ml PbTx. </li><li> Preparar 50% de glicerol / PBS. Mezcle partes iguales de fondo 2x PBS y 100% de glicerol. Almacenar a temperatura ambiente. </li></ol> 2. Fijación de embriones <ol><li> . (. Por ejemplo, β-actina: CAAX-GFP) Obtener la cría de tipo salvaje (AB y Wik) o transgénicos embriones a través de la monta natural. Si se desea, inyectar embriones con constructos o morfolinos, como se describe 15,16. </li><li> Elevar embriones. Recoger los embriones y se incuba a 28,5 ° C en presencia de 0,003% de 1-fenil-2-tiourea (PTU) para bloquear la pigmentación como se describe. 17 </su<html> p&gt; </li><li> Fijar. Cuando los embriones han alcanzado la etapa de desarrollo deseado, 18, &#160; anestesiar a los embriones utilizando MESAB, eliminar tanta agua como sea posible del sistema y añadir fresca PFA al 4% / PBS durante 3 - 4 horas a temperatura ambiente. NOTA: En tiempos de menos de 20 h después de la fecundación (HPF), antes de fijar dechorionation. A veces después de 20 hpf, dechorionate antes de la fijación utilizando dos pares de pinzas de punta fina bajo un microscopio de disección. </li><li> Cambio de soluciones. Transferencia de embriones utilizando una amplia pipeta de diámetro interior, tal como se describe. 17 Cambio de soluciones en 1,5 ml tapados tubos de microcentrífuga, simplemente permitiendo que los embriones se asienten por gravedad y sin centrifugación. </li><li> Después de post-fijador. 3 - 4 horas, retire PFA / PBS, reemplace con metanol al 100% y se almacena a -20 ° C durante al menos 48 horas. NOTA: Para la máxima inmunorreactividad P-CaMK-II, limitar el tiempo total de almacenamiento de metanol a una semana. </li></ol>&quot;&gt; 3. La inmunotinción Total embriones <ol><li> Coloque un mínimo de 10 embriones para cada condición experimental en cubiertas tubo de microcentrífuga de 1,5 ml, y etiquetarlos. </li><li> Permitir que los embriones se asienten. Retirar y desechar el metanol y rehidratar con lavados progresivas que contiene 0,5 ml de concentraciones decrecientes de etanol, como se indica en el siguiente paso. Cada paso es un lavado de 5 min con agitación. </li><li> Progresivamente rehidratar con estos 5 soluciones: 66% de etanol / 33% PbTx, 33% de etanol / 66% PbTx, 100% PbTx, 100% PbTx (esta es la primera repetición de PbTx), 100% PbTx (esta es la segunda repetición de PbTx). </li><li> Retire último lavado PbTx. Añadir 0,5 ml de 10% NGS / PbTx. Incubar durante al menos 1 hora a temperatura ambiente con agitación suave y luego retirar y desechar la solución. </li><li> Preparar la solución de anticuerpo primario mediante la dilución en el 10% de NGS / PbTx. Si co-inmunotinción, utilice anticuerpo de afinidad más alta primero. Para este estudio, se incuba con el anticuerpo de la tubulina monoclonal de ratón anti-acetilado diluido 1: 500. Por ejemploe, si hay 10 muestras, combinar 6 l de la solución madre de anticuerpo con 3 ml de 10% NGS / PbTx y luego distribuir 0,3 ml de esta en cada tubo. </li><li> Añadir 0,2 - 0,5 ml del anticuerpo primario diluido a cada tubo para sumergir todos los embriones. Incubar con suave O / N de balanceo a temperatura ambiente. </li><li> Por la mañana, retirar y desechar la solución de anticuerpo. Añadir 0,5 ml de 2% NGS / PbTx para quitar el exceso de anticuerpo primario. Agite suavemente durante 5 minutos. Retire y deseche la solución. Repetir dos veces. </li><li> Dim las luces del techo y diluir el anticuerpo secundario conjugado con fluorescencia apropiada en el 10% de NGS / PbTx de manera que cada condición contiene al menos 0,5 ml. Con el anticuerpo primario anti-tubulina de ratón acetilado, utilizar el colorante fluorescente verde cabra anti-IgG de ratón en una dilución 1: 500. </li><li> Incubar anticuerpo secundario durante 4 h con agitación suave a temperatura ambiente en la oscuridad. A partir de ahora, se procesan las muestras bajo las luces se apagaron y se incuban en un recipiente oscuro o envolviéndolos en papel de aluminio. </li><li>Al final de la incubación, retirar y desechar la solución de anticuerpo secundario. Añadir 0,5 ml de 2% NGS / PbTx para lavar. Agite suavemente durante 5 minutos. Retire y deseche la solución. Repetir dos veces. </li><li> Si co-inmunotinción, obtener el segundo anticuerpo primario de una especie diferente que el primer anticuerpo primario. En estos estudios, seguir el anticuerpo CaMK-II de conejo anti-fosfo por el tinte conjugado de cabra anti-IgG de conejo anticuerpo secundario rojo fluorescente. </li><li> Diluir conejo anti-fosfo-II CaMK anticuerpo 1:20. Para cada tubo de embriones, suspender en 0,3 ml 10% NGS / PbTx, a continuación, añadir 15 l de la solución madre de anticuerpo. </li><li> Asegúrese