Name: induction of hypoxia in living frog and zebrafish embryos
Uploaded: 2017-06-26T14:00:00.000+00:00
Duration: 8 min 1 s
Description: Watch this Scientific Journal Video about Induction of Hypoxia in Living Frog and Zebrafish Embryos at JoVE.com

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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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=Dissection,+Culture,+and+Analysis+of+Xenopus+laevis+Embryonic+Retinal+Tissue.">Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue.</a> JoVE. (70), (2012).</li></ol>

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><tbody><tr><td>Sodium chloride</td><td>Sigma</td><td>S7653</td><td>NaCl/0.1x MBS, Embryo medium, 10x TBST</td></tr><tr><td>Potassium chloride</td><td>Sigma</td><td>P9333</td><td>KCl/0.1x MBS, Embryo mediu,</td></tr><tr><td>Sodium bicarbonate</td><td>Sigma</td><td>S5761</td><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;/0.1x MBS</td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375</td><td>0.1x MBS</td></tr><tr><td>Magnesium sulfate</td><td>Sigma</td><td>M7506</td><td>MgSO&lt;sub&gt;4&lt;/sub&gt;/0.1x MBS, Embryo medium</td></tr><tr><td>Calcium nitrate</td><td>Sigma</td><td>202967</td><td>Ca(NO&lt;sub&gt;3&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;/0.1x MBS</td></tr><tr><td>Calcium chloride</td><td>Sigma</td><td>C1016</td><td>CaCl&lt;sub&gt;2&lt;/sub&gt;/0.1x MBS, Embryo medium</td></tr><tr><td>Methylene blue</td><td>Sigma</td><td>M9140</td><td>Embryo medium</td></tr><tr><td>Pregnant mare serum gonadotropin (PMSG)</td><td>Sigma</td><td>CG10</td><td>Frog fertilization</td></tr><tr><td>Zebrafish breeding tank</td><td>Carolina</td><td>161937</td><td>Gas chamber construction</td></tr><tr><td>24-well plate</td><td>Thermo Scientific</td><td>142475</td><td>Nunclon Delta Surface, for gas chamber construction</td></tr><tr><td>Epoxy resin</td><td>RS Components UK</td><td>Kit 199-1468</td><td></td></tr><tr><td>Gas distributor valve</td><td>WPI Luer Valves</td><td>Kit 14011</td><td>Aquatic tank attachment (Schema 1, &lt;strong&gt;H&lt;/strong&gt;)</td></tr><tr><td>High precision gas valve</td><td>BOC&amp;nbsp;</td><td>200 bar HiQ C106X/2B</td><td>Gas tank attachment (Schema 1, &lt;strong&gt;I&lt;/strong&gt;)</td></tr><tr><td>5% oxygen and 95% N&lt;sub&gt;2&lt;/sub&gt; gas tank</td><td>BOC</td><td>226686-L</td><td>Hypoxic gas mixture</td></tr><tr><td>ceramic disc diffuser</td><td>CO&lt;sub&gt;2&lt;/sub&gt; Art&amp;nbsp;</td><td>Glass CO2 Nano Aquarium Diffuser, DG005DG005</td><td>Schema 1, &lt;strong&gt;J&lt;/strong&gt;</td></tr><tr><td>silicone grease</td><td>Scientific Laboratory Supplies</td><td>VAC1100</td><td>Schema 1, &lt;strong&gt;K&lt;/strong&gt;</td></tr><tr><td>oxymeter</td><td>Oxford Optronix&amp;nbsp;</td><td>Oxylite, CP/022/001</td><td>Hypoxic chamber setup</td></tr><tr><td>fibre-optic dissolved oxygen sensor</td><td>Oxford Optronix</td><td>HL_BF/OT/E</td><td>Hypoxic chamber setup</td></tr><tr><td>plastic pasteur pipette</td><td>Sterilin</td><td>STS3855604D</td><td>For embryo transfer</td></tr><tr><td>MS222&amp;nbsp;</td><td>Sigma Aldrich</td><td>E10521-50G</td><td>Embryo anesthetic</td></tr><tr><td>RIPA buffer&amp;nbsp;</td><td>Sigma</td><td>R0278-50ML</td><td>Tissue homogenization</td></tr><tr><td>Protease inhibitor</td><td>Sigma</td><td>P8340</td><td>Tissue homogenization</td></tr><tr><td>Tris</td><td>Sigma</td><td>77-86-1</td><td>4x Laemmli loading buffer, 10x TBST</td></tr><tr><td>Glycerol</td><td>Sigma</td><td>G5516</td><td>4x Laemmli loading buffer</td></tr><tr><td>Sodium Dodecyl Sulfate (SDS)</td><td>Sigma</td><td>L3771</td><td>4x Laemmli loading buffer, 5x Running buffer</td></tr><tr><td>beta-Mercaptoethanol&amp;nbsp;</td><td>Sigma</td><td>M6250</td><td>4x Laemmli loading buffer</td></tr><tr><td>Bromophenol