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.&#160;Remove from heat and add 100 ml 2x phosphate buffered saline (PBS). Bring volume to 200 ml&#160;with distilled H2O. Cool to RT&#160;and confirm pH of 7.4 before use. Store in the dark at 4 &#176;C but use within one week.</li>
	<li>Use 100% methanol without any supplements. Use 95% ethanol without any supplements.</li>
	<li>Prepare Phosphate-buffered Tween (PBT). Supplement Phosphate-buffered saline (PBS) with 0.1% Tween-20. Add 0.5 ml of a 20% Tween-20 stock solution to 100 ml&#160;1x PBS. Store at RT.</li>
	<li>Prepare Phosphate-buffered Triton-X (PBTx). Supplement PBS with 0.1% Triton X-100. Add 0.5 ml of a 20% Triton X-100 stock to&#160;100 ml of PBS. Store at RT.</li>
	<li>Prepare 10% NGS/PBTx. Normal goat serum (NGS) blocks non-specific binding sites. Store NGS in aliquots at -20 &#176;C. To use, add 1 ml to 9 ml PBTx.</li>
	<li>Prepare 50% glycerol/PBS. Mix thoroughly equal parts of 2x PBS and 100% glycerol. Store at RT.</li>
</ol>
2. Embryo Fixation
<ol>
	<li>Breeding.&#160;Obtain wild type (AB and WIK) or transgenic (e.g., &#946;-actin:CAAX-GFP) embryos through natural matings. If desired, inject embryos with constructs or morpholinos, as described.15,16</li>
	<li>Raise embryos.&#160;Collect embryos and incubate at 28.5 &#176;C in the presence of 0.003% 1-phenyl-2-thiourea (PTU) to block pigmentation as described.17</li>
	<li>Fix.&#160;When embryos have reached the desired developmental stage,18,&#160;anesthetize the embryos using MESAB, remove as much system water as possible and add fresh 4% PFA/PBS for 3 - 4 hr&#160;at RT.&#160;NOTE: At times less than 20 hr&#160;post fertilization (hpf), fix prior to dechorionation. At times after 20 hpf, dechorionate prior to fixation using two pairs of fine point forceps under a dissecting microscope.</li>
	<li>Changing solutions.&#160;Transfer embryos using a wide bore pipet, as described.17 Change solutions in 1.5 ml capped microcentrifuge tubes, simply by allowing embryos to settle by gravity without centrifugation.</li>
	<li>Post-fixative.&#160;After 3 - 4 hr, remove PFA/PBS, replace with 100% methanol and store at -20 &#176;C for at least 48 hr. NOTE: For maximal P-CaMK-II immunoreactivity, limit total methanol storage time to one week.</li>
</ol>
3. Immunostaining Whole Embryos
<ol>
	<li>Place a minimum of 10 embryos for each experimental condition in capped 1.5 ml microcentrifuge tube, and label them.</li>
	<li>Allow the embryos to settle. Remove and discard methanol and rehydrate with progressive washes containing 0.5 ml of decreasing concentrations of ethanol, as indicated in the next step. Each step is a 5 min wash with rocking.</li>
	<li>Progressively rehydrate with these 5 solutions: 66% ethanol/33% PBTx, 33% ethanol/66% PBTx, 100% PBTx, 100% PBTx (this is the first repeat of PBTx), 100% PBTx (this is the second repeat of PBTx).</li>
	<li>Remove last PBTx wash. Add 0.5 ml 10% NGS/PBTx. Incubate for at least 1 hr at RT&#160;with gentle rocking and then remove and discard solution.</li>
	<li>Prepare primary antibody solution by diluting in 10% NGS/PBTx. If co-immunostaining, use higher affinity antibody first. For this study, incubate with the mouse anti-acetylated tubulin monoclonal antibody diluted 1:500. For example, if there are 10 samples, combine 6 &#956;l of the stock antibody solution with 3 ml&#160;of 10% NGS/PBTx and then distribute 0.3 ml of this into each tube.</li>
	<li>Add 0.2 - 0.5 ml of the diluted primary antibody to each tube to immerse all embryos. Incubate with gentle rocking O/N&#160;at RT.</li>
	<li>In the morning, remove and discard antibody solution. Add 0.5 ml 2% NGS/PBTx to wash away excess primary antibody. Gently rock for 5 min. Remove and discard solution. Repeat twice.</li>
	<li>Dim overhead lights and dilute the appropriate fluorescently-conjugated secondary antibody in 10% NGS/PBTx so that each condition contains at least 0.5 ml. With the mouse anti-acetylated tubulin primary antibody, use the green-fluorescent dye goat anti-mouse IgG at a 1:500 dilution.</li>
	<li>Incubate secondary antibody for 4 hr with gentle rocking at RT in the dark. From now on, process the samples under dimmed lights and incubate in a dark container or by wrapping in aluminum foil.</li>
	<li>At the end of the incubation, remove and discard the secondary antibody solution. Add 0.5 ml 2% NGS/PBTx to wash. Gently rock for 5 min. Remove and discard solution. Repeat twice.</li>