de que los embriones se sumergen y luces se atenúan. Incubar con suave balanceo O / N a temperatura ambiente en la oscuridad. </li><li> Por la mañana bajo luces tenues, retirar y desechar la solución de anticuerpo. Añadir 0,5 ml 2% NGS / PbTx. Agite suavemente durante 5 minutos. Retire y deseche la solución. Repetir dos veces. </li><li> Mantenga las luces del techo tenue y diluir lo suficiente de la fluorescencia apropiada conjuanticuerpo secundario cerrada de modo que cada condición contiene al menos 0,5 ml. En este estudio, se diluye el colorante conjugado de cabra anti-conejo IgG anticuerpo rojo fluorescente secundario (canal rojo) 1: 500 en 10% NGS / PbTx. </li><li> Incubar segundo anticuerpo secundario durante 4 h con agitación suave a temperatura ambiente en la oscuridad. </li><li> Al final de esta incubación y bajo luces tenues, retirar y desechar la solución de anticuerpo secundario. Añadir 0,5 ml PbTx. Agite suavemente durante 5 minutos. Retire y deseche la solución. Repetir dos veces. Tienda, ya sea en PbTx o 50% de glicerol / PBS, dependiendo del procedimiento de formación de imágenes. </li></ol> 4. Imagen confocal y Procesamiento <ol><li> Monte embriones para la imagen. Coloque 1 - 5 embriones en un portaobjetos de vidrio. Crear una cámara entre el cubreobjetos y el portaobjetos principal utilizando fragmentos cubreobjetos en cada lado de la cámara. Típicamente, cuatro # 1 cubreobjetos se utilizan para hacer estas pilas spacer sin sellar. NOTA: No es necesario un medio de montaje. </li><li> Use un aceite de inmersión 100Xobjetivo de poner solo embrión en el foco con luz transmitida. Apaga la luz. Encienda el microscopio confocal. Asegurarse de que los láseres apropiados (verde y / o rojo) se enciende y se accessary enfoque remoto está empeñada. </li><li> Encienda el programa confocal. En la &quot;barra Adquirir,&quot; seleccionar el objetivo adecuado. En la barra de &quot;XY Básica&quot;, establecer el tamaño de la imagen para 1024 haciendo clic en el botón de 1.024. </li><li> En el &quot;láser y el detector&quot; barra, haga clic en el cuadro 488 de color rojo y verde de la caja 568 para activar el láser y el detector para cada canal. Conjunto del agujero de alfiler a medio y ajustar la ganancia en la barra de &quot;ganancia&quot; de cada canal de visualizar. </li><li> En el bar &quot;Adquirir Configuración&quot;, haga clic en &quot;vivo&quot; para iniciar la adquisición de imágenes. En la barra de &quot;Configuración de la vista&quot;, desmarca la casilla &quot;fuerza integral Zoom&quot; para el centro de la ventana &quot;en vivo&quot;. </li><li> En la barra de &quot;Obtener configuración&quot;, en la pestaña &quot;Z&quot;, el paso a través de las capas de la imagen. Después de seleccionar el numbre de secciones ópticas para incorporar en la imagen, se mueve a un punto en el &quot;centro&quot; del plano z. </li><li> En la pestaña &quot;Z&quot;, haga clic en el cuadro pequeño &quot;de referencia&quot; de color rojo. Esto cero la RFA en el punto elegido como el &quot;centro&quot;. En la casilla &quot;Tamaño Paso&quot;, seleccionar el espesor de las capas (entre 0,25 micras y 2,0 micras es lo ideal). Prestar atención a la casilla &quot;Tamaño de archivo&quot; y tratar de limitar a 1 GB. NOTA: Por lo general, hasta 40 secciones ópticas de 0,5 a 1,0 micras se obtienen, pero el número de secciones puede ser determinada empíricamente. </li><li> Para encontrar el extremo superior de la imagen, haga clic en el círculo al lado de &quot;Inicio&quot; y moverse a través de las capas z. Ahora haga clic en el círculo al lado de &quot;inferior&quot;, el equipo tomará la imagen a la capa como el centro. moverse de nuevo a través de las capas de encontrar el extremo inferior de la imagen. </li><li> Haga clic en &quot;Live&quot; de nuevo en el &quot;Obtener parámetros del&quot; cuadro para detener el láseradquisición. En este panel, haga clic en las casillas de rojos marcados media y Z-pila. Estas cajas se volverá verde. </li><li> En el cuadro &quot;Obtener parámetros&quot;, haga clic en &quot;Single&quot; para adquirir la serie completa de imágenes. Usted puede controlar el progreso ya que revisa en ambos canales ya través de todo el z-pila. </li><li> Para guardar, haga clic en la ventana del volumen y haga clic en &quot;guardar como&quot; y el nombre del archivo. Guardar como un archivo &quot;.ids&quot;. Para hacer que el archivo de volumen mientras que el z-pila está todavía abierta, seleccione el menú desplegable de datos y haga clic en &quot;volumen de render&quot;. Guarde el archivo renderizado como un archivo TIFF. Esta es la imagen proyectada que se muestran en esta publicación. </li></ol>