Blue</td><td>Sigma-Aldrich</td><td>B0126</td><td>4x Laemmli loading buffer</td></tr><tr><td>Trizma base&amp;nbsp;</td><td>Sigma</td><td>77-86-1</td><td>5x Running buffer, Transfer buffer</td></tr><tr><td>Glycine</td><td>Sigma</td><td>G8898</td><td>5x Running buffer, Transfer buffer</td></tr><tr><td>Methanol</td><td>Sigma</td><td>34860</td><td>Transfer buffer</td></tr><tr><td>Tween 20</td><td>Sigma</td><td>P2287-500ML</td><td>10x TBST</td></tr><tr><td>skim milk powder</td><td>Sigma</td><td>70166</td><td>Blocking Solution</td></tr><tr><td>Eppendorf microcentrifuge tube</td><td>Sigma</td><td>T9661</td><td></td></tr><tr><td>tissue homogenizer</td><td>Pellet Pestle Motor Kontes</td><td>Z359971</td><td>Tissue homogenization</td></tr><tr><td>pellet pestles</td><td>Sigma</td><td>Z359947-100EA</td><td>Tissue homogenization</td></tr><tr><td>precast 12% gel</td><td>Biorad</td><td>Mini-ProteinTGX, 456-1043</td><td>Western Blot</td></tr><tr><td>protein ladder</td><td>Amersham</td><td>Full-Range Rainbow ladder, RPN800E</td><td>Western Blot</td></tr><tr><td>nitrocellulose membrane (0.45 &amp;micro;m)</td><td>Biorad</td><td>162-0115</td><td>Western Blot</td></tr><tr><td>anti-HIF-1&amp;alpha; antibody</td><td>Abcam</td><td>ab2185</td><td>Western Blot</td></tr><tr><td>anti-&amp;alpha;-tubulin antibody</td><td>Sigma</td><td>T6074</td><td>Western Blot</td></tr><tr><td>goat anti-rabbit antibody</td><td>Abcam</td><td>ab6789</td><td>Western Blot</td></tr><tr><td>goat anti-mouse antibody</td><td>Abcam</td><td>ab97080</td><td>Western Blot</td></tr><tr><td>Pierce ECL 2 reagent&amp;nbsp;</td><td>Thermo Scientific</td><td>80196</td><td>Western Blot</td></tr><tr><td>ECL films Hyperfilm</td><td>GE Healthcare Amersham</td><td>28906837</td><td>Western Blot</td></tr><tr><td>5-Ethynyl-2&amp;prime;-deoxyuridine &amp;nbsp;</td><td>santa cruz</td><td>CAS&amp;nbsp;61135-33-9</td><td>EdU, EdU incorporation</td></tr><tr><td>Phosphate-buffered Saline (PBS)</td><td>Oxoid</td><td>BR0014G</td><td>1x</td></tr><tr><td>Formaldehyde</td><td>Thermo Scientific</td><td>28908</td><td>Fixation solution</td></tr><tr><td>Sucrose</td><td>Fluka</td><td>S/8600/60</td><td>Solution solution</td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T9284-500ML</td><td>PBST</td></tr><tr><td>Heat-inactivated Goat Serum (HiGS)</td><td>Sigma</td><td>G6767-100ml</td><td>Blocking solution (EdU incorporation)</td></tr><tr><td>4&#039;,6-diamidino-2-phenylindole&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>D1306</td><td>DAPI, EdU incorporation</td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Molecular Probes</td><td>C10338</td><td>EdU incorporation</td></tr><tr><td>glass vial</td><td>VWR</td><td>98178853</td><td>EdU incorporation analysis</td></tr><tr><td>Tissue-Plus optimal cutting temperature compound&amp;nbsp;</td><td>Scigen</td><td>4563</td><td>Embedding medium, EdU incorporation analysis</td></tr><tr><td>cryostat Jung Fridgocut 2800E</td><td>Leica&amp;nbsp;</td><td>CM3035S</td><td>EdU incorporation analysis</td></tr><tr><td>microscope slides Super-Frost plus Menzel glass</td><td>Thermo Scientific</td><td>J1800AMNZ</td><td>EdU incorporation analysis</td></tr><tr><td>EdU Click-iT chemistry kit</td><td>Molecular Probes</td><td>C10338</td><td>EdU incorporation analysis</td></tr><tr><td>FluorSave</td><td>Calbiochem</td><td>D00170200</td><td>Mounting medium, EdU incorporation analysis</td></tr><tr><td>coverslips</td><td>VWR</td><td>ECN631-1575</td><td>EdU incorporation analysis</td></tr><tr><td>fluorescent microscope</td><td>Nikon</td><td>Eclipse 80i</td><td>EdU incorporation analysis</td></tr><tr><td>confocal scanning microscope</td><td>Olympus</td><td>Fluoview FV1000</td><td>EdU incorporation analysis</td></tr><tr><td>Volocity software</td><td>PerkinElmer</td><td>Volocity 6.3</td><td>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，?...

