Support in Massimo Santoro lab come from HFSP, Marie Curie Action, Telethon and AIRC. We thank Dafne Gays and Emiliano Panieri for critical reading of the manuscript.

仪器和工作方案1。准备<ol><li>制备的鱼水溶液。使原液通过将2克加入50ml蒸馏水海盐“即时海洋&#39;的。加入1.5毫升储鱼水至1升蒸馏水中，制备准备用鱼水（60微克/毫升海盐最终浓度）。高压灭菌的准备使用前用鱼水。此溶液用作斑马鱼胚胎培养基中。 </li><li>准备甲基纤维素胚胎安装。溶解1.5克甲基纤维素在50毫升无菌水中的鱼。通过使用磁铁上的搅拌盘促进溶解。粉末完全溶解，可能需要几个小时。检查的清晰度和分装成小管的解决方案。储存在-20°C为个月，解冻分装于使用。使用之前离心甲基纤维素在950×g离心5分钟。避免等分的冻融循环。 </li><li>准备加入50ml三卡因/乙酸乙酯3 - 氨基苯甲酸米乙磺酸盐（原液）中溶解200mg的三卡因在100毫升水中，用Tris-盐酸1 M（pH为9），调节pH值至7.0。存储此股票在4℃不超过30天。注意：三卡因是有毒的。使用按照适当的处理准则。 </li><li>诱导氧化应激在斑马鱼胚胎<ol><li>准备通用氧化应激诱导的氧化性溶液：请加入50ml氧化剂溶液中加入H 2 O 2贮备液（过氧化氢​​; 100毫米）鱼水。使用2毫米和100微米之间的最终浓度H 2 O 2。用法前不久准备这个解决方案。氧化剂溶液可以同时应用于整个安装ROS检测和单细胞的ROS检测方法。不要将本解决方案。注意：H 2 O 2是危险的，吸入有害，如果吞食。与可燃物料接触可能会引起火灾。处理在通风橱和穿戴适当的个人ONAL防护装备。 </li><li>准备线粒体源性氧化应激诱导的氧化性溶液：用2毫升二甲基亚砜（DMSO）溶解3.9毫克鱼藤酮制作氧化剂原液（5毫米）。保持该溶液于室温下在黑暗中。 <ol><li>鱼藤酮溶解原液〜10毫升水的鱼，使即用的解决方案。使用鱼藤酮在5-50微米之间的浓度存在。在浓度大于100微米高，不要使用鱼藤酮。以合适的浓度，这氧化剂溶液可以同时应用于整个安装ROS检测和单细胞的ROS检测方法。注意：鱼藤酮是有毒有害的。根据相应的提示语句处​​理。 </li></ol></li><li>通过基因诱导氧化应激击倒：敲倒在斑马鱼胚胎nrf2a基因表达的吗啉注射如先前报道Timme-Laragy A. 等 ，2012 38。 </li><li>诱发OXID组织损伤后ative应力：产生一个伤口边缘在72个高倍视野尾鳍斑马鱼胚胎作为先前由Niethammer 等 ，2009 39描述。 </li></ol></li><li>配制成5毫升的单细胞ROS的检测方法ROS检测的解决方案： <ol><li>解决方法一般ROS的检测：将通用的ROS敏感的分子探针在HBSS（汉克斯平衡盐溶液），以便准备工作溶液（浓度范围：2.5〜10微米）。