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Los autores declaran que no tienen intereses financieros en competencia.

El método / metanol PFA fue desarrollado en este laboratorio con el objetivo principal de la optimización de la inmunolocalización de la fosfo-T 287 -CaMK-II epítopo durante el desarrollo de pez cebra. Este método localiza con éxito P-CaMK-II durante la formación de varios órganos incluyendo el ciliadas KV pez cebra, 12 oído interno 14 y el riñón. 13 En particular, en la etapa de KV, esta técnica era necesario. El éxito de este método es probablemente debido a un...

The techniques described here are the outcome of studies that have sought to define downstream targets of Ca2+ signals during events that occur during early development, including fertilization, gastrulation, somitogenesis and trunk, eye, brain and organ formation.1-3 The original discoveries of embryonic Ca2+ signaling were dependent on the use of natural and engineered Ca2+ indicators, such as aequorin4 and fura-2.5 Even with current technology, the detection of transient elevations of Ca2+ requires cumbersome analytical tools and does not reveal the targets of such Ca2+ signals. 
This laboratory investigates Ca2+ signals that act through the Ca2+/calmodulin-dependent (multifunctional) protein kinase known as CaMK-II, an enzyme that is enriched in the central nervous system and originally identified as a regulator of long-term potentiation.6 CaMK-II is not brain-specific, is widely expressed and highly conserved throughout the entire lifespan and bodies of species throughout the animal kingdom, including invertebrates.7,8 CaMK-II has the unique capability of sustaining its own activity even after Ca2+ levels have diminished due to its ability to autophosphorylate at Thr287. In this autophosphorylated state, CaMK-II remains active in a Ca2+/CaM-independent manner, until dephosphorylated.6 Thus, the localization of phosphorylated CaMK-II (Thr287) can identify cells in which natural, relevant Ca2+ elevations have occurred. 
An antibody against autophosphorylated (P-Thr287) mammalian CaMK-II has been well-characterized and was initially used to localize activated CaMK-II in brain tissue.9 Zebrafish (Danio rerio) have seven CaMK-II genes10,11 whose protein products contain a sequence of MHRQE[pT287]VECLK in this region.10,11 This sequence is very similar to the phosphopeptide antigen used to create this rabbit polyclonal antibody (MHRQE[pT]VDCLK; Upstate/Millipore) and therefore it was not a complete surprise that this antibody cross-reacted with zebrafish CaMK-II. This laboratory showed that this antibody reacts with zebrafish CaMK-II in proportion to autophosphorylation and Ca2+/CaM-independent activity.12 Additional pan-specific CaMK-II antibodies have also been shown to cross-react with zebrafish CaMK-II.13
This antibody has been used to demonstrate that zebrafish CaMK-II is preferentially activated in cells on one side of the zebrafish Kupffer's Vesicle (KV), the ciliated organ necessary for establishment of left/right asymmetry.12 This antibody was used to demonstrate that CaMK-II is transiently activated in four adjacent cells on the left side of the KV during the exact same developmental phase that organ positioning is determined.12 In addition to the Kupffer's Vesicle (KV), autophosphorylated (P-T287) was also located in specific intracellular sites in other ciliated tissues including the kidney, neuromasts, and inner ear.12,13 In the zebrafish kidney, P-T287-CaMK-II is enriched along the apical border of ciliated ductal cells and within cloacal cilia where it influences their assembly.13 Finally, in the developing inner ear, P-T287-CaMK-II is intensely concentrated at the base of cilia and influences cell differentiation through the Delta-Notch signal pathway.14 In summary, the detection of activated CaMK-II has pinpointed sites of intracellular Ca2+ release and illuminated potential new signaling pathways.
These discoveries were completely dependent on developing a sensitive and accurate method to localize activated (P-T287-autophosphorylated) CaMK-II. The methods to fix and immunostain the zebrafish KV, kidney and inner ear are described. The limitations of this technique are also described. These techniques should be useful to any investigator who seeks to obtain high-resolution images in two fluorescent channels of not just phospho-epitopes, but any epitope, during early vertebrate development.

