这项工作得到了维康信托SIA奖100329 / Z / 12 / Z至WAH的支持和授予香港的DFG奖学金KH 376 / 1-1的支持

该协议遵循剑桥大学的动物护理指南。 动物维护<ol><li> 青蛙胚胎 注意:可以根据动物和实验室设施养殖和维护胚胎。这里描述动物维护的例子。 <ol><li>制备0.1x修饰的Barth溶液（MBS）溶液:0.88mM NaCl，10μMKCl，24μMNaHCO 3，100μMHEPES，8.2μMMgSO 4，3.3μMCa（NO 3 ） 2和4.1μMCaCl 2 ，pH 7.6 。 </li><li>通过体外受精获得非洲爪蟾胚胎。 <ol><li>在产卵前一周（60 IU）和12 h（500 IU）注射怀孕母马血清促性腺激素，在成年雌性非洲爪蛙（ 非洲爪蟾）中诱导排卵。 </li><li>收集卵母细胞并用体外均匀的雄性睾丸进行体外受精 。 </li><li>清莱在16 - 18°C的0.1x MBS胚胎。根据非洲爪蟾的正常表14阶段胚胎。仔细选择整个和活的胚胎进行实验。 </li></ol></li></ol></li><li> 斑马鱼胚胎 注意:可以根据动物和实验室设施养殖和维护胚胎。这里描述动物维护的例子。 <ol><li>准备胚胎培养基:5mM NaCl，0.17mM KCl，0.33mM CaCl 2，0.33mM MgSO 4和0.00001％亚甲基蓝。 </li><li>在26.5°C保持和繁殖WT AB菌株Danio rerio 。 </li><li>在受精早晨收集胚胎，在28℃下在如上所述制备的胚胎培养基中培养至受精后4天（dpf），如前所述选择和分期。 </li></ol></li></ol> 2. 体内缺氧诱导</p&gt; <ol><li> 气体孵化室的建造 <ol><li>使用通常在鱼设施中用于缺氧室建造的养殖水体箱（ 图1A ）。这个坦克应该适合一个24孔板。 </li><li>准备一个网眼24孔板， 如图1所示 。拆下24孔板的塑料底部（ 图1B ）， 例如 ，为此目的使用铣床。 </li><li>用环氧树脂填充孔的塑料和板的壁之间的空间。将网眼直径为0.1-0.2毫米的尼龙过滤器（ 图1C ）连接到树脂涂层的塑料上，并使板完全干燥。 </li><li>一旦干燥，将三根或四条直径为10 mm，直径为5 mm的隧道（ 图1D ）钻入树脂和网眼涂层的板壁。连接塑料或特氟隆棒（ 图1E ），直径相当于隧道长30mm，并将板放入所选择的槽中。 </li><li>将网眼24孔板放入选择的水槽中。使用硅胶管（ 图1F ），使用合适的气体调节器（I）（BOC 200）将气罐（ 图1G ）与所需的O 2 / N 2混合物或其他选择的气体混合物连接到分配阀（ 图1H ）酒吧）。这里使用含有5％氧气和95％N 2的混合物的气罐在这里提供的缺氧实验中。 </li><li>将室介质中的氧气流速调节至每秒0.0017-0.0019立方米。在这种气体流量下，在板的孔中不应该看到介质的干扰。 <ol><li>为此，使用高纯度两级气体调节器。设置调节器以降低内部压力e气罐（1000 - 2500 PSI），在整个实验中输送压力为10 PSI。因此，根据该气体调节器的规格，在整个实验中实现了每秒0.00189立方米的流量。 </li></ol></li><li>将硅胶管和分配器阀连接到水箱内的陶瓷盘扩散器（ 图1J ）。关闭水箱，小心地密封盖子，涂上硅脂（ 图1K ）（与图1相比 ）。如果需要特定的非室温，请将缺氧室放入需要温度的实验室培养箱中。 </li><li>根据实验中使用的胚胎类型，用合适的胚胎培养基填充缺氧室，开始通过陶瓷盘扩散器扩散气体混合物。平衡10-15分钟</li><li>与th平衡后e所需的氧气混合物，使用氧计或氧气探针测量水中的氧浓度。在这里，为了这个目的，使用光纤溶解氧和温度传感器的氧化计。 </li></ol></li><li> 诱导胚胎缺氧 <ol><li>仔细选择实验的胚胎，使用全部和活的胚胎进行缺氧孵育。在这里，我们在这里提出的实验中，将第38阶段或斑马鱼胚胎4 dpf的青蛙胚胎。 </li><li>将胚胎盘放入含有气体孵育室的培养箱（ 图1K ）中，让它们平衡到培养箱的温度。 </li><li>使用塑料移液器将胚胎小心地转移到气体孵育室的网状24孔板（ 图1B ）的孔中。在整个协议中始终使用塑料移液器进行胚胎移植。 注意:该板的每个孔都可以包含到5个第38阶段的青蛙胚胎或4个dpf的多达7个斑马鱼胚胎。如果使用不同的基因型，请小心地标记井。 </li><li>保持气体孵育室恒温输注，选择气体混合物5小时或更长时间适合实验。在这里使用5％O 2和95％N 2的混合物。 </li><li>仔细地将胚胎从气体培养箱转移回到对应于胚胎类型的正常氧培养基，并立即进行步骤3或4。 </li><li>如果需要进一步的常氧处理，请仔细将胚胎转移回对应于胚胎类型的正常氧培养基。 </li></ol></li></ol> 3.通过监测HIF-1α水平控制成功的低氧诱导<ol><li> 准备工作 <ol><li>在0.1x MBS中制备0.4 mg / mL MS222溶液。 </li><li>准备均质缓冲液:购买放射免疫沉淀测定缓冲液（RIPA buffer），用于组织匀浆，并补充使用蛋白酶抑制剂进行实验所需的缓冲液的量至终浓度1:100 v / v。 </li><li>准备用于Western印迹的溶液。 <ol><li>准备4x Laemmli凝胶加载缓冲液:425mM Tris pH 8.0,40％v / v甘油，8％v / v SDS，4％v / vβ-巯基乙醇，0.5％w / v溴苯酚蓝，10mL总体积ddH 2 O. </li><li>准备5倍运行缓冲液:15克Trizma碱，72克甘氨酸，5克SDS，1升总体积ddH 2 O. </li><li>准备转移缓冲液:2.66g Trizma碱，14.4g甘氨酸，200mL 100％甲醇，1L总体积ddH 2 O. </li><li>制备10x TBST:5 mL 1 M Tris pH 8.0,30 mL 5M NaCl，2.5 mL吐温20,1L总体积H 2 O. </li><li>准备封闭溶液:1％TBST中4％w / v脱脂奶粉。 </li></ol></li></ol></li><li> 收集组织 <ol><li>通过沐浴在0.4mg / mL MS222溶液中麻醉胚胎16 和 17，并使用塑料移液管将它们转移到装有均化缓冲液的塑料反应管中。每个胚胎使用20μL缓冲液。请注意，需要至少5个第4阶段的第38或6个斑马鱼的青蛙胚胎，以确定HIF1α蛋白水平的显着变化。 </li><li>使用合适的组织匀浆器或超声波仪均匀化组织。通过离心（15分钟; 13,000×g; 4℃）除去碎屑。 <ol><li>或者，从麻醉的青蛙胚胎17中分离视网膜并如上所述进行均质化。每10个视网膜使用20μL匀浆缓冲液。 </li></ol></li><li>使用移液管取上清液，在85℃变性5分钟，并用Laemmli凝胶加载缓冲液补充至终浓度为1×v / v（ 例如 ，将5μL的4x Laemmli缓冲液加至15μL匀浆）。将这种上清液用于Wes将样品在-20℃下进行印迹或冷冻。 </li></ol></li><li> 蛋白质印迹 <ol><li>使用1x v / v运行缓冲液将在步骤3.2中所述制备的样品在电泳系统中的预制的12％凝胶上载入。使用蛋白质梯子进行蛋白质大小测定。根据制造商的方案，将蛋白质转移到转移缓冲液中的硝酸纤维素膜（0.45μm膜）上，在4℃的冰上包装1小时。 </li><li>阻断非特异性结合位点，将膜在阻滞缓冲液中孵育60分钟。在1x TBST中洗涤3×10分钟。 </li><li>在隔膜溶液中分别在兔抗HIF-1α抗体和1:100 v / v和1:5000 v / v的小鼠抗α-微管蛋白溶液中孵育膜2.5小时。在1x TBST中洗涤3×10分钟。 </li><li>为了检测，在封闭溶液中以1:1000v / v稀释的山羊抗兔和山羊抗小鼠HRP缀合的抗体孵育1小时。随后在1×TBST中洗涤3×10分钟，并使用商业试剂盒和选择的显影机显现HRP染色。 </li><li>使用数字量化量化HIF1α蛋白的量。 </li></ol></li></ol> 4.监测缺氧中的细胞增殖<ol><li> 准备工作 <ol><li>在选择的胚胎培养基中制备5 mM EdU溶液。该溶液可以立即使用或等分，并在-20°C下储存长达6个月。 </li><li>准备磷酸盐缓冲盐水（PBS）或从商业资源购买PBS。在115°C高压灭菌10分钟。 </li><li>准备检测EdU并入的解决方案。 <ol><li>准备固定溶液:4％v / v的16％甲醛储备溶液在PBS中。 </li><li>制备蔗糖溶液:PBS中30％w / v蔗糖。 </li><li>准备PBST:0.1％v / v Triton X-100在PBS中。 </li><li>准备阻断溶液:10％v / v热灭活的山羊血清（HIGS）。 </li><li>在PBST中制备DAPI溶液:1:1000 v / v 4&#39;，6-二脒基-2-苯基吲哚（DAPI）。 </li></ol></li></ol></li><li> 胚胎在缺氧条件下孵育用于EdU并入 <ol><li>向气体培养室中加入400-500mL补充有1％v / v DMSO的5mM EdU溶液。在实验前用实验中使用的相同气体混合物预平衡溶液15分钟。请注意，通过将较短的杆（ 图1E ）连接到网眼底板，可以最小化孵育溶液的体积。 </li><li>将气体管道连接到含有5％O 2和95％N 2的气体混合物的气瓶。孵育溶液15分钟。 </li><li>将胚胎转移到缺氧室，将它们均匀分布在各孔之间并孵育2小时。每个孔可以配合多达5个第38阶段的青蛙胚胎或最多7个斑马鱼胚胎4 dpf。 </li><li>分析胚胎为EdU注册。 </li></ol></li><li> 缺氧胚胎孵化的时间过程和EdU结合 <ol><li>向气体孵育室填充500 mL胚胎培养基。将气体管道连接到含有5％O 2和95％N 2气体混合物的气瓶中，并将介质与气体混合物平衡15分钟。 </li><li>将胚胎转移到缺氧室，将它们均匀分布在各孔之间，孵育达48小时所需时间。在最后1小时内，用补充有1％v / v DMSO的5mM EdU溶液代替胚胎培养基。 </li><li>将胚胎转移回常规菜，并分析EdU的结合情况。 </li></ol></li><li> EdU并入检测和分析 <ol><li>通过沐浴在0.4mg / mL MS222溶液中麻醉胚胎。 </li><li>将胚胎转移到适当的玻璃小瓶中，通过在固定溶液中孵育2 h进行修复t RT或O / N。随后在PBS中洗涤3×10分钟。小心地将胚胎转移到蔗糖溶液中进行组织冷冻保护。孵育4小时或直到胚胎下沉到玻璃小瓶的底部。 </li><li>将胚胎嵌入嵌入培养基（最佳切割温度复合物）。立即用胚胎进行冷冻切块，或在-80℃下储存14 d。 </li><li>使用低温恒温器冷冻含有胚胎的块。在显微镜载玻片上收集12μm（斑马鱼胚胎）或16μm（青蛙胚胎）厚度的部分，使其干燥。立即过程冷冻切片或-20°C储存长达3个月。 </li><li>使用商业化学试剂盒通过免疫荧光染色检测EdU掺入。准备实验中冷冻切片数量所需的EdU反应混合物，如制造商所述。 <ol><li>对于500μL的EdU反应混合物，使用430μL的1X EdU反应缓冲液，20μLCuSO 4，1.2μLAlexa Fluor叠氮化物，50μL1X EdU缓冲添加剂。 </li></ol></li><li>用PBS洗涤一次冷冻切片以除去包埋培养基，并用PBST 3×5分钟透化组织。在封闭溶液中孵育冷冻切片30分钟。 </li><li>用EdU反应混合物孵育1小时的冷冻切片，然后在PBST中洗涤3×10分钟。用DAPI溶液冷冻切片15分钟，然后在PBST中洗涤3×10分钟。将载玻片上的染色冷冻切片放在安装介质中，盖上盖玻片，待荧光显微镜分析后放置干燥，或在4℃下储存长达1年。 </li><li>在反相共焦扫描显微镜下用抗EdU染色分析冷冻切片，并使用数字定量测定EdU阳性细胞的数量。 </li></ol></li></ol>