	<li>If co-immunostaining, obtain the second primary antibody from a different species than the first primary antibody. In these studies, follow the rabbit anti-phospho CaMK-II antibody by the red-fluorescent dye-conjugated goat anti-rabbit IgG secondary antibody.</li>
	<li>Dilute rabbit anti-phospho CaMK-II antibody 1:20. For each tube of embryos, suspend in 0.3 ml 10% NGS/PBTx, then add 15 &#956;l of the stock antibody solution.</li>
	<li>Ensure that embryos are immersed and lights are dimmed. Incubate with gentle rocking O/N at RT in the dark.</li>
	<li>In the morning under dim lights, remove and discard antibody solution. Add 0.5 ml 2% NGS/PBTx. Gently rock for 5 min. Remove and discard solution. Repeat twice.</li>
	<li>Keep overhead lights dim and dilute enough of the appropriate fluorescently-conjugated secondary antibody so that each condition contains at least 0.5 ml. In this study, dilute the red-fluorescent dye-conjugated goat anti-rabbit IgG secondary antibody (red channel) 1:500 in 10% NGS/PBTx.</li>
	<li>Incubate second secondary antibody for 4 hr with gentle rocking at RT in the dark.</li>
	<li>At the end of this incubation and under dim lights, remove and discard the secondary antibody solution. Add 0.5 ml PBTx. Gently rock for 5 min. Remove and discard solution. Repeat twice. Store in either PBTx or 50% glycerol/PBS depending on the imaging procedure.</li>
</ol>
4. Confocal Imaging and Processing
<ol>
	<li>Mount embryos for imaging. Place 1 - 5 embryos on a glass slide. Create a chamber between the main coverslip and the slide using coverslip fragments on each side of the chamber. Typically, four #1 coverslips are used to make these spacer stacks without sealing. 
	NOTE: No mounting medium is necessary.</li>
	<li>Use a 100X oil immersion objective to bring single embryo into focus using transmitted light. Turn off light. Turn on confocal microscope. Ensure that appropriate lasers (green and/or red) are turned on and remote focus accessary is engaged.</li>
	<li>Turn on the confocal program. In the &#34;Acquire bar,&#34; select the proper objective. In the &#34;XY Basic&#34; bar, set image size to 1,024 by clicking the 1,024 button.</li>
	<li>In the &#34;Laser and Detector&#34; bar, click on the red 488 box and the green 568 box to turn on laser and detector for each channel. Set pinhole to medium and adjust the gain in the &#34;Gain&#34; bar of each channel to visualize.</li>
	<li>In the &#34;Acquire Settings&#34; bar, click &#34;Live&#34; to begin acquiring images. In the &#34;View Settings&#34; bar, uncheck the &#34;Force Integral Zoom&#34; box to center in the &#34;Live&#34; window.</li>
	<li>In the &#34;Acquire Settings&#34; bar, under the &#34;Z&#34; tab, step through the layers of the image. After selecting the number of optical sections to incorporate into the image, move to some point in the &#34;center&#34; of the z-plane.</li>
	<li>Under the &#34;Z&#34; tab, click the small red &#34;Reference&#34; box. This will zero the RFA at the point chosen as the &#34;center&#34;. In the box labeled &#34;Step Size&#34;, select the thickness of the layers (between 0.25 &#181;m and 2.0 &#181;m is ideal). Pay attention to the &#34;File Size&#34; box and try to limit to 1GB. 
	NOTE: Typically, up to 40 optical sections of 0.5 - 1.0 &#956;m are obtained, but the number of sections can be determined empirically.</li>
	<li>To find the top extreme of the image, click the circle next to &#34;Top&#34; and move through the z layers. Now click the circle next to &#34;Bottom&#34;, the computer will take the image back to the layer as the center. Again move through the layers to find the bottom extreme of the image.</li>
	<li>Click on &#34;Live&#34; again in the &#34;Acquire Settings&#34; box to stop the laser acquisition. In this panel, click the red boxes labeled Average and Z-stack. These boxes will turn green.</li>
	<li>In the &#34;Acquire Settings&#34; box, click &#34;Single&#34; to acquire the entire series of images. You can monitor the progress as it scans in both channels and through the entire z-stack.</li>
	<li>To save, click on the volume window and click &#34;save as&#34; and name the file. Save as an &#34;.ids&#34; file. To volume render the file while the z-stack is still open, select the Data pull down menu and click on &#34;volume render&#34;. Save the rendered file as a tiff file. This is the projected image that are shown in this publication.</li>
</ol>

