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

Ce travail a été soutenu par la National Science Foundation subvention IOS-0817658.

Les procédures de poisson zèbre dans ce protocole ont été approuvés par la Commission institutionnelle animal soin et l&#39;utilisation (IACUC) à l&#39;Université Virginia Commonwealth. 1. Préparation des réactifs <ol><li> 4% PFA / PBS. Peser 8 g de paraformaldehyde (PFA) dans la hotte. Alors qu&#39;il était encore dans la hotte, dissoudre le PFA sec ~ 80 ml ​​de H 2 O distillée sous agitation et chauffage à 50 ° C. Tout en remuant, ajouter 3 - 10 gouttes de 1N ...

<ol>
	<li>Webb, S. E., Miller, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+signalling+during+embryonic+development.">Calcium signalling during embryonic development.</a> Nat Rev Mol Cell Biol. 4, 539-551 (2003).</li><li>Webb, S. E., Miller, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca2++signalling+and+early+embryonic+patterning+during+zebrafish+development.">Ca2+ signalling and early embryonic patterning during zebrafish development.</a> Clin Exp Pharmacol Physiol. 34, 897-904 (2007).</li><li>Whitaker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+at+fertilization+and+in+early+development.">Calcium at fertilization and in early development.</a> Physiol. Rev. 86, 25-88 (2006).</li><li>Yuen, M. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+Ca(2+)+signaling+in+the+external+yolk+syncytial+layer+during+the+late+blastula+and+early+gastrula+periods+of+zebrafish+development.">Characterization of Ca(2+) signaling in the external yolk syncytial layer during the late blastula and early gastrula periods of zebrafish development.</a> Biochim Biophys Acta. 1833, 1641-1656 (2013).</li><li>Tombes, R. M., Borisy, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+free+calcium+and+mitosis+in+mammalian+cells:+anaphase+onset+is+calcium+modulated,+but+is+not+triggered+by+a+brief+transient.">Intracellular free calcium and mitosis in mammalian cells: anaphase onset is calcium modulated, but is not triggered by a brief transient.</a> J. Cell Biol. 109, 627-636 (1989).</li><li>Hudmon, A., Schulman, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+Ca2+/Calmodulin-Dependent+Protein+Kinase+II:+The+Role+of+Structure+and+Autoregulation+in+Cellular+Function.">Neuronal Ca2+/Calmodulin-Dependent Protein Kinase II: The Role of Structure and Autoregulation in Cellular Function.</a> Annu. Rev. Biochem. 71, 473-510 (2002).</li><li>Tombes, R. M., Faison, M. O., Turbeville, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+and+Evolution+of+Multifunctional+Ca2+/CaM-dependent+Protein+Kinase+(CaMK-II).">Organization and Evolution of Multifunctional Ca2+/CaM-dependent Protein Kinase (CaMK-II).</a> Gene. 322, 17-31 (2003).</li><li>Braun, A. P., Schulman, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Multifunctional+Calcium/Calmodulin-Dependent+Protein+Kinase:+From+Form+to+Function.">The Multifunctional Calcium/Calmodulin-Dependent Protein Kinase: From Form to Function.</a> Annu. Rev. Physiol. 57, 417-445 (1995).</li><li>Rich, R. C., Schulman, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substrate-directed+function+of+calmodulin+in+autophosphorylation+of+Ca2+/calmodulin-dependent+protein+kinase+II.">Substrate-directed function of calmodulin in autophosphorylation of Ca2+/calmodulin-dependent protein kinase II.</a> J Biol Chem. 273, 28424-28429 (1998).