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作者宣称他们没有竞争的经济利益。

在这里，我们提出了一种简单而强大的新方法来诱导缺氧，可用于青蛙和斑马鱼胚胎，但也适用于其他水生生物。这种方法的主要优点在于其简单性和成本效益。然而，用这种方法实现的结果是非常强大的。我们已经表明，在整个胚胎以及特定组织（这里是视网膜）中，室内都可以有效诱导缺氧。为了确定缺氧诱导的有效性，我们监测了全胚胎和视网膜裂解物中HIF-1α蛋白的水平。根据我们的结果，缺氧诱导可以被认为是成功的，如果与正常氧对照相比，在缺氧室中1小时可以观察到HIF-1α水平的增加，并且与全球蛋白质水平正常化， 例如 ，使用α-微管蛋白水平作为对照。我们通过监测干细胞增殖物证实组织中缺氧的活性根据我们先前发表的结果，可以显示使用这里提出的设置的缺氧导致青蛙和斑马鱼胚胎中视网膜干细胞增殖的减少。 该协议的关键步骤之一是确保缺氧室被正确密封。这是通过在罐盖上使用适当的密封来实现的，这是我们的情况下的硅脂。此外，放置在孵化器内将有助于避免大气氧气泄漏进入室，通过提供额外的屏障。用适当的探针或电极测量氧气浓度也可确保低氧氧含量保持稳定且无波动。另外，在实验之间交换溶液或样品时，应注意长时间避开室开放。 给予快速平衡缺氧建立时间为10 - 15 min，需要少量的气体混合物和较低的气体流速来维持系统，甚至比代表性结果（24 h）所述的时间更长。应在实验中使用的溶液预先平衡，以在孵化室前达到缺氧15分钟（如4.2.1所述）。我们在48小时的恒定缺氧期间使用了缺氧室，没有任何错误，从我们的氧浓度测量可以看出（ 表1 ）。应该注意的是，在较长的孵育时间内必须控制正确的气体扩散。最后，在将胚胎放入孔中时应小心（步骤2.2.1。），以避免来自不同突变动物/实验条件的胚胎混合。这是相当简单的实现给定板的单井的相对较高的壁。 尽管是一个简单但有效的和强壮的缺氧诱导系统，这里描述的室的使用对于需要使用昂贵的孵化溶液和培养基的实验具有一个缺点。腔室中介质的最小体积不应低于400 mL。然而，可以进行调整以适应较低的体积:我们已经成功地调整连接到板（2.1.4和4.2.1）的棒的长度（ 图1E ），以便我们只需要50 mL的介质/溶液。虽然对于较长的孵育时间不是理想的，但是该设置可以配合相同的气体管道，并且可以适当地密封，以确保充足的缺氧达8小时。 总而言之，我们的气室设置可以用于任何水生模型生物，并通过其简单性，成本效益和鲁棒性与其他类似的商业可用系统分开。一旦掌握，它可以用于直接体内实验，任何给定的时间段和任何欲望d气氛包括缺氧，高氧或其他气体混合物。