该解决方案必须使用不久之前做好准备。 </li><li>溶液为诱导线粒体特异性检测ROS：不久，使用前溶解线粒体活性氧敏感的探针的DMSO 5mM储备溶液。溶解原液在HBSS中，以制备工作溶液（浓度范围：2.5〜10微米）。 注意：避免活性氧检测工作方案的光线和氧气接触及彼等各自的储备液。通过使用保护管的光铝箔。不要存放或溶解在HBSS再利用探针。存储储备溶液在-20℃下一个月。 小心：分子探针和DMSO在通风橱按照适当的指引。 </li></ol></li><li>制备5毫升整个安装ROS检测方法的ROS检测溶液：将通用的ROS敏感探针原液（稳定化的DMSO）的HBSS中以制备工作溶液（浓度范围：2.5-5微米）。避免光线和氧气接触。从光保护管。使用前准备这个解决方案。不要储存或再使用此解决方案。小心：在通风橱分子探针按照适当的指引。 </li><li>使25毫升终止液中加入10胎牛血清毫升到15的PBS 1X中的溶液。保持无菌的解决方案。该存储解决方案，在4°C。 ROS的检测方法 - 这种解决方案只适用于单细胞必需的。 </li><li>将空气中的斑马鱼在孵化28＆＃176; C。 </li><li>为单细胞的ROS检测方法中设置的离心机于4℃。 </li></ol>成人鱼类2。配套和选择斑马鱼的胚胎<ol><li>根据标准协议40建立成年斑马鱼对跨越。根据具体实验需要选择适当的转基因株系。 </li><li>收集鸡蛋，把它们摆放成90毫米的菜有鱼水/胚胎培养基。鸡蛋保持在28°C，直到胚胎会发展壮大到所需的发育阶段（ 例如 ，48 HPF，72 HPF; HPF：小时受精后）。 </li><li>屏幕胚胎发育。排除所有未受精的卵子或胚胎不发达。 </li><li>通过在50毫升水中的鱼加入1ml三卡因（原液）的麻醉胚胎。 </li><li>选择在立体显微镜下的Tg荧光的胚胎。 </li></ol>胚胎3。治疗氧化剂<ol><li>使用48小时之间至少有30胚胎pF和72％不同的条件HPF。为了消除三卡因与新鲜的鱼水冲洗两次胚胎。 </li><li>分裂荧光的胚胎分为三个菜。把不超过30个胚/皿。 </li><li>除去洗涤溶液并加入10 ml的氧化剂溶液或10毫升鱼水作为对照溶液。 </li><li>胚胎孵育10分钟至1小时，在28°C。潜伏期取决于由氧化应激来诱导在特定组织的水平。短期内是最好的，因为受氧化应激细胞逐渐发生细胞死亡。 </li><li>胚胎转移到含有HBSS和冲洗胚胎纷飞一道新菜。预暖的HBSS在28°C。 </li></ol> 4，全山ROS的检测方法<ol><li>氧化剂处理后收集的胚胎，并把不超过10个胚胎中的小管。冲洗用HBSS尽可能。使用铝箔光保护管。 </li><li>加入1 ml的活性氧检测解决方案每管。 </li><li>孵育在黑暗中15分钟胚胎在28℃，以避免光线照射。 </li><li>在孵化时间准备全胚胎分析载玻片上。把300μl的甲基纤维素上的“抑郁症”的载玻片并用移液管尖铺在玻璃表面上。避免气泡，同时释放所述甲基纤维素。 </li><li>在温育时间结束时，立即取出ROS-检测溶液中并用2ml的HBSS洗两次。通过倒转试管几次洗涤胚胎。不要吸管或旋涡。 </li><li>吸出胚胎成一个玻璃巴斯德吸管。附近的移液管的开口部，并轻轻位置胚胎喷射成的甲基纤维素。用细尼龙线适当定向的胚胎。 </li><li>比较控制胚胎处理的胚胎的荧光。可替换地，固定，使所有的胚胎都使用相同的成像的荧光立体显微镜或共焦显微镜的参数图像设置。 </li></ol> 5，单细胞ROS的检测方法<ol><li>启动步骤：3.5。请确保至少有35胚胎每个条件。 </li><li>胚胎转移到一个新的菜。除去鱼水尽可能。加入10毫升冰冷的PBS 1X和400微升的蛋白酶抑制剂鸡尾酒。手动dechorionate胚胎钳，取出蛋黄麻袋用细针。注意：删除黄麻袋，确保干净样品进行以下FACS分析。这一步可以根据FACS仪器加以避免。 </li><li>转移去yolked胚胎成24孔多孔板（15胚胎/孔），并删除所有的鱼水。 </li><li>添加300μl的HBSS中，30微升的胶原酶P，加入50μl胰蛋白酶-EDTA在每个孔中。 </li><li>通过轻轻地上下吹打均匀化的胚胎具有紧吸头（1,000微升）。 </li><li>孵育28℃20分钟，吹打，每5分钟均质样品。 </li><li>检查组织DISRuption通过观察匀浆在复式显微镜等分。吹打促进单细胞悬液。不要胚胎培养超过30分钟。 </li><li>停止反应，加入200微升终止液。轻轻吹打混匀。 </li><li>转移细胞悬液至预冷的管的紧下方。保持管上的冰和避免光线照射。 </li><li>离心5分钟，在250×g离心在4℃下</li><li>轻轻吹打去除上层相和重悬细胞用冰冷的HBSS。 </li><li>计数细胞，并确保你有细胞在所有的样本数相同。不使用小于2×10 6个细胞。 </li><li>离心细胞5分钟，在250×g离心在4℃下</li><li>除去上清液并暂停沉淀用1 ml冰冷的HBSS含有活性氧敏感的探头。 </li><li>孵育所述管在室温下在黑暗中3分钟。根据氧化应激的水​​平发生变化的温育时间和到吨探针的他的灵敏度。 </li><li>细胞转移到FACS管。保持管上的冰和避免FACS分析前曝光。 </li><li>排序细胞用荧光激活细胞分选术（FACS）。波长集合按照Tg的组织荧光（ 例如 ，GFP）和用于检测的ROS（ 例如 ，特定的线粒体活性氧敏感的探针，一般的活性氧敏感的探针）的分子探针的激发/发射光谱。 </li><li>对于每个条件，通过采用相同的设置，流式细胞仪测量所有的样品在相同的实验会话中。计算荧光细胞不同生物学重复的平均值的百分比。分析至少两个重复为每个条件。 </li></ol>