<table><tbody><tr><td>1-phenyl-2-thiourea (PTU)</td><td>Sigma</td><td>P-7629</td><td>0.12% Stock solution. Dilute 1:40 in system water</td></tr><tr><td>Alexa488 anti-mouse IgG</td><td>Life Technologies</td><td>A11001</td><td>Goat polyclonal, use at 1:500</td></tr><tr><td>Alexa488 anti-rabbit IgG</td><td>Life Technologies</td><td>A11008</td><td>Goat polyclonal, use at 1:500</td></tr><tr><td>Alexa488 phalloidin</td><td>Life Technologies</td><td>A12379</td><td>Preferentially binds to F-actin</td></tr><tr><td>Alexa568 anti-mouse IgG</td><td>Life Technologies</td><td>A11004</td><td>Goat polyclonal, use at 1:500</td></tr><tr><td>Alexa568 anti-rabbit IgG</td><td>Life Technologies</td><td>A11011</td><td>Goat polyclonal, use at 1:500</td></tr><tr><td>anti-acetylated &amp;#945;-tubulin</td><td>Sigma</td><td>T7451</td><td>Mouse monoclonal, use at 1:500</td></tr><tr><td>anti-phospho-T&lt;sup&gt;287&lt;/sup&gt; CaMK-II</td><td>EMD Millipore</td><td>06-881</td><td>Rabbit polyclonal, use at 1:20</td></tr><tr><td>anti-total CaMK-II</td><td>BD Biosciences</td><td>611292</td><td>Mouse monoclonal, use at 1:20</td></tr><tr><td>Ethanol</td><td>Fisher</td><td>S96857</td><td>Lab grade, 95% denatured</td></tr><tr><td>Forceps</td><td>Fine Science Tools</td><td>11252-20</td><td>Dumont #5</td></tr><tr><td>Glass coverslips</td><td>VWR</td><td>16004-330</td><td>#1&amp;nbsp; thickness</td></tr><tr><td>Glass microscope slides</td><td>Fisher</td><td>12-550-15</td><td>Standard glass slides</td></tr><tr><td>Methanol</td><td>Fisher</td><td>A411</td><td>Store in freezer</td></tr><tr><td>Microcentrifuge tubes</td><td>VWR</td><td>20170-038</td><td>capped tubes, not sterile</td></tr><tr><td>Normal goat serum</td><td>Life Technologies</td><td>16210-064</td><td>Aliquot 1 ml tubes, store in freezer</td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>P-6148</td><td>Reagent grade, crystalline</td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Quality Biological</td><td>119-069-131</td><td>10x stock solution or made in lab</td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>BP-151</td><td>10% solution in water, store at RT</td></tr><tr><td>Tween-20</td><td>Life Technologies</td><td>85113</td><td>10% solution in water, store at RT</td></tr><tr><td>Compound microscope</td><td>Nikon</td><td>E-600</td><td>Mount on vibration-free table</td></tr><tr><td>C1 Plus two-laser scanning confocal</td><td>Nikon</td><td>C1 Plus</td><td>Run by EZ-C1 program, but upgrades use &quot;Elements&quot;</td></tr></tbody></table>

immunostaining phospho-epitopes in ciliated organs of whole mount zebrafish embryos

