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

这项工作是由美国国家科学基金会资助IOS-0817658的支持。

在此协议的斑马鱼程序已经批准的机构动物护理和使用委员会（IACUC）在弗吉尼亚联邦大学。 1.试剂的制备<ol><li> 4％PFA / PBS中。在通风柜权衡，8g仲甲醛（PFA）。同时仍然在通风柜，溶解干燥的PFA在〜80毫升蒸馏H 2ö搅拌并加热至50℃。边搅拌，加3 - 新鲜的1N NaOH 10滴，直到PFA完全溶解，溶液澄清。从火上移开，并加入100 ml 2倍磷酸盐缓冲液（PBS）。带来体积至200毫升蒸馏水H 2 O冷却至室温，并在使用前确认的7.4 pH值。存放在4℃的暗，但用一个星期内。 </li><li>用100％的甲醇，没有任何补充剂。使用95％乙醇而没有任何补充剂。 </li><li>制备磷酸盐缓冲吐温（PBT）。补充磷酸盐缓冲盐水（PBS）的0.1％吐温-20。添加0。5mL的20％吐温-20原液至100毫升1×PBS中。存放在室温下。 </li><li>制备磷酸盐缓冲的Triton-X（PBTx）。补充的PBS含有0.1％的Triton X-100。加入0.5毫升20％的Triton X-100的股票，以100毫升的PBS。存放在室温下。 </li><li>准备10％NGS / PBTx。正常山羊血清（NGS）嵌段的非特异性结合位点。商店NGS中在-20℃等分试样。使用，将1ml加入9mL的PBTx。 </li><li>制备50％甘油/ PBS中。混合2×PBS中，100％的甘油，完全相等的部分。存放在室温下。 </li></ol> 2.胚胎固定<ol><li> 。（ 如 β-actin的:CAAX-GFP） 育种获得野生型（AB和WIK）或转基因胚胎通过自然交配。如果需要的话，注射用构建体或吗啉代胚胎，如所描述。15,16 </li><li> 提高胚胎。收集胚胎，并如所述在28.5℃下在0.003％1-苯基-2-硫脲（PTU）阻止色素沉着的存在下孵育17 </su<html> p&gt; </li><li> 修正。当胚胎已达到所希望的发育阶段，18个， &#160;麻醉用MESAB胚胎，删除尽可能多的系统中的水可能和补充新鲜4％PFA / PBS 3 - 在室温4小时。注:在时间少于20小时受精后（HPF），修复了之前dechorionation。在经过20 HPF倍，采用在解剖显微镜下2对细点镊子的前固定dechorionate。 </li><li>简单地通过使胚胎通过重力而不离心沉降变化的解决方案。传输使用宽孔移液管的胚胎，如上所述。在加盖的微量离心管1.5毫升17变化的解决方案。 </li><li> 后固定剂后3 - 4小时，除去PFA / PBS中，用100％甲醇和存储替换在-20℃至少48小时。注:对于最大的P-钙调蛋白激酶-II免疫反应，限制总的甲醇储存时间为一个星期。 </li></ol>"&gt; 3。免疫染色全胚胎<ol><li>将最低10胚胎在上限1.5 ml离心管每个实验条件，并贴上标签。 </li><li>允许胚胎定居。除去并弃去甲醇，并用含有0.5毫升递减的乙醇浓度的渐进洗涤再水化，如在接下来的步骤表示。每一步都是一个5分钟洗具摇摆。 </li><li>这些5溶液逐步再水化:66％乙醇/ 33％PBTx，33％乙醇/ 66％PBTx，100％PBTx，100％PBTx（这是PBTx的第一重复），100％PBTx（这是第二个重复PBTx）。 </li><li>删除最后PBTx洗。将0.5ml 10％NGS / PBTx。在室温下孵育至少1小时与温和的摇摆，然后取出并丢弃的解决方案。 </li><li>通过在10％NGS / PBTx稀释制备一级抗体溶液。如果联合免疫，先用较高的亲和力抗体。在这项研究中，孵育稀释1小鼠抗乙酰化微管蛋白的单克隆抗体:500。对于为例即，如果有10个样品中，结合6微升股票抗体溶液用3毫升10％NGS / PBTx的，然后分发0.3ml的此到每个管中。 </li><li>将0.5ml稀释的初级抗体与每个管浸入所有胚胎 - 0.2添加。在室温下伴随温和的摇摆O / N培养。 </li><li>当天上午，删除和丢弃抗体溶液。将0.5ml 2％NGS / PBTx洗去多余的主要抗体。轻轻摇动5分钟。取下并丢弃的解决方案。重复两次。 </li><li>暗淡顶灯和稀适当荧光标记的二抗在10％NGS / PBTx使得每个条件包含至少0.5毫升用小鼠抗微管蛋白乙酰化的第一抗体，使用绿色荧光染料的山羊抗小鼠IgG在1:500的稀释。 </li><li>孵育次级抗体进行4小时以在室温下在黑暗中轻轻摇动。从现在起，处理样品下变暗的灯光，并在黑暗的容器孵化或在铝箔包装。 </li><li>在温育结束时，除去并丢弃二级抗体溶液。将0.5ml 2％NGS / PBTx洗。轻轻摇动5分钟。取下并丢弃的解决方案。重复两次。 </li><li>如果共免疫染色，来自不同物种比第一初级抗体获得第二初级抗体。在这些研究中，通过红色荧光染料缀合的山羊抗兔IgG二级抗体跟随兔子抗磷酸钙调蛋白激酶-Ⅱ抗体。 </li><li>稀释兔抗磷酸钙调蛋白激酶-II抗体1:20。对于胚胎的各管，暂停0.3含10％NGS / PBTx，再加入15微升股票抗体溶液。 </li><li>确保胚胎都沉浸和灯光会变暗。在黑暗中轻轻摇摆O / N孵育RT。 </li><li>在昏暗的灯光上午，删除和丢弃抗体溶液。加入0.5毫升2-％NGS / PBTx。轻轻摇动5分钟。取下并丢弃的解决方案。重复两次。 </li><li>保持顶灯暗，淡化相应的荧光conju足够门控的第二抗体，使每个条件包含至少0.5毫升在这项研究中，淡化红色荧光染料缀合的山羊抗兔IgG二级抗体（红色通道）1:500在10％NGS / PBTx。 </li><li>孵育第二次级抗体进行4小时以在室温下在黑暗中轻轻摇动。 </li><li>在此孵化，并在昏暗的灯光年底，捞出弃去二抗溶液。将0.5ml PBTx。轻轻摇动5分钟。取下并丢弃的解决方案。重复两次。商店在任PBTx或50％甘油/ PBS中根据成像过程。 </li></ol> 4.共焦成像和处理<ol><li>摩胚胎成像。将1 - 5胚胎在玻片上。创建主盖玻片并用在腔室的每一侧盖玻片片段滑动之间的腔室。通常，四＃1盖玻片被用来制造这些垫片堆栈而不封口。 注:没有安装介质是必要的。 </li><li>使用100X油浸客观上使单个胚胎成焦点采用透射光。关闭灯光。打开共聚焦显微镜。确保适当的激光器（绿色和/或红色）开启和远程对焦附带从事。 </li><li>打开共聚焦程序。在"采集栏，"选择合适的目标。在"XY基本"栏，点击1,024按钮设置的图像大小为1024。 </li><li>在"激光器和探测器"栏，点击红色框488和568的绿色盒子打开激光器和探测器每个通道。针孔设置为中，并调整每个通道以可视化的"增益"栏的增益。 </li><li>在"获取设置"栏中，单击"活着"开始采集图像。在"视图设置"栏中，取消选中"强制积分变焦"框中心"实时"窗口。 </li><li>在"获取设置"栏中的"Z"选项卡下，逐步完成图像的图层。选择后，NU光学切片的MBER掺入到图象，将移动到某一点在z平面的"中心"。 </li><li>在"Z"选项卡，单击红色的小"引用"对话框。这将归零RFA在选定为"中心"的地步。在标有"步长"的方框中，选择图层（0.25和2.0微米之间微米是理想的）厚度。需要注意的是"文件大小"框，并试图限制到1GB。 注意:通常情况下，可达0.5 40光学切片 - 为1.0μm而得到的，但可以根据经验来确定区段的数量。 </li><li>要找到图像的顶部极端，点击旁边的圆圈为"top"，并通过Z轴移动图层。现在点击圆圈旁边的"底"，计算机将图像回层为中心。再次通过层移动到找到图像的底部极端。 </li><li>点击在"获取设置""活"再次框停止激光收购。在此面板中，单击标平均红色框和Z堆栈。这些框变成绿色。 </li><li>在"获取设置"框中，单击"单次"收购整个系列的图像。因为它在两个信道，并通过在整个Z堆叠扫描可以监视进展情况。 </li><li>若要保存，单击该卷窗口中，点击"另存为"，并命名该文件。另存为".ids"文件。体积渲染文件，而Z堆栈仍然是开放的，选择数据下拉菜单，点击"批量渲染"。保存文件渲染为TIFF文件。这是本出版物中显示的投影图像。 </li></ol>

