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

This work was supported by the National Science Foundation grant IOS-0817658.

The zebrafish procedures in this protocol have been approved by the Institutional Animal Care and Use Committee (IACUC) at Virginia Commonwealth University.
1. Preparation of Reagents
<ol>
	<li>4% PFA/PBS. Weigh 8 g of paraformaldehyde (PFA) in the fume hood. While still in the fume hood, dissolve the dry PFA in ~80 ml distilled H2O with stirring and heating to 50 &#176;C. While stirring, add 3 - 10 drops of fresh 1N NaOH until PFA is completely dissolved and solution clarifies.&#...

<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. 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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>

The PFA/methanol method was developed in this laboratory with the primary objective of optimizing the immunolocalization of the phospho-T287-CaMK-II epitope during zebrafish development. This method successfully localized P-CaMK-II during the formation of several ciliated organs including the zebrafish KV,12 inner ear14 and kidney.13 Particularly at the KV stage, this technique was necessary. The success of this method is likely due to a combination of a) minimization of autofl...

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

The rapid proliferation of cells, the tissue-specific expression of genes and the emergence of signaling networks characterize early embryonic development of all vertebrates. The kinetics and location of signals - even within single cells - in the developing embryo complements the identification of important developmental genes. Immunostaining techniques are described that have been shown to define the kinetics of intracellular and whole animal signals in structures as small as primary cilia. The techniques for fixing, imaging and processing images using a laser-scanning confocal compound microscope can be completed in as few as 36 hr.
Zebrafish (Danio rerio) is a desirable organism for investigators who seek to conduct studies in a vertebrate species that is affordable and relevant to human disease. Genetic knockouts or knockdowns must be confirmed by the loss of the actual protein product. Such confirmation of protein loss can be achieved using the techniques described here. Clues into signaling pathways can also be deciphered by using antibodies that are reactive with proteins that have been post-translationally modified by phosphorylation. Preserving and optimizing the phosphorylated state of an epitope is therefore critical to this determination and is accomplished by this protocol.
This study describes techniques to fix embryos during the first 72 hr of development and co-localize a variety of relevant epitopes with cilia in the Kupffer&#39;s Vesicle (KV), the kidney and the inner ear. These techniques are straightforward, do not require dissection and can be completed in a relatively short period of time. Projecting confocal image stacks into a single image is a useful means of presenting these data.

Optimal Conditions for Visualizing Phospho-epitopes
Methods describing the immunolocalization of protein epitopes in zebrafish embryos have been relatively sparse compared to those for localizing mRNAs via in situ hybridization. Fixatives used in localizing protein epitopes in ciliated cells of zebrafish embryos have included 4% PFA/PBS and Dent&#39;s fixative, which is a mixture of methanol and DMSO.<s...

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 are described to immunostain phospho-epitopes in whole zebrafish embryos and then conduct two-color fluorescent confocal localization in cellular structures as small as primary cilia. The techniques for fixing and imaging can define the location and kinetics of the appearance or activation of specific proteins.

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.

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

Research

JoVE Journal

Chemistry

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.

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 ...

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

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular identity and function. Each of the seven PtdInsPs varies in their distribution and abundance, which are tightly regulated by specific kinases and phosphatases. The abundance of PtdInsPs can change abruptly in response to various signaling events or disturbance of the regulatory machinery. To understand how these events lead to changes in the amount of PtdInsPs and their resulting impact, it is important to quantify PtdInsP levels before and after a signaling event or between control and abnormal conditions. However, due to their low abundance and similarity, quantifying the relative amounts of each PtdInsP can be challenging. This article describes a method for quantifying PtdInsP levels by metabolically labeling cells with 3H-myo-inositol, which is incorporated into PtdInsPs. Phospholipids are then precipitated and deacylated. The resulting soluble 3H-glycero-inositides are further extracted, separated by high-performance liquid chromatography (HPLC), and detected by flow scintillation. The labeling and processing of yeast samples is described in detail, as well as the instrumental setup for the HPLC and flow scintillator. Despite losing structural information regarding acyl chain content, this method is sensitive and can be optimized to concurrently quantify all seven PtdInsPs in cells.