The authors have nothing to disclose.

关键步骤对氧化应激的检测在本文中所描述的斑马鱼胚胎的过程包括两个不同的方法。整个安装ROS检测方法主要是定性测定活性氧检测用，而单 ​​细胞ROS的检测方法允许更具体的定量测量（ 图1）。这两种方法提供了一种快速简便的方法来评估对斑马鱼胚胎的体内活性氧检测。然而，他们都提出了一些关键步骤。 关键步骤相关的方法我<...

氧化应激被专门定义为一个条件，结果从不平衡细胞的氧化还原状态。这通常发生在细胞内复杂的氧化还原反应确定细胞的氧化还原状态。氧化还原反应包括的所有化学反应，包括在电子生物分子产生减少和分子（ 即氧化还原反应）的氧化原子之间的转移。这些反应是通过电子方式激活产物（ 即亲氧化性物质），其特征在于，一个极端的结构不稳定性和其与邻近的生物分子交换不平衡的电子的自发活化催化。这些不规则的反应结果放入DNA损伤，蛋白羧化和脂质过氧化，最终导致细胞死亡1。氧化应激水平的增加已经与衰老和不同的病理状态2的进展相关。氧化应激有据报道，负责血管改变在糖尿病和心血管疾病3,4。它还在神经元变性阿尔茨海默病的关键作用和帕金森氏病5。另外，氧化应激已被证明是在管癌的进展和转移的事件6,7的一个关键因素。此外，炎症和免疫反应可能引发进一步的支持氧化应激8。 在活细胞中，亲氧物种是来自氧（ROS，活性氧物种）或氮（RNS;活性氮）。活性氧包括羟基自由基，超氧阴离子（OH）（O 2 - ）和过氧化氢 ​​（H 2 O 2）。主要RNS是一氧化二氮（NO。）可以通过自发的相互作用来产生betwee一系列继发反应物种的ÑROS和RNS或游离金属离子9。例如，超氧阴离子自由基与氮氧化物发生反应，形成peroxynitrate（ONOO - ），而H 2 O 2反应，以Fe 2 +产生的羟自由基。 ROS和RNS，由于其与几种生物分子发生反应的能力，被认为是生理氧化还原状态10维护一个危险的威胁。以保持氧化还原状态的细胞都配备了一系列解毒抗氧化剂分子和酶。超氧化物歧化酶（SOD），过氧化氢 ​​酶，谷胱甘肽过氧化物酶和Peroxiredoxins基本上构成了抗氧化酶法，阿森纳，从亲氧化物质提供细胞保护包括H 2 O 2，OH和过氧化亚硝酸盐- 11。还抗氧化分子，如维生素C和E，多酚和辅酶Q10（辅酶Q10）是至关重要的淬火ROS和他们的危险去衍生参数12,13。然而，过量生产的ROS和RNS，或者在抗氧化剂系统功能障碍，为转移细胞的氧化还原状态的朝向氧化应激14。 除了他们的贬义，活性氧可以在不同来源的细胞发挥不同的生理作用。正常细胞产生的ROS作为信号分子介导正常的生理活动，如宿主防御和修复创面15-17。反应性物质是响应信号因子，生长因子，和钙的水平18,19的胞内波动通常生产中的细胞通过细胞内的酶，如氮氧化物（NADPH氧化酶）和XO（黄嘌呤氧化酶）。据报道，活性氧可能差异调节的重要核因子如p53或细胞组分，如ATM激酶的DNA损伤反应20的主调节器的活性。类似的ROS强烈调解日影响细胞信号Ê氧化和蛋白质酪氨酸磷酸酶酶（PTPs），其被建立为信号转导21的关键调节剂失活。此外，基于蛋白质组学的方法证明RNS还负责特定的蛋白质修饰和分子信号的改变。 RNS反应与半胱氨酸的巯基修饰成S-nitrothiols（SNO）和触发伴随着病理状态，如炎症和自身免疫性疾病，22,23的分子途径。 由于细胞培养实验中只是部分地重现了众多的演技在体内的因素，它是非常感兴趣的动物模型24,25进行氧化还原的研究。为了实现这一目标，在斑马鱼一直被认为是合适的脊椎动物的动物模型来研究氧化应激动力学26。斑马鱼是给予几个优势，在脊椎动物的开发研究细胞和基因活动的新模式系统elopment和疾病。可以生成和提供每周一次实验需要大型集群胚胎。此外斑马鱼胚胎的非凡的光学清晰度，以及它们的小尺寸，使单细胞成像和动态跟踪在整个生物体27。在过去的十年中，已经产生了相当多的斑马鱼突变体来模拟人类的病理状况，如癌症和遗传疾病28-31。最重要的是，大量的转基因株系已经产生，允许基因和生物操纵32的广泛的机会。例如，转基因的组织特异性的斑马鱼线被经常用于体内研究。这些行表示一个选择的启动子的控制下的荧光蛋白质，提供识别单个细胞在体内的功能，以及它们含有的解剖结构。 几个毒理学研究已经采用t他斑马鱼来评估化学品对氧化还原平衡在体内的作用，提示这种脊椎动物的适用性作为动物模型，药物发现和氧化应激33-35的领域。即使一些荧光探针已经过测试，以监测氧化应激的斑马鱼幼虫36,37，有没有既定的分析检测和测量氧化应激在斑马鱼组织中的水平和活细胞。在这里，我们描述了用于在体内量化氧化应激在活斑马鱼胚胎细胞的过程。成像工具，流式细胞仪分选，荧光探针和促氧化条件都结合在一起，生成一个简单的化验检测和氧化物质的定量分析在斑马鱼胚胎和组织。