La rápida proliferación de las células, la expresión específica de tejido de genes y la aparición de las redes de señalización caracterizan el desarrollo embrionario temprano de todos los vertebrados. La cinética y la ubicación de las señales - incluso dentro de las células individuales - en el embrión en desarrollo complementa la identificación de genes de desarrollo importantes. técnicas de inmunotinción se describen que se han demostrado para definir la cinética de señales intracelulares y animales enteros en estructuras tan pequeñas como cilios primarios. Las técnicas de fijación, de imágenes y procesamiento de imágenes utilizando un microscopio compuesto confocal de escaneo láser se puede completar en tan sólo 36 horas. El pez cebra (Danio rerio) es un organismo deseable para los investigadores que buscan llevar a cabo estudios en una especie de vertebrados que es asequible y relevante para las enfermedades humanas. knockouts o caídas genéticos deben ser confirmadas por la pérdida de la proteína producto real. Dicha confirmación de la pérdida de proteínasse puede lograr usando las técnicas descritas aquí. Pistas sobre las vías de señalización también se pueden descifrar mediante el uso de anticuerpos que son reactivos con proteínas que han sido modificados después de la traducción mediante fosforilación. Preservar y optimizar el estado fosforilado de un epítopo tanto, es crítico para esta determinación y se logra mediante este protocolo. Este estudio describe las técnicas para solucionar los embriones durante las primeras 72 h de desarrollo y co-localizar una variedad de epítopos relevantes con los cilios en la vesícula Kupffer (KV), el riñón y el oído interno. Estas técnicas son sencillas, no requieren de disección y se puede completar en un período relativamente corto de tiempo. Proyección de pilas de imágenes confocal en una sola imagen es un medio útil para la presentación de estos datos.

Condiciones óptimas para la visualización de fosfo-epítopos Métodos que describen la inmunolocalización de epítopos de proteínas en embriones de pez cebra han sido relativamente escaso en comparación con aquellos para la localización de los ARNm mediante hibridación in situ. Los agentes de fijación utilizados en la localización de los epítopos de la proteína en las células ciliadas de e...

Watch this Scientific Journal Video about Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos at JoVE.com

Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos

Se describen las técnicas de inmunotinción fosfo-epítopos en embriones de pez cebra enteros y luego llevar a cabo la localización de dos colores confocal de fluorescencia en las estructuras celulares tan pequeños como los cilios primarios. Las técnicas para la fijación y formación de imágenes pueden definir la ubicación y la cinética de la aparición o la activación de proteínas específicas.

Inmunotinción fosfo-epítopos en ciliadas Órganos de todo el montaje embriones de pez cebra

The rapid proliferation of cells, the tissue-specific expression of genes and the emergence of signaling networks characterize early embryonic development ...

immunostaining-phospho-epitopes-ciliated-organs-whole-mount-zebrafish

Universidad de Cartagena UDEC

Research

JoVE Journal

Chemistry

8.0K vistas.  Virginia Commonwealth University.  Se describen las técnicas de inmunotinción fosfo-epítopos en embriones de pez cebra enteros y luego llevar a cabo la localización de dos colores confocal de fluorescencia en las estructuras celulares tan pequeños como los cilios primarios. Las técnicas para la fijación y formación de imágenes pueden definir la ubicación y la cinética de la aparición o la activación de proteínas específicas. 

Video: Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos

High Precision Zinc Isotopic Measurements Applied to Mouse Organs

We present a procedure to measure with high precision zinc isotope ratios in mouse organs. Zinc is composed of 5 stable isotopes (64Zn, 66Zn, 67Zn, 68Zn and 70Zn) which are naturally fractionated between mouse organs. We first show how to dissolve the different organs in order to free the Zn atoms; this step is realized by a mixture of HNO3 and H2O2. We then purify the zinc atoms from all the other elements, in particular from isobaric interferences (e.g., Ni), by anion-exchange chromatography in a dilute HBr/HNO3 medium. These first two steps are performed in a clean laboratory using high purity chemicals. Finally, the isotope ratios are measured by using a multi-collector inductively-coupled-plasma mass-spectrometer, in low resolution. The samples are injected using a spray chamber and the isotopic fractionation induced by the mass-spectrometer is corrected by comparing the ratio of the samples to the ratio of a standard (standard bracketing technique).&#160;This full typical&#160;procedure produces an isotope ratio with a 50 ppm (2 s.d.) reproducibility.