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

Universidad San Sebastian

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

Microbead Implantation in the Zebrafish Embryo

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic conservation, as well as similarities in cellular, molecular, and physiological processes, with other vertebrates including humans. During early ontogeny, zebrafish embryos are optically transparent, allowing researchers to visualize the dynamics of organogenesis using a simple stereomicroscope. Microbead implantation is a method that enables tissue manipulation through the alteration of factors in local environments. This allows researchers to assay the effects of any number of signaling molecules of interest, such as secreted peptides, at specific spatial and temporal points within the developing embryo. Here, we detail a protocol for how to manipulate and implant beads during early zebrafish development.

The zebrafish is an excellent model system for genetic and developmental studies. Bead implantation is a valuable tissue manipulation technique that can be used to interrogate developmental mechanisms by introducing alterations in local cellular environments. This protocol describes how to perform microbead implantation in the zebrafish embryo.

微珠植入斑马鱼胚胎

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic ...

科研

发育生物学

Visualizing the Developing Brain in Living Zebrafish using Brainbow and Time-lapse Confocal Imaging

Development of the vertebrate nervous system requires a precise coordination of complex cellular behaviors and interactions. The use of high resolution in vivo imaging techniques can provide a clear window into these processes in the living organism. For example, dividing cells and their progeny can be followed in real time as the nervous system forms. In recent years, technical advances in multicolor techniques have expanded the types of questions that can be investigated. The multicolor Brainbow approach can be used to not only distinguish among like cells, but also to color-code multiple different clones of related cells that each derive from one progenitor cell. This allows for a multiplex lineage analysis of many different clones and their behaviors simultaneously during development. Here we describe a technique for using time-lapse confocal microscopy to visualize large numbers of multicolor Brainbow-labeled cells over real time within the developing zebrafish nervous system. This is particularly useful for following cellular interactions among like cells, which are difficult to label differentially using traditional promoter-driven colors. Our approach can be used for tracking lineage relationships among multiple different clones simultaneously. The large datasets generated using this technique provide rich information that can be compared quantitatively across genetic or pharmacological manipulations. Ultimately the results generated can help to answer systematic questions about how the nervous system develops.

In vivo imaging is a powerful tool that can be used to investigate the cellular mechanisms underlying nervous system development. Here we describe a technique for using time-lapse confocal microscopy to visualize large numbers of multicolor Brainbow-labeled cells in real time within the developing zebrafish nervous system.

使用"彩虹"和"延时共聚焦成像"可视化活斑马鱼中发育中的脑

Development of the vertebrate nervous system requires a precise coordination of complex cellular behaviors and interactions. The use of high resolution in ...

Two-Photon-Based Photoactivation in Live Zebrafish Embryos

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic development, cellular signaling and adult physiology. In this respect, the use of multi-photon microscopy enables quantitative photoactivation of a given light responsive agent in deep tissues at a single cell resolution. As zebrafish embryos are optically transparent, their development can be monitored in vivo. These traits make the zebrafish a perfect model organism for controlling the activity of a variety of chemical agents and proteins by focused light. Here we describe the use of two-photon microscopy to induce the activation of chemically caged fluorescein, which in turn allows us to follow cell's destiny in live zebrafish embryos. We use embryos expressing a live genetic landmark (GFP) to locate and precisely target any cells of interest. This procedure can be similarly used for precise light induced activation of proteins, hormones, small molecules and other caged compounds.

Multiphoton microscopy allows control of low energy photons with deep optical penetration and reduced phototoxicity. We describe the use of this technology for live cell labeling in zebrafish embryos. This protocol can be readily adapted for photo-induction of various light-responsive molecules.

在现场的斑马鱼胚胎的基于双光子Photoactivati​​on

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic ...

