The authors would like to thank Drs. Hilary Clay, Stephen Wilson, Anna Payne-Tobin, and James Smyth for helpful discussions. Microscopy was done at the Cardiovascular Research Institute Imaging Core at the University of California, San Francisco. This work was supported by NIH K08 HL105657 (LDW) and NIH HL65590 (SRC).

注:所有实验程序批准了加州大学旧金山分校机构动物护理和使用委员会。 1.冷冻和胚胎小鼠心脏的固定。 1.1）管理单元冷冻胚胎的心<ol><li>填写3.5公分培养皿和7毫米cryomolds与最佳切削温度（OCT）中（见材料表）。在化学罩，冷却2-甲基丁烷在液氮中。 </li><li>分配30毫升磷酸盐缓冲盐水（PBS）的成10毫米培养皿中，将10毫升PBS中的3.5毫米培养皿中，并放置在冰上所有培养皿中。准备1个10 cm培养皿，每个孕鼠数3.5厘米菜肴。 </li><li>隔离胚胎如前所述15，执行在冰冷的PBS解剖。 </li><li>简单地说，使用安乐死二氧化碳麻醉和颈椎脱位怀孕的女性。 <ol><li>使在腹部切口，解剖出子宫通过切断船只ALO纳克子宫的内曲率以及子宫转移到含有冰PBS 10cm的培养皿。 </li><li>切子宫中每个胚与转印之间含有冰冷的PBS个体3.5厘米培养皿。隔离每个胚胎所描述的15。 </li><li>使用细镊子打开心包腔，通过切割在主动脉，下腔静脉，肺静脉取出心脏远离肺和脉管，并转移到含有OCT中3.5厘米培养皿。 </li><li>让华侨城的心脏平衡几秒钟，然后将心脏转移到含有华侨城的7毫米模具。定向心脏的前壁到模具的底部。 </li></ol></li><li>轻轻地放在模具放入液氮冷却的2-甲基丁烷。小心不要让2甲基丁烷液体接触OCT或心脏。冻结，直到华侨城是白色固体，然后将模具转移到一个冰桶含干冰。移动到下一个胚胎。 </li><li> WRAP cryomolds在铝箔和储存在-80°C，直到准备cryosectioning。 </li></ol> 1.2）备选:多聚甲醛（PFA）固定，cryoprotecting和OCT嵌入胚胎的心<ol><li>填充一个12孔组织培养板的孔用4％PFA中的1×PBS。 </li><li>解剖出胚胎心如1.1.3所述。将每个心脏成孔含有4％PFA和在4℃CO / N固定。 </li><li>冷冻保护:使用塑料移液管，将每个心脏含有1.5毫升15％蔗糖的PBS中的1.5 ml离心管，轻轻搅动在4℃，直至心脏沉到试管底部（数小时到O / N ）。每个心脏转移到在PBS中30％蔗糖的含有1.5毫升1.5 ml离心管，轻轻搅动在4℃再次直到心脏沉到试管底部（数小时到O / N）。 </li><li>用塑料移液管，将冷冻保护心脏进入十月，让它平衡几分钟，以除去过量的蔗糖，然后将心脏转移到含有OCT中7毫米模具。定向心脏的前壁到模具的底部。 </li><li>通过将模具中任一液氮冷却的2-甲基丁烷或干冰冻结在OCT中的心脏。 </li><li>包装在铝箔和储存cryomolds在-80°C，直到准备cryosectioning。 </li></ol> 2. Cryosectioning <ol><li>设定恒温器的​​温度为-17℃。 </li><li>地方cryomolds进入低温恒温室和平衡至温度15-20分钟。 </li><li>反转cryomold并用温和的压力，从模具排出心脏传导阻滞。定向心脏的前壁到模塑组织块的顶部。 </li><li>将大降华侨城到吸盘，并安装心脏传导阻滞到十月下降冻结到夹头。保持的取向，使得心脏的前壁是最远从卡盘。 </li><li>加载的卡盘和安装，他艺术块到低温恒温器对象持有人。调整，以使叶片的角度为3-5°相对于样品。 </li><li>收集10微米的部分上已预先用带正电的涂料显微镜载玻片（见材料表）。允许储存在-80℃之前完全干燥。 </li></ol> 3.免疫<ol><li>为快速冷冻切片，固定在丙酮透组织用于在通风橱在RT 10分钟。 </li><li>为快速冷冻和PFA固定部分，孵育在PBS-0.1％的Triton X-100进行20分钟以除去OCT和通透的PFA固定部分。 </li><li>块45分钟在1×封闭缓冲液，在PBS中稀释。 </li><li>如果使用在小鼠中产生的第一抗体，孵育在驴或山羊抗小鼠IgG（H + L）的单价Fab片段1:100稀释在PBS-0.1％Tween 20的45分钟，在RT（见讨论）。 </li><li>孵育在初级或多种抗体在RT或稀释在1×封闭缓冲液2小时O / N在4°C（见表材料/设备的特定稀释）。 </li><li>在1×PBS中在RT下10分钟洗涤切片三次。 </li><li>在封闭缓冲液中进行2小时，在室温，避光500:在孵育的Alexa稀释1氟 - 共轭次级抗体。 </li><li>在1×PBS中进行10分钟，在室温，避光洗部分三次。 </li><li>可选:在孵育赫希斯特染料稀释1:2000在PBS在室温（避光）标记细胞核，然后用PBS。 </li><li>定位后在1％PFA中标记的部分在RT下1分钟。 </li><li>摩滑动在抗褪色培养基（用DAPI细胞核如果尚未标记）放置在滑动的各端两滴培养基，然后覆盖盖玻片。密封盖玻片指甲油。商店避光，在4℃，直到准备图像。 </li></ol> 4.共焦成像和图像分析<ol><li>打开相应的激光波长，摄像头，和共聚焦显微镜INC泸定阶段先行者和z电机。启动成像软件程序。使用405，488和561的激光波长成像Hoechst-，Alexa的488和Alexa 568染色切片，分别。 注:请参阅材料表为我们的硬件和软件规格。 </li><li>安装在幻灯片阶段控制幻灯片（盖玻片下来倒置显微镜）。 </li><li>使用4X目标（见材料表），发现样品和感兴趣的领域。图像捕捉为一个地图时，在高放大倍率成像使用。 </li><li>去除幻灯片，使最低限度的调整，以幻灯片的阶段。切换到60X油浸物镜（见材料表），放置一小滴油的目标，并更换幻灯片（盖玻片下）到幻灯片阶段。 </li><li>再次找到样本。设置激光功率，曝光时间，并分级为每个信道的所需的水平。 注意:我们一般使用0.8激光功率，为100毫秒相机的曝光时间，以及2分级（SEË材料表的硬件和软件规格）;最佳设置需要凭经验每个实验来确定。 <ol><li>一旦最佳设置的确定，使用相同的设置实验中的所有组织切片。使用强度直方图要注意的最佳强度范围为每个通道（该信息将用于分析）。 </li></ol></li><li>生成使用获取功能AZ堆栈:选择适当的激光信道，那么选择的Z堆叠的上限和下限。选择AZ堆栈步长大小是由软件提供的光学片的厚度的二分之一的值。点击"运行"来收集图像。 </li><li>使用斐济16或图像分析可比的程序。在斐济，打开使用自定义色彩模式选项，并分成不同的窗口通道的Z堆栈文件。打开从图像下拉菜单中的"调节亮度/对比度"的工具;每个通道内，SET的4.5.1最佳直方图强度范围确定的。应用这些通道范围为所有的z栈被分析。 </li><li>合并单个通道成一个单一的复合图像使用图像 - &gt;颜色下拉菜单。 </li><li>创建复合图像使用图像 - &gt; Stacks-&gt;以Z项目菜单扁平ž堆栈。这个图像会比三维图像显著更亮;调整直方图强度范围为对照样品，以避免过饱和，并应用相同的设置，以对实验扁平ž栈。 </li><li>要生成3D图像，先用图像- &gt; Stacks-&gt; 3D菜单中的项目17。选择其中的x轴或旋转的y轴。设置切片间距为微米作为Z堆栈步长相同数量。选择所需的总旋转，设置旋转角度增量为1。然后在插件下拉菜单中打开的Image J 3D查看器。选择在4.8产生的显示为体积的合成图像，并设置了Re采样因子为1或2。 </li></ol>