缺氧研究有很多应用。这些包括调查发病机理和开发治疗缺氧1和急性高原病2症状的医疗状况。低氧应激导致需要氧气的所有生物体中的主要代谢变化。低氧应激也影响胎儿生长发育及几种人类疾病的发病机制，包括子宫内生长受限3 。低氧应激不仅可以导致出生体重，胎儿和新生儿死亡率降低，而且还可能导致成人生活中的许多并发症，如心血管疾病，2型糖尿病，肥胖症和高血压4 。当实体肿瘤发展期间，当肿瘤组织超过其血液供应时，也经常观察到低氧应激。因此，能够在体内和直接研究缺氧的作用至关重要<html> yonic发展。 用于研究发育过程中缺氧影响的最为众所周知的方法是在生长培养基中使用氯化钴或在低氧室中孵育生物体。氯化钴在正常氧浓度下人工诱导缺氧反应，由于其通过防止其蛋白质降解5,6,7而在低氧诱导因子-1α（HIF-1α）的稳定中起作用。然而，作为方便的方法8 ，使用氯化钴以及其它类似的化学缺氧模拟物可能对细胞和组织具有非特异性有害的作用， 例如细胞凋亡9 。因此，缺氧室是通过正常发育过程诱导活体中"天然缺氧"的更好方法。 我们专注于开发一种诱导水生动物胚胎缺氧的系统，青蛙和斑马鱼现在已经成为信息丰富的脊椎动物模型生物，用于许多生物过程的研究，以及各种人类疾病的模型青蛙和斑马鱼胚胎另外，快速发展的过程使得可以实时地操纵环境因素并观察器官形成的表型变化，此外，主要信号转导途径的许多成分被高度保守这些模型生物体已经被大量文献详细描述，使用青蛙和斑马鱼胚胎研究缺氧对脊椎动物发育的影响的主要优点是可以直接监测所有过程，因为氧气很快穿透胚胎。因此，在青蛙和斑马鱼中，与其他模型生物体相反小鼠胚胎，可以在感兴趣的组织中研究特定氧浓度的影响，而不考虑功能性脉管系统的存在或缺乏。 用于缺氧孵育的大多数商业可用的装置具有相当大的并具有相应高的运行成本的缺点。除了高起始成本和气体消耗之外，普通缺氧室的平衡和维持需要维持恒定的低氧气氛，抵抗由于其较大尺寸和/或生物呼吸而自然发生在这些腔室中的气体梯度。这需要使用气体风扇和冷却系统，这增加了额外的必要设备的数量，阻碍了研究人员的灵巧性和整体降低了实验程序的简单性。相比之下，我们在这里提出的设置是相当强大的，但是非常具有成本效益，小巧，易于建立并允许f天然气平衡，稳定的低氧气氛和室内材料和溶液的简单交换。我们的系统可以用于任何感兴趣的水生模型生物体。 我们构建了一个方便小的缺氧室，因此可以放置在普通的实验室培养箱内，这样可以容易地在任何特定的温度下进行实验。提供方便的温度控制以及介质中的氧浓度，我们的系统针对市售的缺氧培养箱的优势在于其小型化和成本效益。因此，我们的设置可以使用可用于大多数研究实验室的一般实验室用品来建立，并且不需要任何昂贵的材料。此外，我们的设置不会产生热量，不同于市售的缺氧培养箱，并允许在低于室温的温度下使用放置在培养箱中。拉st对于与冷血生物（如青蛙和鱼）的工作特别重要，其中发育和代谢速率依赖于温度。 我们的气体孵化室非常具有成本效益，易于构建，但是在建立各种缺氧或高氧条件方面是非常通用的，并且能够为广泛的实验条件快速方便地管理不同的介质和解决方案。此外，我们的系统使用24孔板代替常用的餐具或实验室罐10,11,12 ，可以一次观察和实验几种突变条件。 为了控制正常的缺氧诱导，我们通过蛋白质印迹检测监测了HIF-1α蛋白的水平。此外，孵化前后的增殖细胞数量可以使用缺氧室中的n来确定组织中是否已经诱导了缺氧。该方法基于我们先前发表的结果13 ，显示胚胎视网膜干细胞生态位的增殖在诱导缺氧时减少。因此，我们通过向胚胎培养基中加入5-乙炔基-2&#39;-脱氧尿苷（EdU）并测量其结合到新增殖细胞的DNA中，来监测视网膜干细胞增殖水平。