生物学

研究斑马鱼光遗传学信号转导操作的发育

Optogenetic Signaling Activation in Zebrafish Embryos

Signaling pathways orchestrate fundamental biological processes, including development, regeneration, homeostasis, and disease. Methods to experimentally manipulate signaling are required to understand how signaling is interpreted in these wide-ranging contexts. Molecular optogenetic&#160;tools can provide reversible, tunable manipulations of signaling pathway activity with a high degree of spatiotemporal control and have been applied in vitro, ex vivo, and in vivo. These tools couple light-responsive protein domains, such as the blue light homodimerizing light-oxygen-voltage sensing (LOV) domain, with signaling effectors to confer light-dependent experimental control over signaling. This protocol provides practical guidelines for using the LOV-based bone morphogenetic protein (BMP) and Nodal signaling activators bOpto-BMP and bOpto-Nodal in the optically accessible early zebrafish embryo. It describes two control experiments: A quick phenotype assay to determine appropriate experimental conditions, and an immunofluorescence assay to directly assess signaling. Together, these control experiments can help establish a pipeline for using optogenetic tools in early zebrafish embryos. These strategies provide a powerful platform to investigate the roles of signaling in development, health, and physiology.

作者聚焦：操纵斑马鱼胚胎中的信号转导以解码细胞命运决定 

Optogenetic manipulation of signaling pathways can be a powerful strategy to investigate how signaling is decoded in development, regeneration, homeostasis, and disease. This protocol provides practical guidelines for using light-oxygen-voltage sensing domain-based Nodal and bone morphogenic protein (BMP) signaling activators in the early zebrafish embryo.

斑马鱼胚胎中的光遗传学信号激活

Signaling pathways orchestrate fundamental biological processes, including development, regeneration, homeostasis, and disease. Methods to experimentally ...

Live Imaging of Cell Extrusion from the Epidermis of Developing Zebrafish

Homeostatic maintenance of epithelial tissues requires the continual removal of damaged cells without disrupting barrier function. Our studies have found that dying cells send signals to their live neighbors to form and contract a ring of actin and myosin that ejects it out from the epithelial sheet while closing any gaps that might have resulted from its exit, a process termed cell extrusion1. The optical clarity of developing zebrafish provides an excellent system to visualize extrusion in living epithelia. Here we describe a method to induce and image extrusion in the larval zebrafish epidermis. To visualize extrusion, we inject a red fluorescent protein labeled probe for F-actin into one-cell stage transgenic zebrafish embryos expressing green fluorescent protein in the epidermis and induce apoptosis by addition of G418 to larvae. We then use time-lapse imaging on a spinning disc confocal microscope to observe actin dynamics and epithelial cell behaviors during the process of apoptotic cell extrusion. This approach allows us to investigate the extrusion process in live epithelia and will provide an avenue to study disease states caused by the failure to eliminate apoptotic cells.

Dying cells are extruded from epithelial tissues by concerted contraction of neighboring cells without disrupting barrier function. The optical clarity of developing zebrafish provides an excellent system to visualize extrusion in living epithelia. Here we describe methods to induce and image extrusion in the larval zebrafish epidermis at cellular resolution.

发展中国家斑马鱼的表皮细胞挤压实时成像

Homeostatic maintenance of epithelial tissues requires the continual removal of damaged cells without disrupting barrier function.  Our studies have found ...

Triggering Cell Stress and Death Using Conventional UV Laser Confocal Microscopy

Using a standard confocal setup, a UV ablation method can be utilized to selectively induce cellular injury and to visualize single-cell responses and cell-cell interactions in the CNS in real-time. Previously, studying these cell-specific responses after injury often required complicated setups or the transfer of cells or animals into different, non-physiological environments, confounding immediate and short-term analysis. For example, drug-mediated ablation approaches often lack the specificity that is required to study single-cell responses and immediate cell-cell interactions. Similarly, while high-power pulsed laser ablation approaches provide very good control and tissue penetration, they require specialized equipment that can complicate real-time visualization of cellular responses. The refined UV laser ablation approach described here allows researchers to stress or kill an individual cell in a dose- and time-dependent manner using a conventional confocal microscope equipped with a 405-nm laser. The method was applied to selectively ablate a single neuron within a dense network of surrounding cells in the zebrafish spinal cord. This approach revealed a dose-dependent response of the ablated neurons, causing the fragmentation of cellular bodies and anterograde degeneration along the axon within minutes to hours. This method allows researchers to study the fate of an individual dying cell and, importantly, the instant response of cells-such as microglia and astrocytes-surrounding the ablation site.

Targeted manipulations to cause directed stress or death in individual cells have been relatively difficult to accomplish. Here, a single-cell-resolution ablation approach to selectively stress and kill individual cells in cell culture and living animals is described based on a standard confocal UV laser.

