Name: evaluation of zebrafish kidney function using a fluorescent clearance assay
Uploaded: 2015-02-20T13:00:00.000+00:00
Duration: 8 min 13 s
Description: Watch this Scientific Journal Video about Evaluation of Zebrafish Kidney Function Using a Fluorescent Clearance Assay at JoVE.com

由Jaipreet Bharj提供的技术援助。这项工作是由欧盟第七框架计划（SYSCILIA -241955）和荷兰肾脏基金会（CP11.18）资助。

伦理学声明:动物保养，畜牧业和程序是由动物（科学程序）所有的动物实验已经进行了下符合生物授予内政大臣（PIL号七千八百九十二分之七十零）许可证法案1986年界定和控制服务管理组和生物服务伦理委员会，SGUL，伦敦，英国。所作的所有努力，以减少使用动物的数量，并以尽量减少痛苦，提高福利完善两个程序和畜牧业。 1.制备仪器，麻醉，并且荧光染料<ol><li>使用微量拉马和硼硅标准毛细血管壁（无灯丝）拉的适当长度针显微注射（ 图1A）。 </li><li>使琼脂糖模具胚胎易于注射的取向入心包（ 图1B）。胶约10玻璃显微镜SL新娘子在一起，形成抵消幻灯片的"楼梯"。为了达到最佳效果，请使用每张幻灯片的中心一滴快速设定环氧树脂胶。 </li><li>切出约78毫米长到一半使用本生灯加热的手术刀两个10毫升的塑料吸管的一段，取出吸管结束适当。附上和胶（环氧树脂）吸管部分，以​​堆叠的载玻片，以提供稳定性，以允许模具浸成90毫米的培养皿。尽一切努力，确保滑动角对准垂直于培养皿地板。允许胶时间来设置。 </li><li>浇铸模具中鱼水制成2％的琼脂糖溶液（来自水族馆获得）。倾琼脂糖成90毫米的培养皿，并放置在模具上开培养皿的顶部，允许堆叠滑刃淹没在琼脂糖表面下的角度。离开实验室长凳上设置30分钟。 </li><li>去除幻灯片演员和覆盖滑印琼脂糖新鲜的鱼水包含三卡因麻醉（见步骤1.9）在1:25的比例。 </li><li>使罗丹明右旋糖酐（RD）染料溶液。重悬罗丹明乙万兆瓦标记的葡聚糖在蒸压超纯水中，使得一个储备浓度为50毫克/毫升。对于显微注射，进一步稀释股票为5毫克/毫升在高压灭菌超纯水的最终浓度。 注:色素细胞开始在斑马鱼从24小时受精后（HPF）区分;这些可以在图像采集过程中模糊的荧光标记物。 </li><li> 用正-Phenylthiourea（PTU），其中块通过抑制酪氨酸酶的抑制黑色素生成黑色素的形成。使PTU的0.003％储备溶液通过在60℃下在水族馆的水，热溶液溶解PTU粉末完全溶解。 </li><li>孵育亚甲基蓝溶液，温和的杀菌剂斑马鱼胚胎，以防止霉菌大量繁殖，提高存活率。加入2毫升亚甲蓝原液，含0.1％亚甲蓝以Ultrapure水，至1升的水族馆的水和作为一个标准的胚胎培养基。 </li><li>补15mM的储备浓度三卡因/ 3-氨基苯甲酸乙酯甲磺酸盐中的说明"斑马鱼书"23。麻醉胚胎，从而固定它们以执行注射过程。 </li><li>麻醉胚胎后，通过使用甲基纤维素的无毒性和粘性定向它们，用于成像。使200毫升制备3％的甲基纤维素，寒意130毫升水于-20℃下30分钟，并置于冰上。热70ml水至80℃，在一个玻璃烧杯中，加入6克甲基纤维素，和搅拌用玻璃棒，直到所有的颗粒润湿和均匀地分散。添加冰冷的水，混合，并允许准备在4℃等分到50ml试管前冷却30分钟。 注:降低温度使甲基纤维素变成可溶性的。该解决方案将变得更浓作为粉末水合物。 3％的甲基纤维素可存储无细菌生长一个星期的时间很短，在4℃以上，在-20℃下。确保使用前甲基达到RT。 </li><li>以执行荧光染料注射入斑马鱼使用标准的显微注射设置（ 图1C）。