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

作者宣称，他们没有竞争的经济利益。

煤灰/甲醇方法，在这个实验室使用斑马鱼发育期间优化磷酸-T 287 -CaMK-II型表位的免疫定位的主要目的开发的。这种方法的几个纤毛器官，包括斑马鱼KV，12内耳14和肾脏。13特别是在对KV阶段，这种技术是必要的形成过程中成功地本地化的P-钙调蛋白激酶-II该方法的成功可能是由于一个）的自体荧光的最小化，b）该磷钙调蛋白激酶-II型表位的保存和c）的表位的抗体?...

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

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

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

细胞迅速增殖，基因的组织特异性表达和信令网络的出现表征所有脊椎动物的早期胚胎发育。即使在单个细胞 - - 动力学和信号的位置，在胚胎发育补充重要的发育基因的鉴定。免疫染色技术描述已被证明以限定小至初级纤毛结构的细胞内和整个动物的信号的动力学。使用激光扫描共聚焦显微镜复合固定，成像和图像处理技术可以在短短36小时内完成。 斑马鱼（ 斑马鱼 ）是谁寻求脊椎动物物种是负担得起的和相关的人类疾病进行研究调查一个理想的有机体。遗传击倒或击倒，必须通过实际的蛋白质产物的损失来确认。蛋白质丢失这样的确认可以使用这里描述的技术来实现。线索到信号通路，也可以通过使用与已经由磷酸化的翻译后修饰的蛋白反应的抗体破译。维护和优化表位的磷酸化状态，因此这一决定的关键，并通过该协议来实现的。 这项研究的发展和第一72小时期间介绍了技术修复的胚胎共定位的各种与在枯否囊泡（KV）纤毛相关的表位，肾脏和内耳。这些技术是简单的，不需要清扫，并可以在一个相对较短的时间内完成。焦投影图像堆栈成一个单一的形象呈现这些数据的有效手段。

可视化的磷酸化表位条件最优 描述蛋白抗原决定簇在斑马鱼胚胎的免疫组化方法相比，通过原位杂交本地化的mRNAs一直比较稀疏。在定位在斑马鱼胚胎的纤毛细胞蛋白的表位使用的固定剂包括4％PFA / PBS中并登特的固定剂，它是甲醇和DMSO。19定位RNA的由整体原位杂交（WISH）的混合物中，通常?...