<ol>
	<li>Risebro, C. A., Riley, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+the+ventricles.">Formation of the ventricles.</a> The Scientific World Journal. 6, 1862-1880 (2006).</li><li>Ji, R. P., 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=Onset+of+cardiac+function+during+early+mouse+embryogenesis+coincides+with+entry+of+primitive+erythroblasts+into+the+embryo+proper.">Onset of cardiac function during early mouse embryogenesis coincides with entry of primitive erythroblasts into the embryo proper.</a> Circulation research. 92, 133-135 (2003).</li><li>Bennett, P. M., Maggs, A. M., Baines, A. J., Pinder, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transitional+junction:+a+new+functional+subcellular+domain+at+the+intercalated+disc.">The transitional junction: a new functional subcellular domain at the intercalated disc.</a> Molecular biology of the cell. 17, 2091-2100 (2006).</li><li>Sparrow, J. C., Schock, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+initial+steps+of+myofibril+assembly:+integrins+pave+the+way.+Nature+reviews.">The initial steps of myofibril assembly: integrins pave the way. Nature reviews.</a> Molecular cell biology. 10, 293-298 (2009).</li><li>Kastner, P., 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=Vitamin+A+deficiency+and+mutations+of+RXRalpha,+RXRbeta+and+RARalpha+lead+to+early+differentiation+of+embryonic+ventricular+cardiomyocytes.">Vitamin A deficiency and mutations of RXRalpha, RXRbeta and RARalpha lead to early differentiation of embryonic ventricular cardiomyocytes.</a> Development. 124, 4749-4758 (1997).</li><li>Zhang, W., Chen, H., Qu, X., Chang, C. P., Shou, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanism+of+ventricular+trabeculation/compaction+and+the+pathogenesis+of+the+left+ventricular+noncompaction+cardiomyopathy+(LVNC).">Molecular mechanism of ventricular trabeculation/compaction and the pathogenesis of the left ventricular noncompaction cardiomyopathy (LVNC).</a> American journal of medical genetics Part C, Seminars in medical genetics. 163C, 144-156 (2013).</li><li>Kim, Y. 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=Cellular+localization+of+alpha3beta1+integrin+isoforms+in+association+with+myofibrillogenesis+during+cardiac+myocyte+development+in+culture.">Cellular localization of alpha3beta1 integrin isoforms in association with myofibrillogenesis during cardiac myocyte development in culture.</a> Cell adhesion and communication. 7, 85-97 (1999).</li><li>Lu, M. H., 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=The+vinculin/sarcomeric-alpha-actinin/alpha-actin+nexus+in+cultured+cardiac+myocytes.">The vinculin/sarcomeric-alpha-actinin/alpha-actin nexus in cultured cardiac myocytes.</a> The Journal of cell biology. 117, 1007-1022 (1992).</li><li>Schultheiss, 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=Differential+distribution+of+subsets+of+myofibrillar+proteins+in+cardiac+nonstriated+and+striated+myofibrils.">Differential distribution of subsets of myofibrillar proteins in cardiac nonstriated and striated myofibrils.</a> The Journal of cell biology. 110, 1159-1172 (1990).</li><li>Samarel, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Costameres,+focal+adhesions,+and+cardiomyocyte+mechanotransduction.+American+journal+of+physiology.">Costameres, focal adhesions, and cardiomyocyte mechanotransduction. American journal of physiology.</a> Heart and circulatory physiology. 289, H2291-H2301 (2005).</li><li>Sinn, H. W., Balsamo, J., Lilien, J., Lin, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+the+novel+Xin+protein+to+the+adherens+junction+complex+in+cardiac+and+skeletal+muscle+during+development.">Localization of the novel Xin protein to the adherens junction complex in cardiac and skeletal muscle during development.</a> Developmental dynamics : an official publication of the American Association of Anatomists. 225, 1-13 (2002).</li><li>Lu, S., Borst, D. E., Horowits, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-RAP+expression+during+mouse+heart+development.">N-RAP expression during mouse heart development.</a> Developmental dynamics : an official publication of the American Association of Anatomists. 233, 201-212 (2005).</li><li>Hirschy, A., Schatzmann, F., Ehler, E., Perriard, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+cardiac+cytoarchitecture+in+the+developing+mouse+heart.">Establishment of cardiac cytoarchitecture in the developing mouse heart.</a> Developmental biology. 289, 430-441 (2006).</li><li>Whitman, S. A., 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=Desmoplakin+and+talin2+are+novel+mRNA+targets+of+fragile+X-related+protein-1+in+cardiac+muscle.">Desmoplakin and talin2 are novel mRNA targets of fragile X-related protein-1 in cardiac muscle.</a> Circulation research. 109, 262-271 (2011).</li><li>Kalaskar, V. K., Lauderdale, 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=Mouse+embryonic+development+in+a+serum-free+whole+embryo+culture+system.">Mouse embryonic development in a serum-free whole embryo culture system.</a> Journal of visualized experiments : JoVE. , (2014).</li><li>Schindelin, J., 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=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature. 9, 676-682 (2012).</li><li>Schmid, B., Schindelin, J., Cardona, A., Longair, M., Heisenberg, 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+high-level+3D+visualization+API+for+Java+and+ImageJ.">A high-level 3D visualization API for Java and ImageJ.</a> BMC. 11, 274 (2010).</li><li>Swope, D., Cheng, L., Gao, E., Li, J., Radice, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+cadherin-binding+proteins+beta-catenin+and+plakoglobin+in+the+heart+leads+to+gap+junction+remodeling+and+arrhythmogenesis.">Loss of cadherin-binding proteins beta-catenin and plakoglobin in the heart leads to gap junction remodeling and arrhythmogenesis.</a> Molecular and cellular biology. 32, 1056-1067 (2012).</li><li>Riedl, J., 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=Lifeact+mice+for+studying+F-actin+dynamics.">Lifeact mice for studying F-actin dynamics.</a> Nature. 7, 168-169 (2010).</li><li>Shi, S. R., Shi, Y., Taylor, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen+retrieval+immunohistochemistry:+review+and+future+prospects+in+research+and+diagnosis+over+two+decades.">Antigen retrieval immunohistochemistry: review and future prospects in research and diagnosis over two decades.</a> The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society. 59, 13-32 (2011).</li><li>North, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seeing+is+believing?+A+beginners'+guide+to+practical+pitfalls+in+image+acquisition.">Seeing is believing? A beginners' guide to practical pitfalls in image acquisition.</a> The Journal of cell biology. 172, 9-18 (2006).</li><li>Risebro, C. A., 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=Prox1+maintains+muscle+structure+and+growth+in+the+developing+heart.">Prox1 maintains muscle structure and growth in the developing heart.</a> Development. 136, 495-505 (2009).</li><li>Ehler, E., Rothen, B. M., Hammerle, S. P., Komiyama, M., Perriard, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myofibrillogenesis+in+the+developing+chicken+heart:+assembly+of+Z-disk,+M-line+and+the+thick+filaments.">Myofibrillogenesis in the developing chicken heart: assembly of Z-disk, M-line and the thick filaments.</a> Journal of cell science. 112 (Pt 10), 1529-1539 (1999).</li></ol>