<ol>
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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. 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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 authors declare that they have no competing financial interests.

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 autofluorescence, b) preservation of the phospho-CaMK-II epitope and c) enhanced accessibility of the epitope to antibodies.
A primary limitation of this approach is the time of storage in methanol. While immunoreactivity of P-CaMK-II is maximal after two days in methanol at -20&#176;C, the reactivity of this epitope is lost after one week of storage in methanol. It is not clear whether at this time the phosphate group is hydrolyzed or further denaturation occurs that decreases immunoreactivity. Methanol/EGTA at -20 &#176;C is a traditionally rapid fixative for preserving tubulin-rich structures during the development of early embryos.22 However, methanol alone is not desirable for preserving morphology or even structures such as the actin cytoskeleton and must be preceded by PFA.
Additional advantages of this approach are that it also preserves other epitopes such as F-actin and acetylated tubulin and does not eliminate the native fluorescence of GFP. Other fixatives, such as Dent&#39;s fixative, are superior for other epitopes13 and remain compatible with P-CaMK-II staining, although P-CaMK-II staining is weaker in Dent&#39;s fixative than PFA/methanol. Since phospho-proteins are prevalent in signaling pathways that are active during early development, Dent&#39;s fixative and PFA/methanol are recommended as two fixatives for other phospho-epitopes in zebrafish embryos. In developing applications with this technique for other proteins and their antibodies, it has been helpful to have antibodies that are also reactive on immunoblots and to have cloned and overexpressed proteins with which antibodies can be validated.12
It is worth the effort to optimize techniques for immunolocalization because it a) can verify gene suppression at the protein level and b) enables the development of models of action. In this laboratory, the results of these assays have allowed the formulation of models through which CaMK-II functions. For example, its location in the KV is transient and asymmetric, like the role of this organ. In the kidney, its location on the apical side of pronephric ductal cells suggests roles that respond to signals from the duct. Finally, the activation of this enzyme in specific intense puncta at the base of ear kinocilia suggests a localized Ca2+ signal. The methods described here should also be useful to any investigator who seeks to obtain high-resolution images of any embryonic cell type in two fluorescent channels.