We present the technique to measure with high precision zinc isotope ratios in mouse organs.

Altas medidas de precisión de zinc isotópicos aplicados a Ratón Órganos

We present a procedure to measure with high precision zinc isotope ratios in mouse organs. Zinc is composed of 5 stable isotopes (64Zn, 66Zn, 67Zn, 68Zn ...

Investigación

Química

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and optical transparency. In particular, the zebrafish embryo has become an important organism to study vertebrate kidney organogenesis as well as multiciliated cell (MCC) development. To visualize MCCs in the embryonic zebrafish kidney, we have developed a combined protocol of whole-mount fluorescent in situ hybridization (FISH) and whole mount immunofluorescence (IF) that enables high resolution imaging. This manuscript describes our technique for co-localizing RNA transcripts and protein as a tool to better understand the regulation of developmental programs through the expression of various lineage factors.

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

Cilia development is vital to proper organogenesis. This protocol describes an optimized method to label and visualize ciliated cells of the zebrafish.

Visualización de las células Multiciliated en el pez cebra a través de un protocolo combinado de todo Monte fluorescente In Situ hibridación e inmunofluorescencia

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and ...

Biología del Desarrollo

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

Pez cebra todo el montaje de alta resolución doble fluorescente In Situ La hibridación

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

Biología

Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. Although many immunohistochemistry protocols have been optimized for mammalian tissue and tissue sections, these protocols often require modification and optimization for non-mammalian model organisms. Zebrafish are increasingly used as a model system in basic, biomedical, and translational research to investigate the molecular, genetic, and cell biological mechanisms of developmental processes. Zebrafish offer many advantages as a model system but also require modified techniques for optimal protein detection. Here, we provide our protocol for whole-mount fluorescence immunohistochemistry in zebrafish embryos and larvae. This protocol additionally describes several different mounting strategies that can be employed and an overview of the advantages and disadvantages each strategy provides. We also describe modifications to this protocol to allow detection of chromogenic substrates in whole mount tissue and fluorescence detection in sectioned larval tissue. This protocol is broadly applicable to the study of many developmental stages and embryonic structures.

Here, we present a protocol for fluorescent antibody-mediated detection of proteins in whole preparations of zebrafish embryos and larvae.

Inmunohistoquímica de montaje completo en embriones y larvas de pez cebra

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. ...

Single Cell Fate Mapping in Zebrafish

The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, lymphatic, and blood vessel lineages arise in developing embryos requires fate mapping and lineage tracing of undifferentiated precursor cells. Recently, photoactivatable proteins which include: Eos1, 2, PAmCherry3, Kaede4-7, pKindling8, and KikGR9, 10 have received wide interest as cell tracing probes. The fluorescence spectrum of these photosensitive proteins can be easily converted with UV excitation, allowing a population of cells to be distinguished from adjacent ones. However, the photoefficiency of the activated protein may limit long-term cell tracking11. As an alternative to photoactivatable proteins, caged fluorescein-dextran has been widely used in embryo model systems7, 12-14. Traditionally, to uncage fluorescein-dextran, UV excitation from a fluorescence lamp house or a single photon UV laser has been used; however, such sources limit the spatial resolution of photoactivation. Here we report a protocol to fate map, lineage trace, and detect single labeled cells. Single cells in embryos injected with caged fluorescein-dextran are photoactivated with near-infrared laser pulses produced from a titanium sapphire femtosecond laser. This laser is customary in all two-photon confocal microscopes such as the LSM 510 META NLO microscope used in this paper. Since biological tissue is transparent to near-infrared irradiation15, the laser pulses can be focused deep within the embryo without uncaging cells above or below the selected focal plane. Therefore, non-linear two-photon absorption is induced only at the geometric focus to uncage fluorescein-dextran in a single cell. To detect the cell containing uncaged fluorescein-dextran, we describe a simple immunohistochemistry protocol16 to rapidly visualize the activated cell. The activation and detection protocol presented in this paper is versatile and can be applied to any model system. 
Note: The reagents used in this protocol can be found in the table appended at the end of the article.