Phosphoinositides are signaling lipids whose relative abundance rapidly changes in response to various stimuli. This article describes a method to measure the abundance of phosphoinositides by metabolically labeling cells with 3H-myo-inositol, followed by extraction and deacylation. Extracted glycero-inositides are then separated by high-performance liquid chromatography and quantified by flow scintillation.

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular ...

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical (Methylation) and Physical (Mass Spectrometry, Nuclear Magnetic Resonance) Techniques

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans. De-starched wheat endosperm cell walls were isolated as an alcohol-insoluble residue (AIR)1 and sequentially extracted with water (W-sol Fr) and 1 M KOH containing 1% NaBH4 (KOH-sol Fr) as described by Ratnayake et al. (2014)2. Two different approaches (see summary in Figure 1) are adopted. In the first, intact W-sol AXs are treated with 2AB to tag the original RE backbone chain sugar residue and then treated with an endoxylanase to generate a mixture of 2AB-labelled RE and internal region reducing oligosaccharides, respectively. In a second approach, the KOH-sol Fr is hydrolyzed with endoxylanase to first generate a mixture of oligosaccharides which are subsequently labelled with 2AB. The enzymically released ((un)tagged) oligosaccharides from both W- and KOH-sol Frs are then methylated and the detailed structural analysis of both the native and methylated oligosaccharides is performed using a combination of MALDI-TOF-MS, RP-HPLC-ESI-QTOF-MS and ESI-MSn. Endoxylanase digested KOH-sol AXs are also characterized by nuclear magnetic resonance (NMR) that also provides information on the anomeric configuration. These techniques can be applied to other classes of polysaccharides using the appropriate endo-hydrolases.

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical Methylation and Physical Mass Spectrometry, Nuclear Magnetic Resonance Techniques

This protocol describes the specific techniques used for the structural characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans by tagging the RE with 2 aminobenzamide prior to enzymatic (endoxylanase) hydrolysis and then analysis of the resultant oligosaccharides using mass spectrometry (MS) and nuclear magnetic resonance (NMR).

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of ...

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation.
We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine - maleimide coupling of proteins to other proteins, dyes, drugs or polymers.

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

This protocol details the important steps required for the bioconjugation of a cysteine containing protein to a maleimide, including reagent purification, reaction conditions, bioconjugate purification and bioconjugate characterization.

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

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

A reliable, intermediate scale preparation of 1,2,3,4,5-pentamethylcyclopentadiene (Cp*H) is presented, based on modifications of existing protocols that derive from initial 2-bromo-2-butene lithiation followed by acid mediated dienol cyclization. The revised synthesis and purification of the ligand avoids the use of mechanical stirring while still permitting access to significant quantities (39 g) of Cp*H in good yield (58%). The procedure offers other additional benefits, including a more controlled quench of excess lithium during the production of the intermediate heptadienols and a simplified isolation of Cp*H of sufficient purity for metallation with transition metals. The ligand was subsequently used to synthesize [Cp*MCl2]2 complexes of both iridium and ruthenium to demonstrate the utility of the Cp*H prepared and purified by our method. The procedure outlined herein affords substantial quantities of a ubiquitous ancillary ligand support used in organometallic chemistry while minimizing the need for specialized laboratory equipment, thus providing a simpler and more accessible entry point into the chemistry of 1,2,3,4,5-pentamethylcyclopentadiene.

Reliable, intermediate scale preparation of 1,2,3,4,5-pentamethylcyclopentadiene (Cp*H) is presented. The revised protocol for the synthesis and purification of the ligand minimizes the need for specialized laboratory equipment while simplifying reaction workups and product purification. Use of Cp*H in the synthesis of [Cp*MCl2]2 complexes (M = Ru, Ir) is also described.

A reliable, intermediate scale preparation of 1,2,3,4,5-pentamethylcyclopentadiene (Cp*H) is presented, based on modifications of existing protocols that ...