</li><li>Rothschild, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tbx5-mediated+expression+of+Ca2+/calmodulin-dependent+protein+kinase+II+is+necessary+for+zebrafish+cardiac+and+pectoral+fin+morphogenesis.">Tbx5-mediated expression of Ca2+/calmodulin-dependent protein kinase II is necessary for zebrafish cardiac and pectoral fin morphogenesis.</a> Dev Biol. 330, 175-184 (2009).</li><li>Rothschild, S. C., Lister, J. A., Tombes, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+of+CaMK-II+genes+during+early+zebrafish+embryogenesis.">Differential expression of CaMK-II genes during early zebrafish embryogenesis.</a> Dev Dyn. 236, 295-305 (2007).</li><li>Francescatto, L., Rothschild, S. C., Myers, A. L., Tombes, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+activation+of+membrane+targeted+CaMK-II+in+the+zebrafish+Kupffer's+vesicle+is+required+for+left-right+asymmetry.">The activation of membrane targeted CaMK-II in the zebrafish Kupffer's vesicle is required for left-right asymmetry.</a> Development. 137, 2753-2762 (2010).</li><li>Rothschild, S. C., Francescatto, L., Drummond, I. A., Tombes, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CaMK-II+is+a+PKD2+target+that+promotes+pronephric+kidney+development+and+stabilizes+cilia.">CaMK-II is a PKD2 target that promotes pronephric kidney development and stabilizes cilia.</a> Development. 138, 3387-3397 (2011).</li><li>Rothschild, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CaMK-II+activation+is+essential+for+zebrafish+inner+ear+development+and+acts+through+Delta-Notch+signaling.">CaMK-II activation is essential for zebrafish inner ear development and acts through Delta-Notch signaling.</a> Dev Biol. 381, 179-188 (2013).</li><li>Yuan, S., Sun, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+mRNA+and+morpholino+antisense+oligonucleotides+in+zebrafish+embryos.">Microinjection of mRNA and morpholino antisense oligonucleotides in zebrafish embryos.</a> J Vis Exp. , e1113 (2009).</li><li>Rosen, J. N., Sweeney, M. F., Mably, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+zebrafish+embryos+to+analyze+gene+function.">Microinjection of zebrafish embryos to analyze gene function.</a> J Vis Exp. , e1115 (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: A guide for the laboratory use of zebrafish (Brachydanio rerio). , (1993).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev. Dyn. 203, 253-310 (1995).</li><li>Obara, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polycystin-2+immunolocalization+and+function+in+zebrafish.">Polycystin-2 immunolocalization and function in zebrafish.</a> J Am Soc Nephrol. 17, 2706-2718 (2006).</li><li>Chitramuthu, B. P., Bennett, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+resolution+whole+mount+in+situ+hybridization+within+zebrafish+embryos+to+study+gene+expression+and+function.">High resolution whole mount in situ hybridization within zebrafish embryos to study gene expression and function.</a> J Vis Exp. , e50644 (2013).</li><li>Thisse, C., Thisse, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+in+situ+hybridization+to+whole-mount+zebrafish+embryos.">High-resolution in situ hybridization to whole-mount zebrafish embryos.</a> Nature. 3, 59-69 (2008).</li><li>Harris, P., Osborn, M., Weber, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+tubulin-containing+structures+in+the+egg+of+the+sea+urchin+Strongylocentrotus+purpuratus+from+fertilization+through+first+cleavage.">Distribution of tubulin-containing structures in the egg of the sea urchin Strongylocentrotus purpuratus from fertilization through first cleavage.</a> J Cell Biol. 84, 668-679 (1980).</li></ol>