A method is described to photoactivate single cells containing a caged fluorescent protein using two-photon absorption from a Ti:Sapphire femtosecond laser oscillator. To fate map the photoactivated cell, immunohistochemistry is used. This technique can be applied to any cell type.

Sola célula de mapeado de destino en el pez cebra

The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, ...

An Efficient Protocol to Assess ERK Activity Modulation in Early Zebrafish Noonan Syndrome Models via Live FRET Microscopy and Immunofluorescence

RASopathies are genetic syndromes caused by ERK hyperactivation and resulting in multisystemic diseases that can also lead to cancer predisposition. Despite a broad genetic heterogeneity, germline gain-of-function mutations in key regulators of the RAS-MAPK pathway underlie the majority of the cases, and, thanks to advanced sequencing techniques, potentially pathogenic variants affecting the RAS-MAPK pathway continue to be identified. Functional validation of the pathogenicity of these variants, essential for accurate diagnosis, requires fast and reliable protocols, preferably in vivo. Given the scarcity of effective treatments in early childhood, such protocols, especially if scalable in cost-effective animal models, can be instrumental in offering a preclinical ground for drug repositioning/repurposing. 
Here we describe step-by-step the protocol for rapid generation of transient RASopathy models in zebrafish embryos and direct inspection of live disease-associated ERK activity changes occurring already during gastrulation through real-time multispectral F&#246;rster resonance energy transfer (FRET) imaging. The protocol uses a transgenic ERK reporter recently established and integrated with the hardware of commercial microscopes. We provide an example application for Noonan syndrome (NS) zebrafish models obtained by expression of the Shp2D61G. We describe a straightforward method that enables registration of ERK signal change in the NS fish model before and after pharmacological signal modulation by available low-dose MEK inhibitors. We detail how to generate, retrieve, and assess ratiometric FRET signals from multispectral acquisitions before and after treatment and how to cross-validate the results via classical immunofluorescence on whole embryos at early stages. We then describe how, via examining standard morphometric parameters, to query late changes in embryo shape, indicative of a resulting impairment of gastrulation, in the same embryos whose ERK activity is assessed by live FRET at 6 h post fertilization.

RASopathies are multisystem genetic syndromes caused by RAS-MAPK pathway hyperactivation. Potentially pathogenic variants awaiting validation emerge continuously while poor preclinical evidence limits therapy. Here, we describe our in vivo protocol to test and cross-validate RASopathy-associated ERK activation levels and its pharmacological modulation during embryogenesis by live FRET imaging in Teen-reporter zebrafish.

Un protocolo eficiente para evaluar la modulación de la actividad de ERK en modelos tempranos de síndrome de Noonan de pez cebra mediante microscopía FRET en vivo e inmunofluorescencia

RASopathies are genetic syndromes caused by ERK hyperactivation and resulting in multisystemic diseases that can also lead to cancer predisposition. ...

Bioquímica

Immunostaining of Dissected Zebrafish Embryonic Heart 

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology and genetics 1,2. About 100-200 embryos are readily available every week from a single pair of adult fish. The transparent embryos that develop ex utero make them ideal for assessing cardiac defects 3. The expression of any gene can be manipulated via morpholino technology or RNA injection 4. Moreover, forward genetic screens have already generated a list of mutants that affect different perspectives of cardiogenesis 5. 
Whole mount immunostaining is an important technique in this animal model to reveal the expression pattern of the targeted protein to a particular tissue 6. However, high resolution images that can reveal cellular or subcellular structures have been difficult, mainly due to the physical location of the heart and the poor penetration of the antibodies. 
 Here, we present a method to address these bottlenecks by dissecting heart first and then conducting the staining process on the surface of a microscope slide. To prevent the loss of small heart samples and to facilitate solution handling, we restricted the heart samples within a circle on the surface of the microscope slides drawn by an immEdge pen. After the staining, the fluorescence signals can be directly observed by a compound microscope. 
Our new method significantly improves the penetration for antibodies, since a heart from an embryonic fish only consists of few cell layers. High quality images from intact hearts can be obtained within a much reduced procession time for zebrafish embryos aged from day 2 to day 6. Our method can be potentially extended to stain other organs dissected from either zebrafish or other small animals.