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

The quantification of small molecules in single cells raises new potentials for better understanding the basic processes that underlie embryonic development. To enable single-cell investigations directly in live embryos, new analytical approaches are needed, particularly those that are sensitive, selective, quantitative, robust, and scalable to different cell sizes. Here, we present a protocol that enables the in situ analysis of metabolism in single cells in freely developing embryos of the South African clawed frog (Xenopus laevis), a powerful model in cell and developmental biology. This approach uses a capillary microprobe to aspirate a defined portion from single identified cells in the embryo, leaving neighboring cells intact for subsequent analysis. The collected cell content is analyzed by a microscale capillary electrophoresis electrospray ionization (CE-ESI) interface coupled to a high-resolution tandem mass spectrometer. This approach is scalable to various cell sizes and compatible with the complex three-dimensional structure of the developing embryo. As an example, we demonstrate that microprobe single-cell CE-ESI-MS enables the elucidation of metabolic cell heterogeneity that unfolds as a progenitor cell gives rise to descendants during development of the embryo. Besides cell and developmental biology, the single-cell analysis protocols described here are amenable to other cell sizes, cell types, or animal models.

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

We describe steps that enable fast in situ sampling of a small portion of an individual cell with high precision and minimal invasion using capillary-based micro-sampling, to facilitate chemical characterization of a snapshot of metabolic activity in live embryos using a custom-built single cell capillary electrophoresis and mass spectrometry platform.

The quantification of small molecules in single cells raises new potentials for better understanding the basic processes that underlie embryonic ...

Photoelectron Imaging of Anions Illustrated by 310 Nm Detachment of F&#8722;

Anion photoelectron imaging is a very efficient method for the study of energy states of bound negative ions, neutral species and interactions of unbound electrons with neutral molecules/atoms. State-of-the-art in vacuo anion generation techniques allow application to a broad range of atomic, molecular, and cluster anion systems. These are separated and selected using time-of-flight mass spectrometry. Electrons are removed by linearly polarized photons (photo detachment) using table-top laser sources which provide ready access to excitation energies from the infra-red to the near ultraviolet. Detecting the photoelectrons with a velocity mapped imaging lens and position sensitive detector means that, in principle, every photoelectron reaches the detector and the detection efficiency is uniform for all kinetic energies. Photoelectron spectra extracted from the images via mathematical reconstruction using an inverse Abel transformation reveal details of the anion internal energy state distribution and the resultant neutral energy states. At low electron kinetic energy, typical resolution is sufficient to reveal energy level differences on the order of a few millielectron-volts, i.e., different vibrational levels for molecular species or spin-orbit splitting in atoms. Photoelectron angular distributions extracted from the inverse Abel transformation represent the signatures of the bound electron orbital, allowing more detailed probing of electronic structure. The spectra and angular distributions also encode details of the interactions between the outgoing electron and the residual neutral species subsequent to excitation. The technique is illustrated by the application to an atomic anion (F&#8722;), but it can also be applied to the measurement of molecular anion spectroscopy, the study of low lying anion resonances (as an alternative to scattering experiments) and femtosecond (fs) time resolved studies of the dynamic evolution of anions.

Photoelectron Imaging of Anions Illustrated by 310 Nm Detachment of F&lt;sup&gt;&amp;#8722;&lt;/sup&gt;

Here, we present a protocol for photoelectron imaging of anionic species. Anions generated in vacuo and separated by mass spectrometry are probed using velocity mapped photoelectron imaging, providing details of anion and neutral energy levels, anion and neutral structure and the nature of the anion electronic state.

Anion photoelectron imaging is a very efficient method for the study of energy states of bound negative ions, neutral species and interactions of unbound ...