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Sodium chloride</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">S7653</td>
			<td style="width:167px;">NaCl / 0.1X MBS, Embryo medium, 10X TBST</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Potassium chloride</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">P9333</td>
			<td style="width:167px;">KCl / 0.1X MBS, Embryo mediu,</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:216px;">Sodium bicarbonate</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">S5761</td>
			<td style="width:167px;">NaHCO3 / 0.1X MBS</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">HEPES</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">H3375</td>
			<td style="width:167px;">0.1X MBS</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">Magnesium sulfate</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">M7506</td>
			<td style="width:167px;">MgSO4 / 0.1X MBS, Embryo medium</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:216px;">Calcium nitrate</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">202967</td>
			<td style="width:167px;">Ca (NO3)2 / 0.1X MBS</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">Calcium chloride</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">C1016</td>
			<td style="width:167px;">CaCl2 / 0.1X MBS, Embryo medium</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Methylene blue</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">M9140</td>
			<td style="width:167px;">Embryo medium</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Pregnant mare serum gonadotropin</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">CG10</td>
			<td style="width:167px;">frog fertilization</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Zebrafish breeding tank</td>
			<td style="width:71px;">Carolina</td>
			<td style="width:119px;">161937</td>
			<td style="width:167px;">gas chamber construction</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">24-well plate</td>
			<td style="width:71px;">Thermo Scientific</td>
			<td style="width:119px;">142475</td>
			<td style="width:167px;">Nunclon Delta Surface, for gas chamber construction</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Epoxy resin</td>
			<td style="width:71px;">RS Components UK</td>
			<td style="width:119px;">Kit 199-1468</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Gas distributor valve</td>
			<td style="width:71px;">WPI Luer Valves</td>
			<td style="width:119px;">Kit 14011</td>
			<td style="width:167px;">aquatic tank attachment (Schema 1, H)</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">High precision gas valve</td>
			<td style="width:71px;">BOC&#160;</td>
			<td style="width:119px;">200 bar HiQ C106X/2B</td>
			<td style="width:167px;">gas tank attachment (Schema 1, I)</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">5% oxygen and 95% N2 gas tank</td>
			<td style="width:71px;">BOC</td>
			<td style="width:119px;">226686-L</td>
			<td style="width:167px;">hypoxic gas mixture</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:216px;">ceramic disc diffuser</td>
			<td style="width:71px;">CO2 Art&#160;</td>
			<td style="width:119px;">Glass CO2 Nano Aquarium Diffuser, DG005DG005</td>
			<td style="width:167px;">Schema 1, J</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:216px;">silicone grease</td>
			<td style="width:71px;">Scientific Laboratory Supplies</td>
			<td style="width:119px;">VAC1100</td>
			<td style="width:167px;">Schema 1, K</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">oxymeter</td>
			<td style="width:71px;">Oxford Optronix&#160;</td>
			<td style="width:119px;">Oxylite, CP/022/001</td>
			<td style="width:167px;">hypoxic chamber setup</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">fibre-optic dissolved oxygen sensor</td>
			<td style="width:71px;">Oxford Optronix</td>
			<td style="width:119px;">HL_BF/OT/E</td>
			<td style="width:167px;">hypoxic chamber setup</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">plastic pasteur pipette</td>
			<td style="width:71px;">Sterilin</td>
			<td>STS3855604D</td>
			<td style="width:167px;">for embryo transfer</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">MS222&#160;</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:119px;">E10521-50G</td>
			<td style="width:167px;">embryo anesthetic</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">RIPA buffer&#160;</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">R0278-50ML</td>
			<td style="width:167px;">tissue homogenization</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Protease inhibitor</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">P8340</td>
			<td style="width:167px;">tissue homogenization</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Tris</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">77-86-1</td>
			<td style="width:167px;">4X Laemmli loading buffer, 10X TBST</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Glycerol</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">G5516</td>
			<td style="width:167px;">4X Laemmli loading buffer</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Sodium Dodecyl Sulfate</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">L3771</td>
			<td style="width:167px;">SDS, 4X Laemmli loading buffer, 5X Running buffer</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">beta-Mercaptoethanol&#160;</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">M6250</td>
			<td style="width:167px;">4X Laemmli loading buffer</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Bromophenol Blue</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">B0126</td>
			<td style="width:167px;">4X Laemmli loading buffer</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Trizma base&#160;</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">77-86-1</td>
			<td style="width:167px;">5X Running buffer, Transfer buffer</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Glycine</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">G8898</td>
			<td style="width:167px;">5X Running buffer, Transfer buffer</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Methanol</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">34860</td>
			<td style="width:167px;">Transfer buffer</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Tween 20</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">P2287-500ML</td>
			<td style="width:167px;">10X TBST</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">skim milk powder</td>
			<td style="width:71px;">Sigma</td>
			<td>70166</td>
			<td style="width:167px;">Blocking Solution</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Eppendorf microcentrifuge tube</td>
			<td style="width:71px;">Sigma</td>
			<td>T9661</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:216px;">tissue homogenizer</td>
			<td style="width:71px;">Pellet Pestle Motor Kontes</td>
			<td style="width:119px;">Z359971</td>
			<td style="width:167px;">tissue homogenization</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">pellet pestles</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">Z359947-100EA</td>
			<td style="width:167px;">tissue homogenization</td>
		</tr>
		<tr height="20">
			<td height="40" style="height:40px;width:216px;">precast 12% gel</td>
			<td rowspan="2" style="width:71px;">Biorad</td>
			<td rowspan="2" style="width:119px;">Mini-ProteinTGX, 456-1043</td>
			<td rowspan="2" style="width:167px;">Western Blot</td>
		</tr>
		<tr height="20">
			<td height="40" style="height:40px;width:216px;">protein ladder</td>
			<td rowspan="2" style="width:71px;">Amersham</td>
			<td rowspan="2" style="width:119px;">Full-Range Rainbow ladder, RPN800E</td>
			<td rowspan="2" style="width:167px;">Western Blot</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">nitrocellulose membrane (0.45 &#181;m)</td>
			<td style="width:71px;">Biorad</td>
			<td style="width:119px;">162-0115</td>
			<td style="width:167px;">Western Blot</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">anti-HIF-1&#945; antibody</td>
			<td style="width:71px;">Abcam</td>
			<td style="width:119px;">ab2185</td>
			<td style="width:167px;">Western Blot</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">anti-&#945;-tubulin antibody</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">T6074</td>
			<td style="width:167px;">Western Blot</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">goat anti-rabbit antibody</td>
			<td style="width:71px;">Abcam</td>
			<td style="width:119px;">ab6789</td>
			<td style="width:167px;">Western Blot</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">goat anti-mouse antibody</td>
			<td style="width:71px;">Abcam</td>
			<td style="width:119px;">ab97080</td>
			<td style="width:167px;">Western Blot</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Pierce ECL 2 reagent&#160;</td>
			<td style="width:71px;">Thermo Scientific</td>
			<td style="width:119px;">80196</td>
			<td style="width:167px;">Western Blot</td>
		</tr>
		<tr height="106">
			<td height="106" style="height:106px;width:216px;">ECL films Hyperfilm</td>
			<td style="width:71px;">GE Healthcare Amersham</td>
			<td style="width:119px;">28906837</td>
			<td style="width:167px;">Western Blot</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">5-Ethynyl-2&#8242;-deoxyuridine &#160;</td>
			<td style="width:71px;">santa cruz</td>
			<td style="width:119px;">CAS&#160;61135-33-9</td>
			<td style="width:167px;">EdU, EdU incorporation</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Phosphate-buffered Saline</td>
			<td style="width:71px;">Oxoid</td>
			<td style="width:119px;">BR0014G</td>
			<td style="width:167px;">1X PBS</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Formaldehyde</td>
			<td style="width:71px;">Thermo Scientific</td>
			<td style="width:119px;">28908</td>
			<td style="width:167px;">Fixation solution</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Sucrose</td>
			<td style="width:71px;">Fluka</td>
			<td style="width:119px;">S/8600/60</td>
			<td style="width:167px;">Solution solution</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Triton X-100</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">T9284-500ML</td>
			<td style="width:167px;">PBST</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Heat-inactivated Goat Serum</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">G6767-100ml</td>
			<td style="width:167px;">HIGS, Blocking solution (EdU incorporation)</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">4&#39;,6-diamidino-2-phenylindole&#160;</td>
			<td style="width:71px;">ThermoFisher Scientific</td>
			<td style="width:119px;">D1306</td>
			<td style="width:167px;">DAPI, EdU incorporation</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Dimethyl sulfoxide</td>
			<td style="width:71px;">Molecular Probes</td>
			<td style="width:119px;">C10338</td>
			<td style="width:167px;">DMSO, EdU incorporation</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">glass vial</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">98178853</td>
			<td style="width:167px;">EdU incorporation analysis</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Tissue-Plus optimal cutting temperature compound&#160;</td>
			<td style="width:71px;">Scigen</td>
			<td style="width:119px;">4563</td>
			<td style="width:167px;">embedding medium, EdU incorporation analysis</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">cryostat Jung Fridgocut 2800E</td>
			<td style="width:71px;">Leica&#160;</td>
			<td style="width:119px;">CM3035S</td>
			<td style="width:167px;">EdU incorporation analysis</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">microscope slides Super-Frost plus Menzel glass</td>
			<td style="width:71px;">Thermo Scientific</td>
			<td style="width:119px;">J1800AMNZ</td>
			<td style="width:167px;">EdU incorporation analysis</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">EdU Click-iT chemistry kit</td>
			<td style="width:71px;">Molecular Probes</td>
			<td style="width:119px;">C10338</td>
			<td style="width:167px;">EdU incorporation analysis</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">FluorSave</td>
			<td style="width:71px;">Calbiochem</td>
			<td style="width:119px;">D00170200</td>
			<td style="width:167px;">mounting medium, EdU incorporation analysis</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">coverslips</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">ECN631-1575</td>
			<td style="width:167px;">EdU incorporation analysis</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">fluorescent microscope</td>
			<td style="width:71px;">Nikon</td>
			<td style="width:119px;">Eclipse 80i</td>
			<td style="width:167px;">EdU incorporation analysis</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">confocal scanning microscope</td>
			<td style="width:71px;">Olympus</td>
			<td style="width:119px;">Fluoview FV1000</td>
			<td style="width:167px;">EdU incorporation analysis</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Volocity software</td>
			<td style="width:71px;">PerkinElmer</td>
			<td style="width:119px;">Volocity 6.3</td>
			<td style="width:167px;">EdU incorporation analysis</td>
		</tr>
	</tbody>
</table>