La méthode / méthanol PFA a été développé dans ce laboratoire avec l&#39;objectif principal d&#39;optimiser la immunolocalisation du -CaMK-II épitope phospho-T 287 au cours du développement du poisson zèbre. Cette méthode localisée succès P-CaMK-II lors de la formation de plusieurs organes ciliées dont le KV de poisson zèbre, 12 oreille interne 14 et les reins. 13 en particulier au stade de KV, cette technique était nécessaire. Le succès de cette méthode est ...

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 border="0" cellpadding="0" cellspacing="0" width="886">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">1-phenyl-2-thiourea (PTU)</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:119px;">P-7629</td>
			<td style="width:347px;">0.12% Stock solution. Dilute 1:40 in system water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Alexa488 anti-mouse IgG</td>
			<td style="width:175px;">Life Technologies</td>
			<td style="width:119px;">A11001</td>
			<td style="width:347px;">Goat polyclonal, use at 1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Alexa488 anti-rabbit IgG</td>
			<td style="width:175px;">Life Technologies</td>
			<td style="width:119px;">A11008</td>
			<td style="width:347px;">Goat polyclonal, use at 1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Alexa488 phalloidin</td>
			<td style="width:175px;">Life Technologies</td>
			<td style="width:119px;">A12379</td>
			<td>Preferentially binds to F-actin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Alexa568 anti-mouse IgG</td>
			<td style="width:175px;">Life Technologies</td>
			<td style="width:119px;">A11004</td>
			<td style="width:347px;">Goat polyclonal, use at 1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Alexa568 anti-rabbit IgG</td>
			<td style="width:175px;">Life Technologies</td>
			<td style="width:119px;">A11011</td>
			<td style="width:347px;">Goat polyclonal, use at 1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">anti-acetylated a-tubulin</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:119px;">T7451</td>
			<td style="width:347px;">Mouse monoclonal, use at 1:500</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:247px;">anti-phospho-T287 CaMK-II</td>
			<td style="width:175px;">EMD Millipore</td>
			<td style="width:119px;">06-881</td>
			<td style="width:347px;">Rabbit polyclonal, use at 1:20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">anti-total CaMK-II</td>
			<td style="width:175px;">BD Biosciences</td>
			<td style="width:119px;">611292</td>
			<td style="width:347px;">Mouse monoclonal, use at 1:20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Ethanol</td>
			<td style="width:175px;">Fisher</td>
			<td style="width:119px;">S96857</td>
			<td style="width:347px;">Lab grade, 95% denatured</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Forceps</td>
			<td style="width:175px;">Fine Science Tools</td>
			<td>11252-20</td>
			<td>Dumont #5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Glass coverslips</td>
			<td style="width:175px;">VWR</td>
			<td style="width:119px;">16004-330</td>
			<td>#1&#160; thickness</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Glass microscope slides</td>
			<td style="width:175px;">Fisher</td>
			<td style="width:119px;">12-550-15</td>
			<td>Standard glass slides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Methanol</td>
			<td style="width:175px;">Fisher</td>
			<td style="width:119px;">A411</td>
			<td>Store in freezer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Microcentrifuge tubes</td>
			<td style="width:175px;">VWR</td>
			<td style="width:119px;">20170-038</td>
			<td>capped tubes, not sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Normal goat serum</td>
			<td style="width:175px;">Life Technologies</td>
			<td style="width:119px;">16210-064</td>
			<td>Aliquot 1ml tubes, store in freezer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Paraformaldehyde</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:119px;">P-6148</td>
			<td>Reagent grade, crystalline</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Phosphate buffered saline (PBS)</td>
			<td style="width:175px;">Quality Biological</td>
			<td style="width:119px;">119-069-131</td>
			<td>10X stock solution or made in lab</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Triton X-100</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:119px;">BP-151</td>
			<td>10% solution in water, store at room temp</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Tween-20</td>
			<td style="width:175px;">Life Technologies</td>
			<td style="width:119px;">85113</td>
			<td>10% solution in water, store at room temp</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Compound microscope</td>
			<td style="width:175px;">Nikon</td>
			<td style="width:119px;">E-600</td>
			<td>Mount on vibration-free table</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">C1 Plus two-laser scanning confocal</td>
			<td style="width:175px;">Nikon</td>
			<td style="width:119px;">C1 Plus</td>
			<td>Run by EZ-C1 program, but upgrades use &#34;Elements&#34;</td>
		</tr>
	</tbody>
</table>