<table border="0" cellpadding="0" cellspacing="0" width="1261">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:301px;">Name of Material/ Equipment</td>
			<td style="width:147px;">Company</td>
			<td style="width:285px;">Catalog Number</td>
			<td style="width:528px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:301px;">Hydrogen peroxide solution</td>
			<td style="width:147px;">SIGMA</td>
			<td style="width:285px;">516813</td>
			<td>DO NOT STORE DILUITIONS</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:301px;">Hank&#39;s Balanced Salt Solution 1X</td>
			<td style="width:147px;">GIBCO</td>
			<td style="width:285px;">14025</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:301px;">Methyl cellulose</td>
			<td style="width:147px;">SIGMA</td>
			<td style="width:285px;">M0387</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:301px;">Instant Ocean Aquarium Sea Salt Mixture</td>
			<td style="width:147px;">INSTANT OCEAN</td>
			<td style="width:285px;">SS15-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tricaine</td>
			<td style="width:147px;">SIGMA</td>
			<td>A5040</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:301px;">Cgeneric ROS-sensitive probe:&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; CellROX Deep Red Reagent</td>
			<td style="width:147px;">INVITROGEN</td>
			<td style="width:285px;">C10422</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:301px;">Mitochondria specific ROS-sensitive probe: MitoSOX&#160;</td>
			<td style="width:147px;">INVITROGEN</td>
			<td style="width:285px;">M36008</td>
			<td>dissolve one vial with 13&#956;l of DMSO</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:301px;">Hydroethidine</td>
			<td style="width:147px;">INVITROGEN</td>
			<td style="width:285px;">D23107</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:301px;">Rotenone</td>
			<td style="width:147px;">SIGMA</td>
			<td style="width:285px;">R8875</td>
			<td>Prepare 5mM stock solution in DMSO.&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:301px;">Dimethyl sulfoxide</td>
			<td style="width:147px;">SIGMA</td>
			<td style="width:285px;">D2650</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:301px;">VAS2870; 3-Benzyl-7-(2-benzoxazolyl)thio-1,2,3-triazolo(4,5-d)pyrimidine</td>
			<td style="width:147px;">EnzoLifeScience</td>
			<td>BML-EI395</td>
			<td>dissolve the powder in DMSO; diluite in fish water</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:301px;">Propidium Iodide&#160;</td>
			<td style="width:147px;">Molecular probes&#160;&#160;&#160;&#160;&#160;&#160; (Life Technologies)&#160;</td>
			<td>P3566</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:301px;">7-aminoactinomycin D (7-AAD)&#160;</td>
			<td style="width:147px;">Molecular probes&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; (Life Technologies)&#160;</td>
			<td>A1310</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:301px;">Nrf2a Morpholino</td>
			<td style="width:147px;">GeneTools</td>
			<td style="width:285px;">5&#39;-CATTTCAATCTCCATCATGTCTCAG-3&#39;</td>
			<td>Ref: Timme-LaLaragy et al; 2012 (PMID: 22174413); Kobayashi et al; 2002(PMID:12167159 )</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Collagenase P</td>
			<td style="width:147px;">ROCHE</td>
			<td>11213857001</td>
			<td>Dissolve the powder at 100mg/ml in sterile HBSS. Store aliquots at -20&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:301px;">Phosphate-Buffered Saline (PBS)</td>
			<td style="width:147px;">GIBCO</td>
			<td style="width:285px;">10010-056</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Bovine Serum&#160;</td>
			<td style="width:147px;">GIBCO</td>
			<td>10082-147</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Complete Protease Inhibitor Cocktail Tablets</td>
			<td style="width:147px;">ROCHE</td>
			<td style="width:285px;"></td>
			<td>Dissolve one tablet in 1ml of water</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.5% Trypsin-EDTA (10x), no phenol red</td>
			<td style="width:147px;">GIBCO</td>
			<td style="width:285px;">15400-054</td>
			<td>Prepare 1X working solution before usage</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Compound microscope&#160;</td>
			<td>ZEISS</td>
			<td style="width:285px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Stereo microscope with fluorescent illumination</td>
			<td>Nikon</td>
			<td style="width:285px;">AZ100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">camera&#160;</td>
			<td>ZEISS</td>
			<td style="width:285px;">AxioCamMRm</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">software for fluorescence image acquisition</td>
			<td>ZEISS</td>
			<td style="width:285px;">ZEN 2011</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluorescence-activated cell sorter</td>
			<td style="width:147px;">BD FACSCalibur</td>
			<td style="width:285px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Centrifuge&#160;</td>
			<td style="width:147px;">Eppendorf</td>
			<td style="width:285px;">5417R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FACS tubes&#160;</td>
			<td style="width:147px;">BD</td>
			<td>342065</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Multiwell Plate&#160;</td>
			<td>BD Falcon</td>
			<td>353047</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:301px;">Sterilized, non treated Petri dishes 90mm</td>
			<td style="width:147px;">VWR</td>
			<td style="width:285px;">391-1915</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:301px;">Confocal microscope</td>
			<td style="width:147px;">Leica</td>
			<td style="width:285px;">Leica SP5</td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of oxidative stress in zebrafish embryos