induction of hypoxia in living frog and zebrafish embryos

在这里，我们介绍一种低氧诱导的新系统，我们开发了研究缺氧在水生生物如青蛙和斑马鱼胚胎中的作用。我们的系统包括一个具有简单设置的室，在任何选择的实验溶液中都能很好地诱导和保持特定的氧气浓度和温度。所提出的系统非常具有成本效益，但功能非常高，它允许诱导和维持缺氧，用于体内直接实验和多达48小时的不同时间段。 为了监测和研究缺氧的影响，我们采用了两种方法 - 在全胚胎或特异性组织中测量缺氧诱导因子1α（HIF-1α）的水平，并通过5-乙炔基-2&#39;-位测定视网膜干细胞增殖，脱氧尿苷（EdU）并入DNA。 HIF-1α水平可作为整个胚胎或组织中的一般缺氧标记的选择，这里是胚胎视网膜。 EdU掺入胚胎视网膜的增殖细胞是缺氧诱导的特异性输出。因此，我们已经表明，缺氧胚胎视网膜祖细胞在青蛙和斑马鱼胚胎的5％氧气下孵育1小时内减少增殖。 一旦掌握，我们的设置可以用于小型水生模型生物体，用于直接体内实验，任何给定的时间段以及在正常的低氧或高氧氧浓度下或在任何其他给定的气体混合物下。

使用我们在这里提供的缺氧室系统允许研究单独和体内的缺氧对整个动物的影响。可以通过将整个青蛙或斑马鱼胚胎放置在缺氧室（ 图1 ）中来诱导缺氧，并在不同的条件组合下进行。我们完成的气室设置的图像如图2所示。我们使用光纤氧传感器（2.1.9）在实验期间的不同时间点监测培养基中的氧浓度。这些数据表明，我们在我们的实验中已经诱导了稳定的缺氧（6-6.5％的溶解氧）（ 表1 ）。 首先，我们评估了在缺氧室诱导的正常氧和低氧中HIF-1α的普遍存在以及视网膜水平。 HIF-1α是在低氧浓度下稳定的蛋白质。我们测量了保持在正常氧浓度或经受5％缺氧的整个青蛙胚胎裂解物中和在其分离的视网膜裂解物中的HIF-1α水平。 如图3所示，HIF-1α在整个胚胎和视网膜中都在缺氧条件下稳定。 HIF-1α的稳定性在网孔培养板的不同孔中实现（ 图S1 ）。 如我们以前所示，缺氧影响视网膜13的睫状边缘区（CMZ）中视网膜干细胞祖细胞的增殖。因此，通过EdU结合监测CMZ中的增殖是成功诱导缺氧的良好标志。我们在保持在5％氧气的缺氧室中孵育胚胎，并按照步骤4.2所述评估视网膜增殖。正如所料正常氧对照视网膜在CMZ中显示出强烈的EdU染色，其中增殖祖细胞驻留在青蛙（ 图4B和4D ）和斑马鱼胚胎中（ 图5A和5C ）。在缺氧室中缺氧后，在两只青蛙（ 图4C-4D ）和斑马鱼胚胎中观察到CMZ祖细胞增殖的强烈降低（图5B，5C）。为了确定效果的程度，我们进行了一个时间过程，在低氧室内孵育长达24小时的胚胎，如步骤4.3所述通过EdU并入监测增殖。我们可以看出，视网膜祖细胞增殖的减少在2小时内是急性和最大的，并持续了许多小时（ 图4D ），而胚胎根据其发育阶段正常发育。该结果表明，我们的系统可以有效地诱导目标体内的缺氧在短期孵化时间以及维持这些条件较长时间，同时支持正常的胚胎发育。 <img alt="图1" class="xfigimg" src="/files/ftp_upload/55710/55710fig1.jpg" /> 图1:实验气室设置示意图。 （ A ）养殖水箱（22（长）×10.5（宽）×10.5厘米（高））（ B ）网底24孔板（12.8×8.6×0.7×1.5厘米孔径）（ C ）尼龙筛网直径为0.1-0.2毫米（ D ）10（长）×5毫米（直径）的隧道（ E ）适当直径和30毫米长（ F ）硅胶管（ G ）气罐的特氟纶或PVC棒所需的氧/ CO 2混合物（ H ）分配阀（ I ）气阀（ J ）光盘扩散器（ K ）罐盖。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55710/55710fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" class="xfigimg" src="/files/ftp_upload/55710/55710fig2.jpg" /> 图2:实验气室设置的图像。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55710/55710fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="表格1" src="/files/ftp_upload/55710/55710table1.jpg" /> 表1: 气室中不同培养时间 的 氧浓度 测量 。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55710/55710table1large.jpg" target="_blank">请点击这里查看此f的较大版本</a><html> igure。 <img alt="图3" class="xfigimg" src="/files/ftp_upload/55710/55710fig3.jpg" /> 图3:HIF-1α水平在非洲爪蟾胚胎及其视网膜中的低氧诱导水平增加。来自全胚胎（ A ）或分离的胚胎视网膜（ B ）的蛋白质裂解物的蛋白质印迹保持在正常氧浓度或缺氧条件下。探针用于HIF-1α亚基和α-微管蛋白。对于每个条件（ n = 5）至少5个胚胎或22个视网膜（ C，D ）分别在（ A，B ）中进行的Western印迹的定量。蛋白质水平的HIF-1α标准化的蛋白质水平的α-微管蛋白。误差条代表实验之间的标准偏差。 * p值&lt;0.05，*** p值&lt;0.001; n = 5。d / 55710 / 55710fig3large.jpg"target ="_ blank"&gt;请点击此处查看此图的较大版本。 <img alt="图4" class="xfigimg" src="/files/ftp_upload/55710/55710fig4.jpg" /> 图4:缺氧会降低青光眼的视网膜干细胞生殖细胞中祖细胞的增殖。 （ A ）通过38阶段非洲爪蟾视网膜的横截面示意图，表明在来自38个阶段的DAPI染色的视网膜中2小时EdU脉冲后测量的睫状边缘区（CMZ）（ B ， C ）EdU掺入的位置青蛙胚胎保持在常氧（ B ）或缺氧室（ C ）内。品红= DAPI染色;灰色= EdU染色。刻度棒=50μm（ D ）在B和C中进行的实验的定量。误差条表示标准偏差。 *** <em&gt; p-value &lt;0.001; n = 7。对于每个条件，定量10-15视网膜（ G ）在缺氧后不同时间（x轴上以分钟表示的时间）内从动物视网膜中加入1小时EdU后，定量EdU阳性细胞。误差条代表两个独立实验的标准偏差（ n = 2）。对于每个条件和时间点，至少定量10个视网膜。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55710/55710fig4large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图5" class="xfigimg" src="/files/ftp_upload/55710/55710fig5.jpg" /> 图5:缺氧降低4 dpf斑马鱼眼视网膜干细胞中祖细胞的增殖。在来自4 dpf老的WTe斑马鱼胚胎的DAPI染色的视网膜中经过2小时EdU脉冲后测量的EdU掺入量保持不变（ A ）或缺氧室（ B ）。 洋红色:DAPI染色;灰色:EdU染色。刻度棒=50μm（ C ）在B和C中进行的实验的定量。误差条表示标准偏差。 *** p值&lt;0.001; n = 7。对于每个条件，定量10-15个视网膜。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55710/55710fig5large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="补充图1" src="/files/ftp_upload/55710/55710S1.jpg" /> 补充图1:HIF-1α水平在板的不同孔中和不同实验之间的低氧诱导下增加。来自整个非洲爪蟾胚胎的蛋白质裂解物的蛋白质印迹保持在正常氧浓度或在实验1（ A ）或实验2（ B ）中，在缺氧诱导下2小时，在缺氧室的两个不同孔中。探针用于HIF-1α亚基和α-微管蛋白。每个条件采集了第38期的5只青蛙胚胎。实验1和2是独立的生物复制（ C，D ）分别在（ A，B ）中进行的蛋白质印迹的定量。蛋白质水平的HIF-1α标准化的蛋白质水平的α-微管蛋白。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55710/55710S1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about Induction of Hypoxia in Living Frog and Zebrafish Embryos at JoVE.com