The authors have nothing to disclose.

最佳组织固定技术和稀释必须为每个抗体凭经验确定。在我们手中，搭扣冷冻优于PFA固定数心肌抗原，包括S-α辅肌动蛋白，β连环蛋白，β1整联，原肌球蛋白，塔林（未示出）和N-钙粘蛋白;与此相反，PFA固定得到优异的结果为粘着斑激酶（未示出）。由PFA形成的蛋白质交联可能掩盖表位和限价抗体结合;抗原修复，可能需要在这样的情况下，以及用于抗原修复方法可在别处20找到。 PFA浓度或固定的长度可以减小，以减少表位掩蔽，以最佳条件对于每个抗体，并在每个发育阶段经验确定。当表征一个新的抗体或心脏突变体，包括免疫前血清作为初级抗体对照和一个"无第一抗体"共同适当的阴性对照，应使用ntrol。使用基因敲除小鼠是一种理想的阴性对照，但早期致死防止它们的使用对许多这里所研究的基因产物。 使用足够量以完全淹没实验组和对照载玻片在同一免疫阻断，抗体，并作为是幻灯片的孵育期间温和的摇摆，以确保各部分的解决方案的均匀曝光洗涤溶液是重要的。这种方法最小化技术变化在一个实验中的章节和幻灯片之间染色。当成本限制了抗体溶液的体积，用巴氏笔来限制流量的解决方案超出了组织切片，并在漫长的孵化保持在湿润的玻片。如果一个单克隆小鼠一次抗体 - 并且因此抗小鼠次级抗体 - 正在被使用，第二阻挡步骤以覆盖内源性小鼠免疫球蛋白将是必要的（步骤3.4），以减少非特异性背景信号。 适当显微镜和图像处理技术是在获得生物准确信息21是至关重要的。所述强度直方图表示像素的各个强度等级的分布（0 - 65535的水平为16位的图像），用于每种颜色。背景，亮度和对比度可以通过设置侧翼直方图峰的强度显示范围进行调整;使条件之间有效的比较，必须使用对照组和实验条件之间的相同的设置。 该协议提供了分析心肌细胞的成熟和发展，在本土胚胎小鼠心脏的可靠方法。而心肌细胞特异性蛋白的免疫荧光常常用于标记发展过程中的心肌细胞，少数研究采用的技术，使肌原纤维结构的高分辨率分析和闰盘和costameres 12-14,22,23的出现。此技术可用于在VIVO评估突变，导致发育心脏缺陷，作为查明改变心肌细胞的成熟，可能揭示结构异常的机制的光的装置。

在开发过程中，心脏收缩开始后不久，心肌细胞移植到中线，形成线性心脏管1,2。肌节是心肌细胞内的基本收缩单位;这个高度组织细胞骨架结构包含锚定于Z盘由肌节α辅肌动蛋白（S-α辅肌动蛋白）和肌球蛋白纤维锚固到M个行（ 图1）的肌动蛋白丝。作为心肌的成熟，肌节组装串联形成肌原纤维跨越小区延伸。肌原纤维由闰盘固定在心肌细胞的端部，包含与Z-盘元素如S-α辅肌动蛋白3，粘着的一个子集的过渡交界处的细胞间交界结构连接蛋白，例如N-钙粘蛋白和β连环蛋白，间隙连接蛋白，和桥粒（ 图1）4。沿着纵向膜，在Z光盘周肌原纤维也附加到经由costameres细胞膜;这些专门粘着斑提供的肌原纤维，质膜，和细胞外基质，以提供对心肌额外的结构支撑之间的锚定（ 图1）4。早在心脏发育，心肌细胞排列在称为小梁手指状突起伸入所述室的空间和包含相对成熟的肌原纤维5。作为心脏发育的进行，在副心外膜区域的心肌细胞增殖，以形成紧凑的心肌，其包括心室壁，但肌节肌原纤维和组件被延迟与小梁心肌5,6相比较。 肌节肌原纤维和组件的模型主要来自免疫荧光研究对培养心肌细胞的7-10，这是简单的，但缺乏的三维环境中，血流量，并与其他的心脏细胞接触S出现在体内，小鼠胚胎心脏免疫高分辨率结构的研究在技术上具有挑战性，而且很少有研究探索闰盘和costameres小鼠心脏发育过程中的出现。该粘着连接蛋白β连环似乎定位于闰盘由胚胎天（E）17.5 11，N-cadherin的本地化到可能代表闰盘由E18.5 12与0 13日龄线性结构，并costameres都在被检测E18.5 14，但这些蛋白显示在早期发育时间点11-13漫射和更连续膜分布。 在这里，我们描述了染色和切片小鼠胚胎心脏的荧光显微镜一个简单的和可重复的方法，允许肌原纤维和心肌细胞的发展进行详细分析，包括intercala的出现泰德早在E12.5和新生costameres在E16.5光盘。该协议可以是用于探测上肌节的形成以及肌原纤维和心肌细胞的成熟突变的影响是有用的。