高含量的活性氧物种（ROS）可能导致细胞的氧化还原状态对氧化应激条件的变化。这种情况会导致分子（脂质，DNA，蛋白质）的氧化，并导致细胞死亡。氧化应激也影响一些病理状况如糖尿病，视网膜病，神经退行性疾病和癌症的进展。因此，重要的是定义工具不仅在单细胞水平，而且在整个生物体的情况下，调查氧化应激的条件。在这里，我们考虑的斑马鱼胚胎在体内系统中一个有用的执行这样的研究，并提出了一个协议来测量体内氧化应激。利用荧光探针活性氧和斑马鱼转基因荧光线，我们开发两种不同的方法来测量氧化应激在体内 ：I）的“全胚胎ROS的检测方法”对氧化应激的定性测量和ii）“单细胞ROS的检测方法“对氧化应激的定量测量。在此，我们证明这些程序的有效性由氧化剂剂和生理或遗传方式增加氧化应激的组织。该协议是服从正向遗传筛选，这将有助于ROS的地址因果关系中的氧化应激相关的疾病，如神经系统疾病和癌症的动物模型。

通过应用这里介绍的方法，我们可以很容易地测量和检测氧化应激（和ROS的水平）在斑马鱼胚胎组织。穿越成年斑马鱼后，鸡蛋收集，并使其在28°C至发展到72小时受精后（HPF）。为了诱导的氧化应激，我们提出了两种不同的方法：胚胎1）具有较强的促氧化试剂处理或2）组织损伤后促进活性氧的形成。 过氧化氢 ​​（H 2 O 2），作为通用的细胞ROS形成剂，和鱼藤酮，?...

Watch this Scientific Journal Video about Analysis of Oxidative Stress in Zebrafish Embryos at JoVE.com

Analysis of Oxidative Stress in Zebrafish Embryos

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.

在斑马鱼胚胎氧化应激分析

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

analysis-of-oxidative-stress-in-zebrafish-embryos

Research

JoVE Journal

Biology

36.9K 次观看.  University of Torino. Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses. 

视频: Analysis of Oxidative Stress in Zebrafish Embryos

Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression studies. In zebrafish (Danio rerio), microinjection of RNA, DNA, proteins, antisense oligonucleotides and other small molecules into the developing embryo provides researchers a quick and robust assay for exploring gene function in vivo. In this video-article, we will demonstrate how to prepare and microinject in vitro synthesized EGFP mRNA and a translational-blocking morpholino oligo against pkd2, a gene associated with autosomal dominant polycystic kidney disease (ADPKD), into 1-cell stage zebrafish embryos. We will then analyze the success of the mRNA and morpholino microinjections by verifying GFP expression and phenotype analysis. Broad applications of this technique include generating transgenic animals and germ-line chimeras, cell-fate mapping and gene screening. Herein we describe a protocol for overexpression of EGFP and knockdown of pkd2 by mRNA and morpholino oligonucleotide injection.

Microinjection is a well-established and effective method for introducing foreign substances into fertilized zebrafish embryos. Here, we demonstrate a robust microinjection technique for performing mRNA overexpression, and morpholino oligonucleotide gene knockdown studies in zebrafish.

显微注射mRNA和斑马鱼的胚胎吗啉反义寡核苷酸。

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression ...

科研

生物学

Microinjection of Zebrafish Embryos to Analyze Gene Function

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic oligonucleotides with antisense complementarity to target RNAs, can be added to the embryo to reduce the expression of a particular gene product.  Conversely, processed mRNA can be added to the embryo to increase levels of a gene product.  The vehicle for adding either mRNA or morpholino to an embryo is microinjection.  Microinjection is efficient and rapid, allowing for the injection of hundreds of embryos per hour.  This video shows all the steps involved in microinjection.  Briefly, eggs are collected immediately after being laid and lined up against a microscope slide in a Petri dish.  Next, a fine-tipped needle loaded with injection material is connected to a microinjector and an air source, and the microinjector controls are adjusted to produce a desirable injection volume.  Finally, the needle is plunged into the embryo's yolk and the morpholino or mRNA is expelled.

This video shows how morpholino or mRNA can be injected into zebrafish embryos at the one-cell stage to decrease or increase the level of specific gene products during subsequent development.

斑马鱼的胚胎显微注射来分析基因的功能

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic ...