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

技术描述于免疫染色在整个斑马鱼胚胎磷酸的表位，然后进行在小至初级纤毛细胞结构双色荧光共焦定位。用于固定和成像技术可以定义位置和特定蛋白质的外观或活化​​的动力学。

免疫染色磷酸化表位整装斑马鱼胚胎纤毛器官

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

Research

JoVE Journal

Chemistry

8.0K 次观看.  Virginia Commonwealth University. 技术描述于免疫染色在整个斑马鱼胚胎磷酸的表位，然后进行在小至初级纤毛细胞结构双色荧光共焦定位。用于固定和成像技术可以定义位置和特定蛋白质的外观或活化​​的动力学。 

视频: 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.

适用于鼠标机关高精度锌同位素测量

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 ...

科研

化学

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.

放射性标记和磷酸肌醇的细胞水平的定量高效液相色谱耦合流动闪烁

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

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

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

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

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

全贴装荧光原位杂交和免疫荧光法在斑马鱼中 Multiciliated 细胞的可视化

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

发育生物学

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

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

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

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

斑马鱼整装高分辨率双荧光原位杂交

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

生物学

Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

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

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

斑马鱼胚胎和幼虫的整个山免疫性化学

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

Single Cell Fate Mapping in Zebrafish

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

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

单细胞在斑马鱼的命运映射

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

Immunostaining of Dissected Zebrafish Embryonic Heart 

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

Immunostaining of Dissected Zebrafish Embryonic Heart

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

免疫染色的解剖斑马鱼胚胎心脏

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology ...

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

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

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

通过实时 FRET 显微镜和免疫荧光评估早期斑马鱼 Noonan 综合征模型中 ERK 活性调节的有效方案

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

生物化学

In Situ Hybridization Combined with Immunohistochemistry in Cryosectioned Zebrafish Embryos

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

In Situ Hybridization Combined with Immunohistochemistry in Cryosectioned Zebrafish Embryos

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

原位 冷冻切片斑马鱼胚胎中的杂交结合免疫组织化学

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

神经科学

Sequential Immunofluorescence and Immunohistochemistry on Cryosectioned Zebrafish Embryos

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

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

冷冻斑马鱼胚胎的连续免疫荧光与免疫组化学

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

免疫染色磷酸化表位整装斑马鱼胚胎纤毛器官

摘要

探索更多视频

此视频中的章节

适用于鼠标机关高精度锌同位素测量

放射性标记和磷酸肌醇的细胞水平的定量高效液相色谱耦合流动闪烁

全贴装荧光原位杂交和免疫荧光法在斑马鱼中 Multiciliated 细胞的可视化

斑马鱼整装高分辨率双荧光原位杂交

斑马鱼胚胎和幼虫的整个山免疫性化学

单细胞在斑马鱼的命运映射

免疫染色的解剖斑马鱼胚胎心脏

通过实时 FRET 显微镜和免疫荧光评估早期斑马鱼 Noonan 综合征模型中 ERK 活性调节的有效方案

原位冷冻切片斑马鱼胚胎中的杂交结合免疫组织化学

冷冻斑马鱼胚胎的连续免疫荧光与免疫组化学

免疫染色磷酸化表位整装斑马鱼胚胎纤毛器官

摘要

探索更多视频

此视频中的章节

适用于鼠标机关高精度锌同位素测量

放射性标记和磷酸肌醇的细胞水平的定量高效液相色谱耦合流动闪烁

全贴装荧光原位杂交和免疫荧光法在斑马鱼中 Multiciliated 细胞的可视化

斑马鱼整装高分辨率双荧光原位杂交

斑马鱼胚胎和幼虫的整个山免疫性化学

单细胞在斑马鱼的命运映射

免疫染色的解剖斑马鱼胚胎心脏

通过实时 FRET 显微镜和免疫荧光评估早期斑马鱼 Noonan 综合征模型中 ERK 活性调节的有效方案

原位 冷冻切片斑马鱼胚胎中的杂交结合免疫组织化学

冷冻斑马鱼胚胎的连续免疫荧光与免疫组化学

原位冷冻切片斑马鱼胚胎中的杂交结合免疫组织化学