<table border="0" cellpadding="0" cellspacing="0" width="946">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Name of Reagent/ Equipment</td>
			<td style="width:167px;">Company</td>
			<td style="width:160px;">Catalog Number</td>
			<td style="width:335px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:285px;">Cryomold, Disposable Base Mold, 7x7 mm</td>
			<td style="width:167px;">Richard-Allen Scientific</td>
			<td style="width:160px;">58949</td>
			<td style="width:335px;">This size not available from Fisher Scientific</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:285px;">Cryomold, Disposable Base Mold, 15x15 mm</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:160px;">22-050-159</td>
			<td style="width:335px;">Also available from Richard-Allen Scientific, catalog number 58950</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:285px;">Tissue-Tek Optimal Cutting Temperature (OCT) medium 4583</td>
			<td style="width:167px;">VWR</td>
			<td style="width:160px;">25608-930</td>
			<td style="width:335px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">2-methyl butane</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:160px;">M32631</td>
			<td style="width:335px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Paraformaldehyde</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:160px;">T353-500</td>
			<td style="width:335px;">Use fresh 4% solution in 1X PBS, pH 7.2-7.4.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Sucrose</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:160px;">S0389</td>
			<td style="width:335px;">Use 15% and 30% solutions in 1X PBS.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Disposable transfer pipette</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:160px;">S30467-1</td>
			<td style="width:335px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:285px;">Superfrost Plus slides</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:160px;">12-550-15</td>
			<td style="width:335px;">&#34;Plus&#34; indicates positively-charged coating and is critical for maintaining tissue adherence to the slide.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Acetone</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:160px;">179124</td>
			<td style="width:335px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Triton X-100</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:160px;">X100</td>
			<td style="width:335px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:285px;">Western Blocking Agent</td>
			<td style="width:167px;">Roche</td>
			<td style="width:160px;">11921673001</td>
			<td style="width:335px;">Blocking buffer for immunofluorescence, when secondary antibodies are from different species. Dilute to 1X in PBS.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Tween 20</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:160px;">P9416</td>
			<td style="width:335px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:285px;">Donkey Anti-mouse IgG (H+L) monovalent Fab fragment</td>
			<td style="width:167px;">Jackson ImmunoResearch</td>
			<td style="width:160px;">715-007-003</td>
			<td style="width:335px;">Goat Anti-mouse IgG (H+L) also available. For blocking endogenous immunoglobulins.</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:285px;">Anti-s-a-actinin antibody</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:160px;">A7811</td>
			<td style="width:335px;">Mouse monoclonal, clone EA53. Dilute 1:400 for snap-frozen/acetone-fixed sections, dilute 1:300 for PFA-fixed sections. Before incubation on mouse tissue, block endogenous mouse immunoglobulins with anti-mouse IgG (H+L) monovalent Fab fragment.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:285px;">Anti-bcatenin antibody</td>
			<td style="width:167px;">Cell Signaling</td>
			<td style="width:160px;">9587s</td>
			<td style="width:335px;">Rabbit polyclonal. Requires snap-freezing/acetone fixation. Dilute 1:400.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:285px;">Anti-b1 integrin antibody</td>
			<td style="width:167px;">R&#38;D</td>
			<td style="width:160px;">AF2405</td>
			<td style="width:335px;">Goat polyclonal. Dilute 1:200. Snap-frozen/acetone-fixed tissue staining is superior to PFA-fixed tissue.</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:285px;">Anti-tropomyosin antibody</td>
			<td>Developmental Studies Hybridoma Bank, University of Iowa</td>
			<td style="width:160px;">CH1</td>
			<td style="width:335px;">Mouse monoclonal. Requires snap-freezing/acetone fixation. Dilute 1:200. Before incubation on mouse tissue, block endogenous mouse immunoglobulins with anti-mouse IgG (H+L) monovalent Fab fragment.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:285px;">Anti-N-cadherin antibody</td>
			<td style="width:167px;">Santa Cruz</td>
			<td style="width:160px;">sc-7939</td>
			<td style="width:335px;">Rabbit polyclonal. Dilute 1:200. Snap-frozen/acetone-fixed tissue staining is superior to PFA-fixed tissue.</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:285px;">Anti-talin antibody</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:160px;">T3287</td>
			<td style="width:335px;">Mouse monoclonal, clone 8d4. Requires snap-freezing/acetone fixation. Dilute 1:200. Before incubation on mouse tissue, block endogenous mouse immunoglobulins with anti-mouse IgG (H+L) monovalent Fab fragment.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:285px;">Anti-phosphotyrosine 397 focal adhesion kinase antibody</td>
			<td style="width:167px;">Invitrogen</td>
			<td style="width:160px;">700255</td>
			<td style="width:335px;">Rabbit monoclonal antibody. Requires PFA fixation. Dilute 1:500.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:285px;">Alexa Fluor 488 Donkey Anti-Mouse IgG (H+L) Antibody</td>
			<td style="width:167px;">Life Technologies</td>
			<td style="width:160px;">A-21202</td>
			<td style="width:335px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:285px;">Alexa Fluor 568 Donkey Anti-Rabbit IgG Antibody</td>
			<td style="width:167px;">Life Technologies</td>
			<td style="width:160px;">A10042</td>
			<td style="width:335px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:285px;">Alexa Fluor 488 Goat Anti-Mouse IgG (H+L) Antibody</td>
			<td style="width:167px;">Life Technologies</td>
			<td style="width:160px;">A-11001</td>
			<td style="width:335px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:285px;">Alexa Fluor 568 Goat Anti-Rabbit IgG (H+L)Antibody</td>
			<td style="width:167px;">Life Technologies</td>
			<td style="width:160px;">A-11011</td>
			<td style="width:335px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Hoechst 33342 dye</td>
			<td style="width:167px;">Life Technologies</td>
			<td style="width:160px;">H3570</td>
			<td style="width:335px;">Nuclear stain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Vectashield</td>
			<td style="width:167px;">Vector labs</td>
			<td style="width:160px;">H-1000</td>
			<td style="width:335px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Coverslips</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:160px;">12-548-5M</td>
			<td style="width:335px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:285px;">MLC 400B Monolithic Laser Combiner Laser box</td>
			<td style="width:167px;">Keysight (formerly Agilent)</td>
			<td style="width:160px;"></td>
			<td style="width:335px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Clara Interline CCD camera</td>
			<td style="width:167px;">Andor</td>
			<td style="width:160px;"></td>
			<td style="width:335px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Eclipse Ti inverted microscope</td>
			<td style="width:167px;">Nikon</td>
			<td style="width:160px;"></td>
			<td style="width:335px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:285px;">CSU-X1 Spinning disk confocal scanner unit</td>
			<td style="width:167px;">Yokogawa</td>
			<td style="width:160px;"></td>
			<td style="width:335px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:285px;">CFI Plan Apochromat 4X air objective</td>
			<td style="width:167px;">Nikon&#160;</td>
			<td style="width:160px;">Numerical aperture (NA) 0.2, Working distance (WD) 15.7 mm</td>
			<td style="width:335px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:285px;">CFI Plan Apochromat VC 60x oil immesion objective (MRD01602)</td>
			<td style="width:167px;">Nikon&#160;&#160;</td>
			<td style="width:160px;">NA 1.4, WD 0.13 mm</td>
			<td style="width:335px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:285px;">NIS Elements Imaging Software</td>
			<td style="width:167px;">Nikon</td>
			<td style="width:160px;">http://nikon.com/products/instruments/lineup/bioscience/img_soft/index.htm</td>
			<td style="width:335px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Image analysis software</td>
			<td style="width:167px;">Fiji</td>
			<td style="width:160px;">http://fiji.sc/Fiji</td>
			<td style="width:335px;"></td>
		</tr>
	</tbody>
</table>

analysis of cardiomyocyte development using immunofluorescence in embryonic mouse heart