Generating Chimeric Zebrafish Embryos by Transplantation

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an organism that contains cells from more than one animal; mosaics are one type of chimera in which cells from more than one genotype are mixed, usually wild-type and mutant. In the zebrafish, chimeras can be readily made by transplantation of cells from a donor embryo into a host embryo at the appropriate embryonic stage. Labeled donor cells are generated by injection of a lineage marker, such as a fluorescent dye, into the one-cell stage embryo. Labeled donor cells are removed from donor embryos and introduced into unlabeled host embryos using an oil-controlled glass pipette mounted on either a compound or dissecting microscope. Donor cells can in some cases be targeted to a specific region or tissue of the developing blastula or gastrula stage host embryo by choosing a transplantation site in the host embryo based on well-established fate maps.

A step-by-step guide to generating targeted chimeric zebrafish embryos by transplantation at the blastula or gastrula stage.

移植嵌合斑马鱼胚胎生成

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an ...

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 ...

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 ...

Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. The whole-mount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning and/or colabeling is ultimately required to pinpoint the exact cell types undergoing apoptosis. The whole-mount Casp3 assay is optimized for analysis of fixed embryos between the 4-cell stage and 32 hr-post-fertilization and is useful for a number of applications, including analysis of zebrafish mutants and morphants, overexpression of mutant and wild-type mRNAs, and exposure to chemicals. Compared to acridine orange staining, which can identify apoptotic cells in live embryos in a matter of hours, Casp3 and TUNEL assays take considerably longer to complete (2-4 days). However, because of the dynamic nature of apoptotic cell formation and clearance, analysis of fixed embryos ensures accurate comparison of apoptotic cells across multiple samples at specific time points. We have also found the Casp3 assay to be superior to analysis of apoptotic cells by the whole-mount TUNEL assay in regard to cost and reliability. Overall, the Casp3 assay represents a robust, highly reproducible assay in which to analyze apoptotic cells in early zebrafish embryos.

Certain genetic perturbations or exposure to toxins can disrupt normal developmental processes leading to death of specific cell types. The analysis of activated Caspase 3 by whole-mount immunofluorescence in zebrafish embryos reveals stage- and tissue-specific localization of cells specifically undergoing apoptosis.

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis ...

Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are frequently used to identify chemicals that modulate gene expression. Chemical screens that assay fluorescence in live zebrafish often rely on expensive, specialized equipment for high content screening. We describe a procedure using a standard epifluorescence microscope with a motorized stage to automatically image zebrafish embryos and detect tissue-specific fluorescence. Using transgenic zebrafish that report estrogen receptor activity via expression of GFP, we developed a semi-automated procedure to screen for estrogen receptor ligands that activate the reporter in a tissue-specific manner. In this video we describe procedures for arraying zebrafish embryos at 24-48 hours post fertilization (hpf) in a 96-well plate and adding small molecules that bind estrogen receptors. At 72-96 hpf, images of each well from the entire plate are automatically collected and manually inspected for tissue-specific fluorescence. This protocol demonstrates the ability to detect estrogens that activate receptors in heart valves but not in liver.

Described here is a protocol for semi-automated imaging of tissue-specific fluorescence in zebrafish embryos.

组织特异性荧光的斑马鱼胚胎半自动成像

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are ...

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for forward genetic analysis of developmental processes due to their external fertilization, embryonic size, rapid ontogeny, and optical clarity &#8211; a constellation of traits that enable the direct observation of events ranging from gastrulation to organogenesis with a basic stereomicroscope. Further, zebrafish embryos can survive for several days in the haploid state. The production of haploid embryos in vitro is a powerful tool for mutational analysis, as it enables the identification of recessive mutant alleles present in first generation (F1) female carriers following mutagenesis in the parental (P) generation. This approach eliminates the necessity to raise multiple generations (F2, F3, etc.) which involves breeding of mutant families, thus saving the researcher time along with reducing the needs for zebrafish colony space, labor, and the husbandry costs. Although zebrafish have been used to conduct forward screens for the past several decades, there has been a steady expansion of transgenic and genome editing tools. These tools now offer a plethora of ways to create nuanced assays for next generation screens that can be used to further dissect the gene regulatory networks that drive vertebrate ontogeny. Here, we describe how to prepare haploid zebrafish embryos. This protocol can be implemented for novel future haploid screens, such as in enhancer and suppressor screens, to address the mechanisms of development for a broad number of processes and tissues that form during early embryonic stages.