触发细胞应激和死亡使用传统的紫外线激光共聚焦显微镜

Using a standard confocal setup, a UV ablation method can be utilized to selectively induce cellular injury and to visualize single-cell responses and ...

神经科学

Metabolic Profile Analysis of Zebrafish Embryos

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these relationships will help to understand the underlying etiology for a range of diseases linked with mitochondrial dysfunction, such as diabetes and obesity. Recent advances in instrumentation, has enabled the monitoring of distinct parameters of mitochondrial function in cell lines or tissue explants. Here we present a method for a rapid and sensitive analysis of mitochondrial function parameters in vivo during zebrafish embryonic development using the Seahorse bioscience XF 24 extracellular flux analyser. This protocol utilizes the Islet Capture microplates where a single embryo is placed in each well, allowing measurement of bioenergetics, including: (i) basal respiration; (ii) basal mitochondrial respiration (iii) mitochondrial respiration due to ATP turnover; (iv) mitochondrial uncoupled respiration or proton leak and (iv) maximum respiration. Using this approach embryonic zebrafish respiration parameters can be compared between wild type and genetically altered embryos (mutant, gene over-expression or gene knockdown) or those manipulated pharmacologically. It is anticipated that dissemination of this protocol will provide researchers with new tools to analyse the genetic basis of metabolic disorders in vivo in this relevant vertebrate animal model.

Zebrafish represent a powerful vertebrate model that has been under-utilised for metabolic studies. Here we describe a rapid way to measure the in vivo metabolic profile of developing zebrafish that allows the comparison of different mitochondrial function parameters between genetically or pharmacologically manipulated embryos, thereby increasing the applicability of this organism.

斑马鱼胚胎的代谢谱分析

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these ...

Imaging and 3D Reconstruction of Cerebrovascular Structures in Embryonic Zebrafish

Zebrafish are a powerful tool to study developmental biology and pathology in vivo. The small size and relative transparency of zebrafish embryos make them particularly useful for the visual examination of processes such as heart and vascular development. In several recent studies transgenic zebrafish that express EGFP in vascular endothelial cells were used to image and analyze complex vascular networks in the brain and retina, using confocal microscopy. Descriptions are provided to prepare, treat and image zebrafish embryos that express enhanced green fluorescent protein (EGFP), and then generate comprehensive 3D renderings of the cerebrovascular system. Protocols include the treatment of embryos, confocal imaging, and fixation protocols that preserve EGFP fluorescence. Further, useful tips on obtaining high-quality images of cerebrovascular structures, such as removal the eye without damaging nearby neural tissue are provided. Potential pitfalls with confocal imaging are discussed, along with the steps necessary to generate 3D reconstructions from confocal image stacks using freely available open source software.

Imaging of cerebrovascular development in larval zebrafish is described. Techniques to facilitate 3D imaging and modify cerebrovascular development using chemical treatments are also provided.

成像和胚胎斑马鱼脑血管结构的三维重建

Zebrafish are a powerful tool to study developmental biology and pathology in vivo.  The small size and relative transparency of zebrafish embryos make ...

诱导生活青蛙和斑马鱼胚胎缺氧

摘要

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此视频中的章节

微珠植入斑马鱼胚胎

使用"彩虹"和"延时共聚焦成像"可视化活斑马鱼中发育中的脑

在现场的斑马鱼胚胎的基于双光子Photoactivation

斑马鱼胚胎中的光遗传学信号激活

斑马鱼胚胎中的光遗传学信号激活

斑马鱼胚胎中的光遗传学信号激活

发展中国家斑马鱼的表皮细胞挤压实时成像

触发细胞应激和死亡使用传统的紫外线激光共聚焦显微镜

斑马鱼胚胎的代谢谱分析

成像和胚胎斑马鱼脑血管结构的三维重建

诱导生活青蛙和斑马鱼胚胎缺氧

摘要

探索更多视频

此视频中的章节

微珠植入斑马鱼胚胎

使用"彩虹"和"延时共聚焦成像"可视化活斑马鱼中发育中的脑

在现场的斑马鱼胚胎的基于双光子Photoactivati​​on

斑马鱼胚胎中的光遗传学信号激活

斑马鱼胚胎中的光遗传学信号激活

斑马鱼胚胎中的光遗传学信号激活

发展中国家斑马鱼的表皮细胞挤压实时成像

触发细胞应激和死亡使用传统的紫外线激光共聚焦显微镜

斑马鱼胚胎的代谢谱分析

成像和胚胎斑马鱼脑血管结构的三维重建

在现场的斑马鱼胚胎的基于双光子Photoactivation