期间心脏发育，心肌特异性的结构和功能单位，其中包括肌节，肌原纤维的收缩，闰盘，并costameres产生需要多个部件的在时间和空间的协调组件。破坏这些组件组装导致发育心脏缺陷。通常使用免疫荧光染色技术在培养的心肌细胞，以探测肌原纤维的成熟，但这种体外方法由程度上限制向其中细胞将充分分化培养心内膜线索，缺乏正常体内机械输入，和不存在。免疫荧光技术来开发小鼠心脏研究中的应用是可取的，但更多的技术上的挑战，和方法往往缺乏足够的灵敏度和分辨率，可视化肌节的心脏发育的早期阶段。在这里，我们描述了一个强大的和可重复的方法共同免疫染色万亩ltiple蛋白质或共同与可视化免疫荧光染色荧光蛋白在小鼠胚胎心脏，用这种方法来分析发展中的肌原纤维，闰盘和costameres。该方法可进一步用于评估突变引起的，导致发育心脏缺陷的心肌的结构变化。

图2到用于在快速冷冻和丙酮固定心脏共染色不同的蛋白质6表示典型的结果。对抗体的S-α辅肌动蛋白再现地标记的Z-光盘和闰盘以高特异性和最小的背景（ 图2A，3A，4A，5A，6A和6C）;图6表明了抗小鼠IgG（H + L）的单价Fab片段有效地阻断内源性小鼠IgG通过抗小鼠二级抗体结合。针对粘着连接蛋白β连环蛋白抗体结合的两个心肌细胞和非心肌细胞的膜，和共定位与S-α辅肌动蛋白发生推定闰盘在E16.5（ 图2C和D）中，从预期在成人心脏β18 catenin的染色模式。 β在胚胎心脏1整联免疫是特别具有挑战性的并且经常不能识别粘着斑14，但在这些研究中β1整合染色显示信号具有相同的周期为s-α辅肌动蛋白-标记的Z-光盘，这可能反映了新生costameres在形成E16.5（ 图3D）。 在E12.5，S-α辅肌动蛋白和肌球蛋白（肌节薄丝蛋白），免疫荧光显示，与常规周期与在这些细胞（ 图4A和5A的S-α辅肌动蛋白肌原纤维的成熟一致小梁心肌细胞染色模式; 图4B对原肌球蛋白）。小梁心肌细胞在E12.5心中的N-钙粘蛋白染色往往与共同定位激烈的S-α辅肌动蛋白染色区（ 图5B - D和图6A - C）可能代表闰盘。在对比于小梁细胞，S-α辅肌动蛋白在紧凑区域比线性多点状，而原肌球蛋白染色是弥漫性而不是线性（ 图4A和4B）。因此，肌节装配以后可能会在紧凑型相比，小梁心肌发生。此外，S-α辅肌动蛋白和肌球蛋白在紧凑的区域差异模式表明，S-α辅肌动蛋白组织成泪点和不成熟的Z-盘初，而原肌球蛋白纳入细丝可能是肌原纤维组装以后的事件。 图7，电影1，电影和2展示出从PFA固定E12.5胚胎的心脏典型结果。在这些例子中，一个LifeAct-RFPruby转基因胚胎用于成像;在LifeAct-RFPruby转基因标签19丝状肌动蛋白，但需要PFA固定。 Z-光盘标记与S-α辅肌动蛋白很容易在大多数领域可视化，但信噪比为decre<html> ASED相比速冻心脏切片（ 图7A）;这个信号是典型的PFA固定的组织，其中的表位可通过蛋白质交联被掩蔽以s-α辅肌动蛋白免疫荧光。 图7B示出了共同的可视化丝状肌动蛋白的免疫标记和内的肌原纤维（箭头）S-α辅肌动蛋白相邻小梁细胞（箭头）的心内膜细胞内和丝状肌动蛋白。三维图像重建显露额外的细节:个体心肌细胞更容易分辨，心肌细胞内的肌原纤维被大致彼此平行的，但个体心肌细胞定向在不同的角度，以彼此（ 图7C和D，电影1，电影和2 ）。心内膜细胞和心肌细胞之间的紧密近似被更好地理解，在三维视图以及。 <html> "&gt; <img alt="图1" src="/files/ftp_upload/52644/52644fig1.jpg" /> 图1.心肌肌节，闰盘，和costameres。Z型光盘锚肌动蛋白丝，而对M行锚球蛋白纤维，其重叠的肌动蛋白丝。肌节包括一个Z-盘 - 米线 - Z-盘单位。串联多个肌节创建一个肌原纤维。的肌原纤维的横向端插入到心肌的横向边界处称为闰盘一个专门的细胞 - 细胞交界结构。周围的肌原纤维连接通过costameres，形成粘着斑与心肌细胞之间的细胞外基质的纵向心肌细胞质膜。 <a href="https://www.jove.com/files/ftp_upload/52644/52644fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> PLOAD / 52644 / 52644fig2.jpg"/&gt; 图2. S-α辅肌动蛋白和β连环蛋白免疫的16.5天胚胎的心脏被切除，快速冷冻，冰冻切片，丙酮固定，并用免疫染色（A）鼠单克隆克隆EA53对S-α辅肌动蛋白的抗体，其中合并标记的心肌细胞的Z盘和闰盘，和对粘着连接蛋白β连环蛋白（B）的兔polycloncal抗体。（C）的图像显示S-α辅肌动蛋白和β连环蛋白染色。（D）的放大的自组C兴趣区;星号标记推测闰盘与合作，本地化S-α辅肌动蛋白和β连环蛋白。图像从周边左心室壁或紧凑心肌得到，与心外膜层的面板的AC左上方。亮度直方图显示范围460-1600（出ÒF可0-65535），同时为S-α辅肌动蛋白/ 488 nm和β连环素/ 561 nm激光通道。比例尺10微米。 <a href="https://www.jove.com/files/ftp_upload/52644/52644fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52644/52644fig3.jpg" /> 图3. S-α辅肌动蛋白和β1整合免疫16.5天胚胎的心脏被切除，快速冷冻，冰冻切片，丙酮固定，并用（A）鼠单克隆克隆EA53对S-α辅肌动蛋白抗体免疫染色和（B）的山羊多克隆抗体对黏着蛋白β1整合。（C）的合并的图像显示在心肌β1整合以及非心肌细胞。请注意这两个弥漫性和点状β1在心肌细胞整合信号（D）的放大的从面板C.注意点状，周期β1整合染色（箭头）与周期性类似附近的S-α辅肌动蛋白染色的Z-光盘兴趣区。这些结构可能代表costameres。图像从左心室心肌紧凑获得。亮度直方图显示范围460-1200（出可能0-65535）用于对β1整合/ 561 nm激光通道的S-α辅肌动蛋白/ 488 nm激光通道和460-600。比例尺10微米。 <a href="https://www.jove.com/files/ftp_upload/52644/52644fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/52644/52644fig4.jpg" /> 图4. S-α辅肌动蛋白和胚胎12.5天原肌球蛋白免疫荧光:肌原纤维组织<html>小梁和紧凑的心肌。从窝胚胎心脏切下，快速冷冻，冰冻切片，丙酮固定，并使用（A）的小鼠单克隆的克隆EA53针对S-α辅肌动蛋白和（B）的小鼠单克隆抗体抗肌纤维细长丝抗体免疫染色蛋白原肌球蛋白（发展研究杂交瘤细胞银行CH1）。小梁（箭头）和紧凑心肌（箭头）表示。注意线性S-α辅肌动蛋白染色用常规的周期性在小梁心肌相比，一系列的染色模式，包括泪点，以及在所述致密层（A）的线性染色。还要注意线性原肌球蛋白染色常规周期性小梁心肌更弥漫染色紧凑的心肌。亮度直方图显示范围460-1,400（出可能0-65535）为原肌球蛋白通道的S-α辅肌动蛋白通道和460-1,000。比例尺为10μm。"https://www.jove.com/files/ftp_upload/52644/52644fig4large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图5" src="/files/ftp_upload/52644/52644fig5.jpg" /> 图5. S-α辅肌动蛋白和N-cadherin的免疫为12.5天胚胎:肌原纤维和闰盘小梁心肌细胞的心脏被切除，快速冷冻，冰冻切片，丙酮固定，并用免疫染色（A）鼠单克隆克隆EA53抗体对S-α辅肌动蛋白和抗粘着蛋白的N-钙粘蛋白（B）的兔多克隆抗体。