Immunostaining of Dissected Zebrafish Embryonic Heart

A rapid way to conduct immunostaining of zebrafish embryonic heart is described. Compared to the whole mount immunostaining approach, this method dramatically increases the penetration of the antibodies, which allows obtaining high resolution images that reveal cellular/subcellular structures in the heart within a much reduced processing time.

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 ...

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression both in terms of tissue distribution and developmental stage. Protocols are described for the use of WISH of zebrafish embryos using antisense RNA probes labeled with digoxigenin. Probes are generated by incorporating digoxigenin-linked nucleotides through in vitro transcription of gene templates that have been cloned and linearized. The chorions of embryos harvested at defined developmental stages are removed before incubation with specific probes. Following a washing procedure to remove excess probe, embryos are incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase. By employing a chromogenic substrate for alkaline phosphatase, specific gene expression can be assessed. Depending on the level of gene expression the entire procedure can be completed within 2-3 days.

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

The zebrafish, a small tropical fish, has become a popular model for studying gene function during vertebrate development and disease. The temporal and spatial expression of target genes can be determined by in situ hybridization. Our improved protocol allows for the detection of low abundant transcripts with low non-specific background signal.

Alta resolución todo el montaje de<em&gt; In Situ</em&gt; La hibridación en embriones de pez cebra para estudiar la expresión génica y la función

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression ...

In Situ Hybridization Combined with Immunohistochemistry in Cryosectioned Zebrafish Embryos

As a vertebrate, the zebrafish has been widely used in biological studies. Zebrafish and humans share high genetic homology, which allows its use as a model for human diseases. Gene function study is based on the detection of gene expression patterns. Although immunohistochemistry offers a powerful way to assay protein expression, the limited number of commercially available antibodies in zebrafish restricts the application of costaining. In situ hybridization is widely used in zebrafish embryos to detect mRNA expression. This protocol describes how to obtain images by combining in situ hybridization and immunohistochemistry for zebrafish embryo sections. In situ hybridization was performed prior to cryosectioning, followed by antibody staining. Immunohistochemistry and the imaging of a single cryosection were performed after in situ hybridization. The protocol is helpful to unravel the expression pattern of two genes, first by in situ transcript detection and then by immunohistochemistry against a protein in the same section.

In Situ Hybridization Combined with Immunohistochemistry in Cryosectioned Zebrafish Embryos

This protocol describes how to obtain images by combining in situ hybridization and immunohistochemistry of zebrafish embryonic sections. In situ hybridization was performed prior to cryosectioning, followed by antibody staining. It is useful to detect the expression patterns of two genes in zebrafish if there is a paucity of antibodies.

In situ Hibridación combinada con inmunohistoquímica en embriones crioseccionados de pez cebra

As a vertebrate, the zebrafish has been widely used in biological studies. Zebrafish and humans share high genetic homology, which allows its use as a ...

Neurociencias

Sequential Immunofluorescence and Immunohistochemistry on Cryosectioned Zebrafish Embryos

Investigation of intercellular interactions often requires discrete labeling of specific cell populations and precise protein localization. The zebrafish embryo is an excellent tool for examining such interactions with an in vivo model. Whole-mount immunohistochemical and immunofluorescence assays are frequently applied in zebrafish embryos to assess protein expression. However, it can be difficult to achieve accurate mapping of co-localized proteins in three-dimensional space. In addition, some studies may require the use of two antibodies that are not compatible with the same technique (e.g., antibody 1 is only suitable for immunohistochemistry and antibody 2 is only suitable for immunofluorescence). The purpose of the method described herein is to perform sequential immunofluorescence and/or immunohistochemistry on individual cryosections derived from early-stage zebrafish embryos. Here we describe the use of sequential rounds of immunofluorescence, imaging, immunohistochemistry, imaging for a single cryosection in order to achieve precise identification of protein expression at the single-cell level. This methodology is suitable for any study in early-stage zebrafish embryos that requires accurate identification of multiple protein targets in individual cells.

This protocol demonstrates sequential immunofluorescence and immunohistochemistry on cryosections from early-stage zebrafish embryos enabling precise colocalization analyses in specific cell populations.

Inmunofluorescencia secuencial e inmunohistoquímica en embriones de pez cebra crioseccionados

Investigation of intercellular interactions often requires discrete labeling of specific cell populations and precise protein localization. The zebrafish ...