Bio-inspired Polydopamine Surface Modification of Nanodiamonds and Its Reduction of Silver Nanoparticles

Surface functionalization of nanodiamonds (NDs) is still challenging due to the diversity of functional groups on the ND surfaces. Here, we demonstrate a simple protocol for the multifunctional surface modification of NDs by using mussel-inspired polydopamine (PDA) coating. In addition, the functional layer of PDA on NDs could serve as a reducing agent to synthesize and stabilize metal nanoparticles. Dopamine (DA) can self-polymerize and spontaneously form PDA layers on ND surfaces if the NDs and dopamine are simply mixed together. The thickness of a PDA layer is controlled by varying the concentration of DA. A typical result shows that a thickness of ~5 to ~15 nm of the PDA layer can be reached by adding 50 to 100 &#181;g/mL of DA to 100 nm ND suspensions. Furthermore, the PDA-NDs are used as a substrate to reduce metal ions, such as Ag[(NH3)2]+, to silver nanoparticles (AgNPs). The sizes of the AgNPs rely on the initial concentrations of Ag[(NH3)2]+. Along with an increase in the concentration of Ag[(NH3)2]+, the number of NPs increases, as well as the diameters of the NPs. In summary, this study not only presents a facile method for modifying the surfaces of NDs with PDA, but also demonstrates the enhanced functionality of NDs by anchoring various species of interest (such as AgNPs) for advanced applications.

A facile protocol is presented to functionalize the surfaces of nanodiamonds with polydopamine.

Surface functionalization of nanodiamonds (NDs) is still challenging due to the diversity of functional groups on the ND surfaces. Here, we demonstrate a ...

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

NMR spectroscopy is often used for the identification and characterization of enzyme inhibitors in drug discovery, particularly in the context of fragment screening. NMR-based activity assays are ideally suited to work at the higher concentrations of test compounds required to detect these weaker inhibitors. The dynamic range and chemical shift dispersion in an NMR experiment can easily resolve resonances from substrate, product, and test compounds. This contrasts with spectrophotometric assays, in which read-out interference problems often arise from compounds with overlapping UV-vis absorption profiles. In addition, since they lack reporter enzymes, the single-enzyme NMR assays are not prone to coupled-assay false positives. This attribute makes them useful as orthogonal assays, complementing traditional high throughput screening assays and benchtop triage assays. Detailed protocols are provided for initial compound assays at 500 &#956;M and 250 &#956;M, dose-response assays for determining IC50 values, detergent counter screen assays, jump-dilution counter screen assays, and assays in E. coli whole cells. The methods are demonstrated using two nucleoside ribohydrolase enzymes. The use of 1H NMR is shown for the purine-specific enzyme, while 19F NMR is shown for the pyrimidine-specific enzyme. The protocols are generally applicable to any enzyme where substrate and product resonances can be observed and distinguished by NMR spectroscopy. To be the most useful in the context of drug discovery, the final concentration of substrate should be no more than 2-3x its Km value. The choice of NMR experiment depends on the enzyme reaction and substrates available as well as available NMR instrumentation.

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

NMR-based activity assays have been developed to identify and characterize inhibitors of two nucleoside ribohydrolase enzymes. Protocols are provided for initial compound assays at 500 &#956;M and 250 &#956;M, dose-response assays for determining IC50 values, detergent counter screen assays, jump-dilution counter screen assays, and assays in E. coli whole cells.

NMR spectroscopy is often used for the identification and characterization of enzyme inhibitors in drug discovery, particularly in the context of fragment ...

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

Single crystal specimens of the actinide compound uranium ditelluride, UTe2, are of great importance to the study and characterization of its dramatic unconventional superconductivity, believed to entail spin-triplet electron pairing. A variety in the superconducting properties of UTe2 reported in the literature indicates that discrepancies between synthesis methods yield crystals with different superconducting properties, including the absence of superconductivity entirely. This protocol describes a process to synthesize crystals that exhibit superconductivity via chemical vapor transport, which has consistently exhibited a superconducting critical temperature of 1.6 K and a double transition indicative of a multi-component order parameter. This is compared to a second protocol that is used to synthesize crystals via the molten metal flux growth technique, which produces samples that are not bulk superconductors. Differences in the crystal properties are revealed through a comparison of structural, chemical, and electronic property measurements, showing that the most dramatic disparity occurs in the low-temperature electrical resistance of the samples.

Here, we present a protocol to synthesize two types of UTe2 crystals: those exhibiting robust superconductivity, via chemical vapor transport synthesis, and those lacking superconductivity, via molten metal flux synthesis.

Single crystal specimens of the actinide compound uranium ditelluride, UTe2, are of great importance to the study and characterization of its dramatic ...