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish is a powerful model system for developmental biology and human disease research due to their genetic similarity with higher vertebrates. This protocol describes a methodology to create haploid zebrafish embryos that can be utilized for forward screen strategies to identify recessive mutations in genes essential for early embryogenesis.

生产单倍体斑马鱼的胚胎通过<em&gt;体外</em&gt;施肥

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for ...

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic human disorders, including cardiovascular and neurodegenerative diseases, diabetes, aging, and cancer. Therefore, it is important to study oxidative stress not only in cell systems but also using whole organisms. C. elegans is an attractive model organism to study the genetics of oxidative stress signal transduction pathways, which are highly evolutionarily conserved.
Here, we provide a protocol to measure oxidative stress resistance in C. elegans in liquid. Briefly, ROS-inducing reagents such as paraquat (PQ) and H2O2 are dissolved in M9 buffer, and solutions are aliquoted in the wells of a 96 well microtiter plate. Synchronized L4/young adult C. elegans animals are transferred to the wells (5-8 animals/well) and survival is measured every hour until most worms are dead. When performing an oxidative stress resistance assay using a low concentration of stressors in plates, aging might influence the behavior of animals upon oxidative stress, which could lead to an incorrect interpretation of the data. However, in the assay described herein, this problem is unlikely to occur since only L4/young adult animals are being used. Moreover, this protocol is inexpensive and results are obtained in one day, which renders this technique attractive for genetic screens. Overall, this will help to understand oxidative stress signal transduction pathways, which could be translated into better characterization of oxidative stress-associated human disorders.

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

C. elegans is an attractive model organism to study signal transduction pathways involved in oxidative stress resistance. Here we provide a protocol to measure oxidative stress resistance of C. elegans animals in liquid phase, using several oxidizing agents in 96 well plates.

测量氧化应激性<em&gt;秀丽隐杆线虫</em&gt;在96孔微量滴定板

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic ...

Comet Assay as an Indirect Measure of Systemic Oxidative Stress

Higher eukaryotic organisms cannot live without oxygen; yet, paradoxically, oxygen can be harmful to them. The oxygen molecule is chemically relatively inert because it has two unpaired electrons located in different pi * anti-bonding orbitals. These two electrons have parallel spins, meaning they rotate in the same direction about their own axes. This is why the oxygen molecule is not very reactive. Activation of oxygen may occur by two different mechanisms; either through reduction via one electron at a time (monovalent reduction), or through the absorption of sufficient energy to reverse the spin of one of the unpaired electrons. This results in the production of reactive oxidative species (ROS). There are a number of ways in which the human body eliminates ROS in its physiological state. If ROS production exceeds the repair capacity, oxidative stress results and damages different molecules. There are many different methods by which oxidative stress can be measured. This manuscript focuses on one of the methods named cell gel electrophoresis, also known as &#8220;comet assay&#8221; which allows measurement of DNA breaks. If all factors known to cause DNA damage, other than oxidative stress are kept constant, the amount of DNA damage measured by comet assay is a good parameter of oxidative stress. The principle is simple and relies upon the fact that DNA molecules are negatively charged. An intact DNA molecule has such a large size that it does not migrate during electrophoresis. DNA breaks, however, if present result in smaller fragments which move in the electrical field towards the anode. Smaller fragments migrate faster. As the fragments have different sizes the final result of the electrophoresis is not a distinct line but rather a continuum with the shape of a comet. The system allows a quantification of the resulting &#8220;comet&#8221; and thus of the DNA breaks in the cell.

Comet assay measures DNA breaks, induced by different factors. If all factors (except oxidative stress) causing DNA damage are kept constant, the amount of DNA damage is a good indirect parameter of oxidative stress. The goal of this protocol is to use comet assay for indirect measurement of oxidative stress.

彗星试验系统性氧化应激的间接测量

Higher eukaryotic organisms cannot live without oxygen; yet, paradoxically, oxygen can be harmful to them. The oxygen molecule is chemically relatively ...