为0.2μm的光学片被收集作为z栈，和z栈被压扁，以产生图像。（C）的合并的扁平堆叠显示小梁心肌内既N-二钙粘蛋白和s-α辅肌动蛋白染色为瓦特ELL作为从C图放大感兴趣的细胞核标记用Hoechst染料（D）区。星号标记闰盘与合作，本地化S-α辅肌动蛋白和N-cadherin表达。强度直方图显示范围470-1,200（在可能0-65535）为Hoechst公司/ 405 nm激光信道和470-2,000关于S-α辅肌动蛋白/ 488纳米和N-钙粘蛋白/ 561纳米的激光通道两者。比例尺10微米。 <a href="https://www.jove.com/files/ftp_upload/52644/52644fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/52644/52644fig6.jpg" /> 图6.抗小鼠IgG（H + L）的单价Fab片段有效地阻断内源性小鼠IgG通过抗小鼠二级抗体结合。胚胎心脏切下的E12.5，快速冷冻，冰冻切片，丙酮固定，并进行免疫染色。 <s仲&gt;（AC）的切片阻断用1×封闭缓冲液，随后通过抗小鼠IgG单价Fab片段，暴露于小鼠单克隆的克隆EA53初级抗体对S-α辅肌动蛋白和兔多克隆第一抗体对于N-钙粘蛋白，洗涤，并暴露到的Alexa Fluor 488抗-小鼠和Alexa Fluor 586的抗兔次级抗体。使用强度直方图显示范围480-2500（A）的合并图像（在可能0-65535）。星号音符区，其中的N-钙粘蛋白的信号被限制为小梁心肌，这可能代表新生闰盘的横向端。（B）中的N-钙粘蛋白，仅使用亮度直方图显示范围480-2,500通道。（℃）S-α辅肌动蛋白，仅使用亮度直方图显示范围480-2,500通道。（DG）将切片阻断用1×封闭缓冲液（不使用抗小鼠IgG单价Fab片段阻断步骤）中，暴露于兔多克隆使用强度直方图显示范围480-2500对于N-钙粘蛋白（不使用小鼠单克隆第一抗体），洗涤，并暴露于的Alexa Fluor 488抗-小鼠和Alexa Fluor 586的抗兔次级抗体。（D）的第一抗体合并图像。 （E）N-钙粘蛋白，仅使用亮度直方图显示范围480-2500通道。（F）的 S-α辅肌动蛋白，仅使用亮度直方图显示范围480-2,500通道。（G）的 S-α辅肌动蛋白，仅使用信道高灵敏度的强度直方图的显示范围480-530，这揭示了背景检测内源性小鼠IgG在不存在抗小鼠IgG单价Fab片段阻断步骤。（香港）将切片阻断用1×封闭缓冲液，随后通过抗小鼠IgG单价Fab片段，暴露于对于N-钙粘蛋白（无小鼠单克隆第一抗体）中，用兔多克隆第一抗体，并暴露于的Alexa Fluor 488抗 - 小鼠和甲LEXA氟586抗兔次级抗体。（H）的使用强度直方图显示范围480-2,500合并图像。（Ⅰ）的N-钙粘蛋白，仅使用亮度直方图显示范围480-2,500通道。（J），S-α-actinin-仅使用信道强度直方图显示范围480-2,500。（K）的 S-α辅肌动蛋白，仅使用高灵敏度的强度直方图的显示范围480-530，这表明缺乏背景内源性小鼠IgG检测通道时的抗小鼠IgG单价Fab片段堵步骤使用。 <a href="https://www.jove.com/files/ftp_upload/52644/52644fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图7" src="/files/ftp_upload/52644/52644fig7.jpg" /> 图7. S-α辅肌动蛋白和骨小梁CARDI肌动蛋白组织omyocytes在12.5天胚胎的LifeAct-RFPruby转基因小鼠系用于可视化丝状肌动蛋白19，而针对S-α辅肌动蛋白的小鼠单克隆的克隆EA53抗体用于标记的Z-光盘和闰盘。胚胎PFA固定。 0.2微米光切片收集AZ栈（A）扁平ž堆栈显示，S-α辅肌动蛋白染色更加分散PFA固定组织中比快速冷冻和丙酮固定部分（ 图2 - 5）。（B ）扁平ž堆栈同时显示丝状肌动蛋白和s-α辅肌动蛋白。内肌原纤维（箭头）Z-盘之间的局部丝状肌动蛋白的荧光。丝状肌动蛋白荧光也见于心内膜细胞排列在小梁细胞（箭头）。（C）的小梁心肌的三维视图，如从堆栈的顶部看。（D）的立体图小梁心肌细胞，如从堆栈的底部看。强度直方图显示范围470-900（总分可能0-65535）同时为488纳米的激光信道，并在A和B的561纳米的激光通道;显示范围460-800的两个通道C和D.比例尺10微米。 <a href="https://www.jove.com/files/ftp_upload/52644/52644fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <a href="https://www.jove.com/files/ftp_upload/52644/Movie1.mov">电影</a> 1。 S-的α辅肌动蛋白和骨小梁心肌细胞在胚胎12.5天的肌动蛋白组织中的360°旋转的3D视图，从图6中的图像堆栈使用斐济图像分析程序中的Image J 3D查看器插件在三维渲染。亮度直方图显示范围470-800（出可能0-65535）为488 nm和561 nm激光通道。&lt;/ P&gt; <a href="https://www.jove.com/files/ftp_upload/52644/Movie2.mov">电影</a> 2。 S-α辅肌动蛋白和肌动蛋白组织中的骨小梁心肌细胞在胚胎12.5天 ，从图6中的图像堆栈中选择3D视图。在三个维度使用斐济图像分析程序中的Image J 3D查看器插件呈现。绕x，y和z轴的小的旋转心肌但大多数心肌细胞之间的对准差的内表现出相对对齐的肌原纤维。小旋转还展示了围绕缺乏心肌S-α辅肌动蛋白细胞心内膜的密切近似值。亮度直方图显示范围470-800（出可能0-65535）为488 nm和561 nm激光通道。

Watch this Scientific Journal Video about Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart at JoVE.com

Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart

Mutations that lead to congenital heart defects benefit from in vivo investigation of cardiac structure during development, but high-resolution structural studies in the mouse embryonic heart are technically challenging. Here we present a robust immunofluorescence and image analysis method to assess cardiomyocyte-specific structures in the developing mouse heart.