Induction of Hypoxia in Living Frog and Zebrafish Embryos

我们引进了一种新颖的低氧室系统，用于水生生物，如青蛙和斑马鱼胚胎。我们的系统简单，健壮，经济实惠，可以在体内诱导和维持缺氧，长达48小时。我们提供2种可重复的方法来监测缺氧的有效性。

诱导生活青蛙和斑马鱼胚胎缺氧

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and ...

induction-of-hypoxia-in-living-frog-and-zebrafish-embryos

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

9.7K 次观看.  University of Cambridge. 我们引进了一种新颖的低氧室系统，用于水生生物，如青蛙和斑马鱼胚胎。我们的系统简单，健壮，经济实惠，可以在体内诱导和维持缺氧，长达48小时。我们提供2种可重复的方法来监测缺氧的有效性。 

视频: Induction of Hypoxia in Living Frog and Zebrafish Embryos

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion (FDAP)

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological systems. In FDAP experiments, the clearance of proteins within living organisms can be measured. A protein of interest is tagged with a photoconvertible fluorescent protein, expressed in vivo and photoconverted, and the decrease in the photoconverted signal over time is monitored. The data is then fitted with an appropriate clearance model to determine the protein half-life. Importantly, the clearance kinetics of protein populations in different compartments of the organism can be examined separately by applying compartmental masks. This approach has been used to determine the intra- and extracellular half-lives of secreted signaling proteins during zebrafish development. Here, we describe a protocol for FDAP experiments in zebrafish embryos. It should be possible to use FDAP to determine the clearance kinetics of any taggable protein in any optically accessible organism.

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion FDAP

Protein levels in cells and tissues are often tightly regulated by the balance of protein production and clearance. Using Fluorescence Decay After Photoconversion (FDAP), the clearance kinetics of proteins can be experimentally measured in vivo.

测量生活中的斑马鱼胚胎蛋白质稳定性使用荧光衰减光转化后（FDAP）

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological ...

科研

发育生物学

Mechanical Vessel Injury in Zebrafish Embryos

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes. External development and optical transparency make these embryos especially amenable to microscopy, and numerous transgenic lines that label specific cell types with fluorescent proteins are available, making the zebrafish embryo an ideal system for visualizing the interaction of vascular, hematopoietic, and other cell types during injury and repair in vivo. Forward and reverse genetics in zebrafish are well developed, and pharmacological manipulation is possible. We describe a mechanical vascular injury model using micromanipulation techniques that exploits several of these features to study responses to vascular injury including hemostasis and blood vessel repair. Using a combination of video and timelapse microscopy, we demonstrate that this method of vascular injury results in measurable and reproducible responses during hemostasis and wound repair. This method provides a system for studying vascular injury and repair in detail in a whole animal model.

This article describes a method for creating a mechanical vessel injury in zebrafish embryos. This injury model provides a platform for studying hemostasis, injury-related inflammation, and wound healing in an organism ideally suited for real-time microscopy.

在斑马鱼胚胎的机械血管损伤

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes.  External development ...

Imaging Subcellular Structures in the Living Zebrafish Embryo

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

Imaging the dynamic behavior of organelles and other subcellular structures in vivo can shed light on their function in physiological and disease conditions. Here, we present methods for genetically tagging two organelles, centrosomes and mitochondria, and imaging their dynamics in living zebrafish embryos using wide-field and confocal microscopy.