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

La prolifération rapide des cellules, l&#39;expression tissu-spécifique des gènes et de l&#39;émergence de réseaux de signalisation caractérisent le développement embryonnaire précoce de tous les vertébrés. La cinétique et l&#39;emplacement des signaux - même à l&#39;intérieur des cellules individuelles - dans l&#39;embryon en développement complète l&#39;identification des gènes du développement importants. immunocoloration techniques sont décrites qui ont été montré pour définir les cinétiques de signaux intracellulaires et des animaux entiers dans des structures aussi petites que les cils primaires. Les techniques de fixation, d&#39;imagerie et de traitement des images en utilisant un laser à balayage composé microscope confocal peut être complété en aussi peu que 36 heures. Le poisson zèbre (Danio rerio) est un organisme souhaitable pour les enquêteurs qui cherchent à mener des études dans une espèce de vertébré qui est abordable et pertinente à la maladie humaine. KO ou passes rabattues génétiques doivent être confirmées par la perte du produit protéique réelle. Cette confirmation de la perte de protéinespeut être réalisée en utilisant les techniques décrites ici. Des indices dans les voies de signalisation peuvent également être déchiffrés en utilisant des anticorps qui sont réactifs avec des protéines qui ont été modifiées après la traduction par phosphorylation. La préservation et l&#39;optimisation de l&#39;état phosphorylée d&#39;un épitope est donc essentiel de cette détermination et est accompli par ce protocole. Cette étude décrit les techniques pour fixer des embryons au cours des premières 72 heures de développement et de co-localiser une variété d&#39;épitopes pertinents avec cils dans des vésicules (KV) de l&#39;Kupffer, le rein et l&#39;oreille interne. Ces techniques sont simples, ne nécessitent pas de dissection et peut être complété en une période relativement courte de temps. Projection des piles d&#39;images confocale en une seule image est un moyen utile de présenter ces données.

Conditions optimales pour la visualisation de phospho-épitopes Des procédés décrivant l&#39;immunolocalisation des epitopes protéiques dans embryons de poisson zèbre ont été relativement peu par rapport à celles de localisation d&#39;ARNm par hybridation in situ. Fixateurs utilisés dans la localisation des épitopes des protéines dans les cellules ciliées des embryons de poisson zèbre ont...

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

Techniques sont décrites à immunocoloration phospho-épitopes dans embryons de poisson zèbre entiers et ensuite procéder à deux couleurs confocal fluorescent localisation dans des structures cellulaires aussi petites que les cils primaires. Les techniques de fixation et d&#39;imagerie peuvent définir l&#39;emplacement et de la cinétique de l&#39;apparition ou de l&#39;activation de protéines spécifiques.

Immunocoloration phospho-épitopes dans ciliées organes de Mont entiers embryons de poissons zèbres