在胚胎小鼠心脏采用免疫荧光心肌发展分析

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated ...

analysis-cardiomyocyte-development-using-immunofluorescence-embryonic

Research

JoVE Journal

Developmental Biology

21.1K 次观看.  Northwestern University. Mutations that lead to congenital heart defects benefit from in vivo investigation of cardiac structure during development, but high-resolution structural studies in the mouse embryonic heart are technically challenging. Here we present a robust immunofluorescence and image analysis method to assess cardiomyocyte-specific structures in the developing mouse heart.

视频: Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart

Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis

The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the cardiac transcriptome from being masked by the global embryonic transcriptome, it is necessary to collect sufficient numbers of hearts for further analyses. Furthermore, as zebrafish cardiac development proceeds rapidly, heart collection and RNA extraction methods need to be quick in order to ensure homogeneity of the samples. Here, we present a rapid manual dissection protocol for collecting functional/beating hearts from zebrafish embryos. This is an essential prerequisite for subsequent cardiac-specific RNA extraction to determine cardiac-specific gene expression levels by transcriptome analyses, such as quantitative real-time polymerase chain reaction (RT-qPCR). The method is based on differential adhesive properties of the zebrafish embryonic heart compared with other tissues; this allows for the rapid physical separation of cardiac from extracardiac tissue by a combination of fluidic shear force disruption, stepwise filtration and manual collection of transgenic fluorescently labeled hearts.

To analyse cardiac gene expression profiles during zebrafish heart development, total RNA has to be extracted from isolated hearts. Here, we present a protocol for collecting functional/beating hearts by rapid manual dissection from zebrafish embryos to obtain cardiac-specific mRNA.

大规模的斑马鱼胚胎的心脏解剖的转录分析

The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the ...

科研

发育生物学

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair cells are aligned in one row of inner hair cells and three rows of outer hair cells that extend along the basal to apical axis of the cochlea. The explant culture technique described here provides an efficient method to isolate and maintain cochlear explants from the embryonic mouse inner ear. Also, the morphology and molecular characteristics of sensory hair cells and nonsensory supporting cells within the cochlear explant cultures resemble those observed in vivo and can be studied within its intrinsic cellular environment. The cochlear explants can serve as important experimental tools for the identification and characterization of molecular and genetic pathways that are involved in cellular specification and patterning. Although transgenic mouse models provide an effective approach for gene expression studies, a considerable number of mouse mutants die during embryonic development thereby hindering the analysis and interpretation of developmental phenotypes. The organ of Corti from mutant mice that die before birth can be cultured so that their in vitro development and responses to different factors can be analyzed. Additionally, we describe a technique for electroporating embryonic cochlear explants ex vivo which can be used to downregulate or overexpress specific gene(s) and analyze their potential endogenous function and test whether specific gene product is necessary or sufficient in a given context to influence mammalian cochlear development1-8.

We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear. We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development.

小鼠胚胎植耳蜗的文化和基因转移通过电穿孔

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair ...

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo development due to their similar developmental process. Although genetic factors are known to affect early embryonic development, the discovery of such factors has been a serious challenge. Microarray technology allows quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. One of the main decisions that have to be made when planning a microarray experiment is whether to use a one- or two-color approach. Two-color design increases technical replication, minimizes variability, improves sensitivity and accuracy as well as allows having loop designs, defining the common reference samples. Although microarray is a powerful biological tool, there are potential pitfalls that can attenuate its power. Hence, in this technical paper we demonstrate an optimized protocol for RNA extraction, amplification, labeling, hybridization of the labeled amplified RNA to the array, array scanning and data analysis using the two-color analysis strategy.

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Microarray technology allows quantitative measurement and gene expression profiling of transcripts on a genome-wide basis. Therefore, this protocol provides an optimized technical procedure in a two-color custom made bovine array using Day 7 bovine embryos to demonstrate the feasibility of using low amount of total RNA.

转录组分析]<em&gt;活体</em&gt;生产牛胚胎植入前胚胎采用双色微阵列平台

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo ...

Assessing Cardiomyocyte Subtypes Following Transcription Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts

Direct reprogramming of one cell type into another has recently emerged as a powerful paradigm for regenerative medicine, disease modeling, and lineage specification. In particular, the conversion of fibroblasts into induced cardiomyocyte-like myocytes (iCLMs) by Gata4, Hand2, Mef2c, and Tbx5 (GHMT) represents an important avenue for generating de novo cardiac myocytes in vitro and in vivo. Recent evidence suggests that GHMT generates a greater diversity of cardiac subtypes than previously appreciated, thus underscoring the need for a systematic approach to conducting additional studies. Before direct reprogramming can be used as a therapeutic strategy, however, the mechanistic underpinnings of lineage conversion must be understood in detail to generate specific cardiac subtypes. Here we present a detailed protocol for generating iCLMs by GHMT-mediated reprogramming of mouse embryonic fibroblasts (MEFs). 
We outline methods for MEF isolation, retroviral production, and MEF infection to accomplish efficient reprogramming. To determine the subtype identity of reprogrammed cells, we detail a step-by-step approach for performing immunocytochemistry on iCLMs using a defined set of compatible antibodies. Methods for confocal microscopy, identification, and quantification of iCLMs and individual atrial (iAM), ventricular (iVM), and pacemaker (iPM) subtypes are also presented. Finally, we discuss representative results of prototypical direct reprogramming experiments and highlight important technical aspects of our protocol to ensure efficient lineage conversion. Taken together, our optimized protocol should provide a stepwise approach for investigators to conduct meaningful cardiac reprogramming experiments that require identification of individual CM subtypes.

This manuscript describes a step-by-step protocol for the generation and quantification of diverse reprogrammed cardiac subtypes using a retrovirus-mediated delivery of Gata4, Hand2, Mef2c, and Tbx5.

评估心肌亚型以下鼠标胚胎成纤维细胞的转录因子介导的重编程

Direct reprogramming of one cell type into another has recently emerged as a powerful paradigm for regenerative medicine, disease modeling, and lineage ...

Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development and organogenesis are two areas of research that have benefitted greatly from these advances, due to the high quality of data that can be obtained directly from imaging pre-implantation embryos or ex vivo organs. While pre-implantation embryos have yielded data with especially high spatial resolution, later stages have been less amenable to three-dimensional reconstruction. Obtaining high-quality 3D or volumetric data for known embryonic structures in combination with fate mapping or genetic lineage tracing will allow for a more comprehensive analysis of the morphogenetic events taking place during embryogenesis.
This protocol describes a whole-mount immunofluorescence approach that allows for the labeling, visualization, and quantification of progenitor cell populations within the developing cardiac crescent, a key structure formed during heart development. The approach is designed in such a way that both cell- and tissue-level information can be obtained. Using confocal microscopy and image processing, this protocol allows for three-dimensional spatial reconstruction of the cardiac crescent, thereby providing the ability to analyze the localization and organization of specific progenitor populations during this critical phase of heart development. Importantly, the use of reference antibodies allows for successive masking of the cardiac crescent and subsequent quantitative measurements of areas within the crescent. This protocol will not only enable a detailed examination of early heart development, but with adaptations should be applicable to most organ systems in the gastrula to early somite stage mouse embryo.

Here, we describe a protocol for whole mount immunofluorescence and image-based quantitative volumetric analysis of early stage mouse embryos. We present this technique as a powerful approach to qualitatively and quantitatively assess cardiac structures during development, and propose that it may be widely adaptable to other organ systems.

小鼠胚胎心肌内祖群的定量全贴免疫荧光分析

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

用扫描电镜和全贴免疫荧光技术可视化小鼠胚胎中的节点和外型板

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

Microinjection-based System for In Vivo Implantation of Embryonic Cardiomyocytes in the Avian Embryo

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a general challenge in developmental biology research. In the embryonic heart, this can be particularly difficult as regional differences in the expression of transcriptional regulators, paracrine/juxtacrine signaling cues, and hemodynamic force are all known to influence cardiomyocyte maturation. A simplified method to alter a developing cardiomyocyte's molecular and biomechanical microenvironment would, therefore, serve as a powerful technique to examine how local conditions influence cell fate and function. To address this, we have optimized a method to physically transplant juvenile cardiomyocytes into ectopic locations in the heart or the surrounding embryonic tissue. This allows us to examine how microenvironmental conditions influence cardiomyocyte fate transitions at single cell resolution within the intact embryo. Here, we describe a protocol in which embryonic myocytes can be isolated from a variety of cardiac sub-domains, dissociated, fluorescently labeled, and microinjected into host embryos with high precision. Cells can then be directly analyzed in situ using a variety of imaging and histological techniques. This protocol is a powerful alternative to traditional grafting experiments that can be prohibitively difficult in a moving tissue such as the heart. The general outline of this method can also be adapted to a variety of donor tissues and host environments, and its ease of use, low cost, and speed make it a potentially useful application for a variety of developmental studies.

In this method, embryonic cardiac tissues are surgically microdissected, dissociated, fluorescently labeled, and implanted into host embryonic tissues. This provides a platform for studying the individual or tissue level developmental organization under ectopic hemodynamic conditions, and/or altered paracrine/juxtacrine environments.

基于微注射的免疫胚胎心肌细胞体内植入系统

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a ...

Analysis of Cardiac Chamber Development During Mouse Embryogenesis Using Whole Mount Epifluorescence

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during heart development using ventricular specific fluorescent reporter knock-in mice (MLC-2v-tdTomato mice). Heart development involves a linear heart tube formation, the heart tube looping, and four chamber septation. These complex processes are highly conserved in all vertebrates. The mouse embryonic heart has been widely used for heart developmental studies. However, due to their extremely small size, dissecting mouse embryonic hearts is technically challenging. In addition, visualization of cardiac chamber formation often needs in situ hybridization, beta-galactosidase staining using LacZ reporter mice, or immunostaining of sectioned embryonic hearts. Here, we describe how to dissect mouse embryonic hearts and directly visualize ventricular chamber formation of MLC-2v-tdTomato mice using whole mount epifluorescent microscopy. With this method, it is possible to directly examine heart tube formation and looping, and four chamber formation without further experimental manipulation of mouse embryos. Although the MLC-2v-tdTomato reporter knock-in mouse line is used in this protocol as an example, this protocol can be applied to other heart-specific fluorescent reporter transgenic mouse lines.

We present the protocols to examine mouse heart development using whole mount epifluorescent microscopy on mouse embryos dissected from ventricular specific MLC-2v-tdTomato reporter knock-in mice. This method allows us to directly visualize each stage of the ventricular formation during mouse heart development without labor-intensive histochemical methods.

全山荧光在小鼠胚胎发生过程中心脏室发育的分析

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during ...

Multiplexed Single Cell mRNA Sequencing Analysis of Mouse Embryonic Cells

Single cell mRNA sequencing has made significant progress in the last several years and has become an important tool in the field of developmental biology. It has been successfully used to identify rare cell populations, discover novel marker genes, and decode spatial and temporal developmental information. The single cell method has also evolved from the microfluidic based Fluidigm C1 technology to the droplet-based solutions in the last two to three years. Here we used the heart as an example to demonstrate how to profile the mouse embryonic tissue cells using the droplet based scRNA-Seq method. In addition, we have integrated two strategies into the workflow to profile multiple samples in a single experiment. Using one of the integrated methods, we have simultaneously profiled more than 9,000 cells from eight heart samples. These methods will be valuable to the developmental biology field by providing a cost-effective way to simultaneously profile single cells from different genetic backgrounds, developmental stages, or anatomical locations.

Here we presented a multiplexed single cell mRNA sequencing method to profile gene expression in mouse embryonic tissues. The droplet-based single cell mRNA sequencing (scRNA-Seq) method in combination with multiplexing strategies can profile single cells from multiple samples simultaneously, which significantly reduces reagent costs and minimizes experimental batch effects.

小鼠胚胎细胞的多路复用单细胞mRNA测序分析

Single cell mRNA sequencing has made significant progress in the last several years and has become an important tool in the field of developmental ...

Analysis of Congenital Heart Defects in Mouse Embryos Using Qualitative and Quantitative Histological Methods

Congenital heart defects (CHD) are the most common type of birth defect in humans, affecting up to 1% of all live births. However, the underlying causes for CHD are still poorly understood. The developing mouse constitutes a valuable model for the study of CHD, because cardiac developmental programs between mice and humans are highly conserved. The protocol describes in detail how to produce mouse embryos of the desired gestational stage, methods to isolate and preserve the heart for downstream processing, quantitative methods to identify common types of CHD by histology (e.g., ventricular septal defects, atrial septal defects, patent ductus arteriosus), and quantitative histomorphometry methods to measure common muscular compaction phenotypes. These methods articulate all the steps involved in sample preparation, collection, and analysis, allowing scientists to correctly and reproducibly measure CHD.

In this protocol, we describe procedures to qualitatively and quantitatively analyze developmental phenotypes in mice associated with congenital heart defects.

使用定性和定量组织方法对小鼠胚胎先天性心脏缺陷的分析

Congenital heart defects (CHD) are the most common type of birth defect in humans, affecting up to 1% of all live births. However, the underlying causes ...