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.19 Localization of RNA by whole mount in situ hybridization (WISH) is typically achieved using PFA followed by storage in 100% methanol.20,21 Our studies revealed that the WISH fixative combination, when limiting the time in methanol to between two and seven days, was vastly superior to any other fixative for immunolocalization with the P-CaMK-II antibody. The signal achieved with this PFA/methanol combination was also significantly greater than when Dent&#39;s fixative, 4% PFA/PBS or methanol were used alone, depending on the developmental time and location within the embryo.
When optimizing the technique described here and for each fixative and developmental time used, a control sample was prepared in which all steps were followed, except that the primary antibody was omitted. Such control samples lacked a fluorescent signal when imaged under the same conditions described above. This control is recommended for other investigators replicating this approach with any antibody.
The PFA/methanol fixative combination yielded the strongest P-CaMK-II signal and was also compatible with immunostaining for acetylated tubulin, which is a standard marker for cilia. Developmentally, the first ciliated organ that emerges and has been imaged in these studies is the Kupffer&#39;s vesicle (KV). The KV is located at the posterior end of the notochord as shown at the 12-somite (15 hpf) stage (Figure 1). The KV is a transient organ and is responsible for left-right asymmetry. It emerges at the 2-somite stage (12 hpf) and disappears around the 18-somite stage. Beating primary cilia generate a circular flow of fluid, which leads to an elevation of Ca2+ in the ciliated cells lining the KV and the activation of CaMK-II in discrete locations (Figure 1). P-CaMK-II reactivity appears and disappears on one side of the KV as long as cilia are beating.12 At this early stage, total embryonic CaMK-II levels are still only half the level as at 24 hpf and one tenth of the level at 3 days of development.11 Perhaps because total CaMK-II expression is relatively low at 15 hpf, any improvement in the immunostaining technique at this stage is especially important.
Between 24 and 72 hpf of development, P-CaMK-II appears on the apical surface of ciliated cells lining specific regions of the pronephric ducts (Figures 2,3). The immunoreactivity of total CaMK-II along the entire pronephric duct and throughout adjacent somites (Figure 2) demonstrates that only a subset of embryonic CaMK-II is activated (P-CaMK-II).
Fixation Conditions are Compatible with Preserving GFP Fluorescence
These fixation techniques are also compatible with retaining green fluorescent protein (GFP) fluorescence without enhancement (Figure 3). In the GFP-tagged CaMK-II construct that is used in this figure, GFP retains sufficient fluorescence though PFA/methanol fixation and subsequent immunostaining. In a high magnification view of cells lining the pronephric duct (Figure 3), the mosaic expression of GFP-CaMK-II can be viewed in cells that have also been immunostained for P-CaMK-II.
The zebrafish embryonic ear is another tissue in which the elevation of Ca2+, acting through CaMK-II, influences its development.14 Zebrafish ears normally appear at around one day of development. At this time, CaMK-II is intensely activated at the base of the kinocilia and at lower levels along the kinocilium (Figure 4). This set of images shows that this fixation and immunolocalization technique can detect staining within cilia, a structure whose cross-section is less than 1 &#956;m. These cilia retain their structure throughout this fixation and immunostaining process.
An additional example of two-color counterstaining in the inner ear at a later time of development was achieved by staining with both Alexa488 phalloidin and anti-acetylated tubulin followed by an Alexa568 tagged secondary antibody (Figure 5). It is noteworthy that the PFA/methanol fixation method preserves both F-actin and tubulin, which is not true of all fixatives. This example is shown as a merged color image for the entire ear and then for sub-regions.14
A final representative example of two-color counterstaining was achieved with a strain of fish that produced embryos with membrane-targeted GFP (Figure 6). Membrane targeted GFP is not attached to any other protein and is lost when extracted with methanol, therefore this image was obtained from fish that were fixed with PFA alone. This result suggests that methanol treatment extracts membranes, so it is not recommended for proteins that may be exclusively membrane bound. The P-CaMK-II signal in this figure is diminished relative to that achieved if methanol were also used, but this demonstrates that at this stage and location (kidney) of the developing embryo, the signal is strong enough to be detected without methanol treatment. That is not true at the KV stage; i.e., methanol is required to visualize P-CaMK-II immunostaining. This example is only shown as a merged image where the kidney&#39;s pronephric duct is adjacent to trunk muscle.
In summary, the PFA/methanol fixation method described here is compatible with the preservation of GFP and with staining for F-actin, acetylated tubulin, total CaMK-II and P-CaMK-II.
<img alt="Figure 1" src="/files/ftp_upload/53747/53747fig1.jpg" /> 
Figure 1. P-CaMK-II Counterstained for Acetylated Tubulin (cilia) in Zebrafish KV. Views of a single live 12 somite stage (12 ss) embryo reveals the posterior location of the KV by the arrowhead (lateral view, A) and the circle within the box (ventral view, B). Embryos at this stage were fixed using PFA/methanol. Embryos were immunostained for acetylated tubulin followed by an Alexa488-labelled secondary antibody (green channel). Next, the rabbit polyclonal antibody against P-CaMK-II was followed by an Alexa568-labelled secondary antibody (red channel). Two-channel fluorescent image projections were acquired as described at progressively higher magnifications (C,D). Scale bar = 10 &#956;m. This figure is modified from a previous publication.12 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53747/53747fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53747/53747fig2.jpg" /> 
Figure 2. P-CaMK-II Counterstained for Total CaMK-II along Zebrafish Kidney. A single dechorionated embryo at 30 hpf was fixed using PFA/methanol. Embryos were then stained for total CaMK-II followed by the Alexa488 labeled secondary antibody (green). Next, the P-CaMK-II primary antibody was followed by the Alexa568 labeled secondary antibody (red). Staining reveals an enrichment of activated CaMK-II (in red) along the apical surface of cells that line the ducts of the pronephric zebrafish kidney whereas total CaMK-II is enriched at the somite boundaries of adjacent muscle tissue and throughout sarcomeres. Scale bar = 50 &#956;m. These images were acquired at a lower magnification than is described in the text in order to visualize the entire kidney. This figure is modified from a previous publication.13 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53747/53747fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53747/53747fig3.jpg" /> 
Figure 3. P-CaMK-II Counter Imaged for GFP in Zebrafish Kidney Cells. This 30 hpf embryo was injected with a cDNA encoding a dominant negative GFP-CaMK-II.13 Such injections typically show mosaic expression. The embryo was fixed using PFA/methanol and then immunostained for P-CaMK-II using an Alexa568 labeled secondary antibody. Imaging reveals that GFP persists through PFA/methanol fixation, rehydration, blocking and immunostaining. Scale bar = 10 &#956;m. This figure is modified from a previous publication.13 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53747/53747fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53747/53747fig4.jpg" /> 
Figure 4. P-CaMK-II Counterstained with Acetylated Tubulin in Zebrafish Inner Ear. This 30 hpf embryo was fixed using PFA/methanol. The anti-acetylated tubulin antibody was followed in order by the Alexa488-labeled secondary antibody, the P-CaMK-II antibody and the Alexa568 labeled secondary antibody. Staining reveals that P-CaMK-II is present along inner ear cilia and co-localizes with acetylated tubulin. Scale bar = 5 &#956;m. This figure is modified from a previous publication.14 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53747/53747fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/53747/53747fig5.jpg" /> 
Figure 5. Actin and Acetylated Tubulin in Zebrafish Inner Ear. A three day old (72 hpf) zebrafish embryo was fixed and immunostained for acetylated tubulin using Alexa568-labeled secondary antibodies, followed by actin visualization using Alexa488 phalloidin. Cilia as well as axonal innervation of the ear is revealed by acetylated tubulin (in red) and F-actin structures include muscle fibers (in green). Two ciliated sensory regions of the zebrafish ear are shown in more detail: the anterior macula (am) and posterior crista (pc). Scale bar = 10 &#956;m. This figure is modified from a previous publication.14 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53747/53747fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/53747/53747fig6.jpg" /> 
Figure 6. P-CaMK-II Counter Imaged for Membrane GFP in Zebrafish Kidney. This single merged image in which the &#946;-actin:CAAX-GFP embryo (30 hpf) was fixed and stained for P-CaMK-II followed by the Alexa568 labeled secondary antibody. Staining reveals an enrichment of activated CaMK-II (in red) along the apical surface of cells that line the ducts of the pronephric zebrafish kidney. These kidney ductal cells are nestled just underneath muscle tissue and both are marked by the expression of membrane targeted GFP (in green). Scale bar = 50 &#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53747/53747fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Chemistry