在活斑马鱼胚胎成像的亚细胞结构

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such ...

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

分离和斑马鱼胚胎单细胞的表征

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Affinity Labeling Detection of Endogenous Receptors from Zebrafish Embryos

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish embryos has been implemented. This technique takes advantage of the selectivity and sensitivity conferred by affinity labeling of a given receptor by its ligand with the specificity of the immunoprecipitation. We have used AFLIP to detect the type III TGF-&#946; receptor (TGFBR3), also know as betaglycan, during early zebrafish development. AFLIP was instrumental in validating the efficacy of a TGFBR3 morphant zebrafish phenotype. In the first step, embryo protein extracts are prepared and used to generate 125I-TGF-&#946;2-TGFBR3 complexes that are purified by immunoprecipitation. Later, these complexes are covalently cross-linked and revealed using SDS-PAGE separation and autoradiography detection. This technique requires the availability of a labeled ligand for, and a specific antibody against, the receptor to be detected, and shall be easily adapted to identify any growth factor or cytokine receptor that meets these requirements.

A novel technique for the detection of low abundance endogenous receptors present in zebrafish embryos is described. We have named it AFLIP because it consists of affinity labeling of the receptor by its ligand linked to immunoprecipitation.

从斑马鱼胚胎内源性受体亲和标记检测

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish ...

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the cells are located. Specifically, they have been developed to analyze the electrical activity of excitable cells, such as spinal neurons. In such a scenario, it is crucial to preserve the integrity of the spinal cell, but it is also important to preserve the anatomy and physiological shape of the systems involved. Indeed, the comprehension of the manner in which the nervous system-and other complex systems-works must be based on a systemic approach. For this reason, the live zebrafish embryo was chosen as a model system, and the spinal neuron membrane voltage changes were evaluated without interfering with the physiological conditions of the embryos. 
Here, an approach combining the employment of zebrafish embryos with a FRET-based biosensor is described. Zebrafish embryos are characterized by a very simplified nervous system and are particularly suited for imaging applications thanks to their transparency, allowing for the employment of fluorescence-based voltage indicators at the plasma membrane during zebrafish development. The synergy between these two components makes it possible to analyze the electrical activity of the cells in intact living organisms, without perturbing the physiological state. Finally, this non-invasive approach can co-exist with other analyses (e.g., spontaneous movement recordings, as shown here).

Protocols described here allow for the study of the electrical properties of excitable cells in the most non-invasive physiological conditions by employing zebrafish embryos in an in vivo system together with a fluorescence resonance energy transfer (FRET)-based genetically encoded voltage indicator (GEVI) selectively expressed in the cell type of interest.

生物传感运动神经元膜电位在活斑马鱼胚胎

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the ...

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

The red flour beetle Tribolium castaneum has become an important insect model organism in developmental genetics and evolutionary developmental biology. The observation of Tribolium embryos with light sheet-based fluorescence microscopy has multiple advantages over conventional widefield and confocal fluorescence microscopy. Due to the unique properties of a light sheet-based microscope, three dimensional images of living specimens can be recorded with high signal-to-noise ratios and significantly reduced photo-bleaching as well as photo-toxicity along multiple directions over periods that last several days. With more than four years of methodological development and a continuous increase of data, the time seems appropriate to establish standard operating procedures for the usage of light sheet technology in the Tribolium community as well as in the insect community at large. This protocol describes three mounting techniques suitable for different purposes, presents two novel custom-made transgenic Tribolium lines appropriate for long-term live imaging, suggests five fluorescent dyes to label intracellular structures of fixed embryos and provides information on data post-processing for the timely evaluation of the recorded data. Representative results concentrate on long-term live imaging, optical sectioning and the observation of the same embryo along multiple directions. The respective datasets are provided as a downloadable resource. Finally, the protocol discusses quality controls for live imaging assays, current limitations and the applicability of the outlined procedures to other insect species.
This protocol is primarily intended for developmental biologists who seek imaging solutions that outperform standard laboratory equipment. It promotes the continuous attempt to close the gap between the technically orientated laboratories/communities, which develop and refine microscopy methodologically, and the life science laboratories/communities, which require 'plug-and-play' solutions to technical challenges. Furthermore, it supports an axiomatic approach that moves the biological questions into the center of attention.

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

Imaging the morphogenesis of insect embryos with light sheet-based fluorescence microscopy has become state of the art. This protocol outlines and compares three mounting techniques appropriate for Tribolium castaneum embryos, introduces two novel custom-made transgenic lines well suited for live imaging, discusses essential quality controls and indicates current experimental limitations.

光片为主的生活的荧光显微镜或固定和染色<em&gt;赤拟谷盗</em&gt;胚胎

The red flour beetle Tribolium castaneum has become an important insect model organism in developmental genetics and evolutionary developmental biology. ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

活完好斑马鱼的单细胞 Photoconversion

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling processes of excitable neuronal and muscular cells and many other biological processes, such as embryonic developmental patterning. However, there is a need for in vivo electrical activity monitoring in vertebrate embryogenesis. The advances of genetically encoded fluorescent voltage indicators (GEVIs) have made it possible to provide a solution for this challenge. Here, we describe how to create a transgenic voltage indicator zebrafish using the established voltage indicator, ASAP1 (Accelerated Sensor of Action Potentials 1), as an example. The Tol2 kit and a ubiquitous zebrafish promoter, ubi, were chosen in this study. We also explain&#160;the processes of Gateway site-specific cloning, Tol2 transposon-based zebrafish transgenesis, and the imaging process for early-stage fish embryos and fish tumors using regular epifluorescent microscopes. Using this fish line, we found that there are cellular electric voltage changes during zebrafish embryogenesis, and fish larval movement. Furthermore, it was observed that in a few zebrafish malignant peripheral nerve sheath tumors, the tumor cells were generally polarized compared to the surrounding normal tissues.

Here, we show the process of creating a cellular electric voltage reporter zebrafish line to visualize embryonic development, movement, and fish tumor cells in vivo.

斑马鱼早期胚胎和肿瘤细胞电活动的可视化研究

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...

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. ...