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

The overall goal of this procedure is to immunolocalize proteins that are activated in the zebrafish embryo. This method can help answer key questions in the developmental and cell biology field, such as the localization and activation of phosphorylated proteins in a whole embryo. The main advantage of this technique is that one can localize calcium dependent signaling in a whole embryo by immunostaining proteins activated by calcium.After obtaining wild type or transgenic embryos according to the text protocol, add 0.003%PTU to the embryos in system water to block pigmentation and incubate to the desired developmental stage. When the embryos have reached the correct stage, add MESAB to the dish to anesthetize the fish. Once the fish are anesthetized, remove as much system water as possible, then add fresh 4%PFA and PBS and incubate at room temperature for three to four hours.After the incubation, remove the PFA and use 100%methanol to replace it. Then, store the embryos at negative 20 degrees Celsius for at least 48 hours. To carry out immunostaining, place a minimum of 10 embryos for each experimental condition in labeled 1.5 millimeter micro-centrifuge tubes.When the embryos have settled to the bottom of the tube, remove and discard the methanol and use progressive washes of decreasing concentrations of ethanol in PBTx to re-hydrate the embryos. Remove the last PBTx wash and add 0.5 milliliters of 10%normal goat serum or NGS in PBTx. Then, incubate at room temperature for at least one hour with gentle rocking.Next, remove the solution and add 0.2 to 0.5 milliliters of a 1 in 5 dilution of mouse anti-acetylated tubulin monoclonal antibody and 10%NGS and PBTx. Incubate the embryos with gentle rocking at room temperature overnight. In the morning, remove and discard the antibody and perform three washes using 0.5 milliliters of 2%NGS and PBTx with gentle rocking for five minutes each.Dim the overhead lights and add 500 microliters to each sample of a 1 in 500 dilution of flourescently conjugated secondary antibody in 10%NGS and PBTx. Incubate in the dark at room temperature for four hours with gentle rocking. After the incubation, wash the embryos three times.To co-immunostain, add a 1 in 20 dilution of anti-phospho CaMKII antibody. Incubate in the dark with gentle rocking overnight. The next morning, after washing the embryos with 2%NGS and PBTx three times, add a 1 in 5 dilution of red 568 fluorescent conjugated goat anti-rabbit IgG and 10%NGS and PBTx.After incubating in the dark at room temperature for four hours, wash the embryos three times and store in PBTx or 50%glycerol in PBS depending on the imaging procedure. To mount the embryos for imaging, place one to five embryos on a glass slide. Use four number one stacked cover slip fragments on either side of the fish to create a chamber.And place a cover slip on top. With the 100X oil immersion lens and transmitted light bring a single embryo into focus. Turn off the light.Turn on the confocal microscope with the appropriate lasers and engage the remote focus. Open the confocal program and in the acquire bar, select the proper objective. Then, in the XY basic bar, click on the 1024 button to set the image size.In the laser and detector bar, click on the red 488 and green 568 boxes to turn on the laser detector for each channel and set the pinhole to medium. Then, in the gain bar, adjust the gain for each channel to visualize it. Next, in the acquire settings bar, click live to begin acquiring images.In the view settings bar, uncheck the force integral zoom box to center in the live window. In the acquire settings bar under the Z tab, step through the layers of the image. After selecting the number of layers to incorporate into the image, move to some point in the center of the Z plane.Under the Z tab, click the small red reference box. This will zero the RFA at the point chosen as the center. In the box labeled step size, select the thickness of the layers.Pay attention to the file size box and try to limit the files to one gigabyte. To find the top extreme of the image, click the circle next to top and move through the Z layers. Now click the circle next to bottom and the computer will take the image back to the layer as center.Then, in the acquire settings box, click on live again to stop the laser acquisition. In this panel, click the red boxes labeled average and Z stack. These boxes will turn green.In the acquire settings box, click single to acquire the entire series of images. Monitor the progress as it scans in both channels and through the entire Z stack. To save, click on the volume window and click save as and name the file.To volume render the file, while the Z stack is still open, select the data pulldown menu and click on volume render. Save the rendered file as a dot tif file. This figure shows immunostaining for P-CaMKII and acetylated tubulin, which is a standard marker for cilia in the zebrafish Kuppfer's vesicle or KV.Feeding primary cilia generate a circular flow of fluid, which leads to an elevation of calcium in the ciliated cells lining the KV and the activation of CaMKII in discrete locations as seen here.These figures show P-CaMKII as it appears on the apical surface of ciliated cells lining specific regions of the perinephric ducts between 24 and 72 hours post-fertilization or HPF. At one day of development, the zebrafish embryonic ear fixed with PFA and methanol retains its structures and allows the detection of CaMKII staining at the base and along the length of the kinocilum. At 72 HPF, the inner ear was counterstained with Alexa 488 phalloidin and anti-acetylated tubulin with Alexa 568.The PFA methanol fixation preserved both F-actin and tubulin. This 30 HPF fish expressed a membrane-targeted GFP, which would be lost with methanol fixation. PFA fixation resulted in a diminished P-CaMKII signal compared to fixation with PFA and methanol.However, at this stage of development, the signal is strong enough to be detected without methanol. Once mastered, this technique can be done in two days if it is performed properly. While attempting this procedure, it's important to remember to use fresh embryos and fresh free agents.After watching this video, you should have a good understanding of how to localize phosphoproteins in zebrafish embryos.

Research

JoVE Journal

Chemistry

High Precision Zinc Isotopic Measurements Applied to Mouse Organs

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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 ...

Recherche

Chimie

Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation

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Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

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Accessing Valuable Ligand Supports for Transition Metals: A Modified, Intermediate Scale Preparation of 1,2,3,4,5-Pentamethylcyclopentadiene

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Microprobe Capillary Electrophoresis Mass Spectrometry for Single-cell Metabolomics in Live Frog (Xenopus laevis) Embryos

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NMR-Based Activity Assays for Determining Compound Inhibition, IC50 Values, Artifactual Activity, and Whole-Cell Activity of Nucleoside Ribohydrolases

NMR-Based Activity Assays for Determining Compound Inhibition, IC50 Values, Artifactual Activity, and Whole-Cell Activity of Nucleoside Ribohydrolases

Tests d'activité basés sur la RMN pour déterminer l'inhibition des composés, les valeurs IC50, l'activité artifactual et l'activité à cellules entières des ribohydrolases nucléoside

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Comparison of Two Different Synthesis Methods of Single Crystals of Superconducting Uranium Ditelluride

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