7.9K Views.  Virginia Commonwealth University. 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.

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.

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.

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 ...

Synthesis and Bioconjugation of Thiol-Reactive Reagents for the Creation of Site-Selectively Modified Immunoconjugates

Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. Yet maleimide-based conjugates display limited stability in vivo because the succinimidyl thioether linkage can undergo a retro-Michael reaction. This, of course, can lead to the release of the radioactive payload or its exchange with thiol-bearing biomolecules in circulation. Both of these processes can produce elevated activity concentrations in healthy organs as well as decreased activity concentrations in target tissues, resulting in reduced imaging contrast and lower therapeutic ratios. In 2018, we reported the creation of a modular, stable, and easily accessible phenyloxadiazolyl methyl sulfone reagent &#8212; dubbed &#8216;PODS&#8217; &#8212; as a platform for thiol-based bioconjugations. We have clearly demonstrated that PODS-based site-selective bioconjugations reproducibly and robustly create homogenous, well-defined, highly immunoreactive, and highly stable radioimmunoconjugates. Furthermore, preclinical experiments in murine models of colorectal cancer have shown that these site-selectively labeled radioimmunoconjugates exhibit far superior in vivo performance compared to radiolabeled antibodies synthesized via maleimide-based conjugations. In this protocol, we will describe the four-step synthesis of PODS, the creation of a bifunctional PODS-bearing variant of the ubiquitous chelator DOTA (PODS-DOTA), and the conjugation of PODS-DOTA to the HER2-targeting antibody trastuzumab.

In this protocol, we will describe the synthesis of PODS, a phenyoxadiazolyl methyl sulfone-based reagent for the site-selective attachment of cargos to the thiols of biomolecules, particularly antibodies. In addition, we will describe the synthesis and characterization of a PODS-bearing bifunctional chelator and its conjugation to a model antibody.

Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. ...

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 ...