这是由一个空气压缩机连接到压力调节器系统，该系统馈入用于与1.0外径毛细管使用的直吸管保持器。吸管持有人应被安置的MM33紧凑的3轴控制显微用磁性支架固定在一个钢制底板内。胚胎可以可视化，操纵和使用立体解剖显微镜注入。 </li></ol> 2.斑马鱼饲养和预注射治疗<ol><li>维持斑马鱼如前所述23。使用一个水族馆建立，提供在28.5℃的恒温供给再循环水分别7.2和450-550μS用碳酸氢钠和即时海洋海盐，电导率 - ，调节至pH为6.8。用野生型或转基因斑马鱼线适当实验，维持男性15例，放养密度为15名女性每8升油箱。鱼设施光周期设置为14小时上午9点之间的日光 - 晚上11点。 </li><li>斑马鱼产卵开始于光周期中，当灯在早上打开的开始。为了确保最大的蛋可在不干扰鱼类收集，收藏前淹没繁殖缸（含网状嵌件，可从斑马鱼专家）为野生型股票坦克的夜晚。 </li><li>对收集的一天，等待30 - 40分钟为鱼产卵，之后收集和冲洗用滤茶和新鲜的水族箱的水蛋。存清洗鸡蛋放在90毫米的培养皿含有胚胎培养基和孵化在28.5℃。 </li><li>对待胚胎PTU抑制黑色素起源。 8 HPF，在最短的液体转移可行的胚胎含有1新鲜培养皿:100 PTU胚胎中，进一步培养在28.5°C，直到72高倍视野。 注:PTU可引起发育缺陷，如果用在早期阶段所以应限制要发表8 HPF阶段，但可以迟至24 HPF处理。晚此外PTU不完全阻塞眼部色素沉着，但一般是成功的，抑制黑色素细胞干线形成。 </li></ol> 3.显微注射注:心包包住心脏，但被流体心包腔内分开保护和方便的心脏运动。这个程序的目的是要注入的RD进入心包腔，这允许所述染料到脉管系统的快速吸收。 <ol><li>负载针与6微升 - 稀释RD，使用微加载提示和安全进入显微的持针器。打破针用细TIPP结束编辑钳（ 图1A）。移动的RD溶液到针的尖端通过最大化脉冲持续到分钟设定，切换回毫秒一次完成。 </li><li>使用一个阶段微米，压力调节器，和改进对针尖长度（使用镊子）来调整排出液滴的大小为100微米的直径，这相当于0.5 NL体积（ 图2A）。 注:当破针，小心不要折断太多关闭否则会令校准注射量困难。如果需要的话，进一步打破针尖以增加墨滴大小然而，保持的针厚度，允许进入心包无需过度弯曲或损坏的组织。 </li><li>注射前，在麻醉胚胎培养基含有三卡因的胚胎。建立含有5ml的胚胎中，标签1&#39;三卡因"和其他&#39;恢复&#39;2×35毫米培养皿。 </li><li>加入200股市微升的三卡因在适当标记的菜。一旦准备就绪注入，选择单个胚胎在72 HPF，并在最短的液体转移到三卡因菜。监测胚胎的活性，测试胚胎被麻醉并用截短微加载尖端通过温和搅拌固定。 </li><li>麻醉胚胎转移到注射模具和定向内琼脂糖槽所以左侧朝上，定位心脏到的视场的左侧的胚胎。 </li><li>到RD注入心脏，刺破用针心包注入1 NL RD入心包的麻醉胚胎（ 图2B，右图）的腔。注射后撤回针。在最小的液体转移注入胚胎到&#39;恢复菜&#39;和监测1分钟。个别胚胎移植至含有1ml新鲜胚胎培养基（含PTU）24孔板并正确标记。 注意:心包是艰难的，但有是那里的心包符合腹，尾咽弓和背侧，喙卵黄囊（ 图2B箭头）的交点一个显着的薄弱点。针头应被定向到该槽。当针到位，手指上的显微顶部的自来水公司将有利于进入心包腔。 </li><li>重复步骤3.2 - 3.6元实验组至少有10胚胎，把每条鱼在一个单独的好，唯一labelto允许单个实验跟进。孵育在28.5℃下进行3小时。 </li></ol> 4.图像采集<ol><li>在3小时后喷射（HPI）麻醉胚胎如前所述并转移到含有3％甲基纤维素35毫米培养皿。轻轻推胚胎到甲基纤维素和定向等侧面进行成像。 </li><li>获得的UV下将胚胎的图像，使用的过滤器的可视化ø˚F发出的光在570nm（ 图2C）。图像采集后，让胚胎在更换指定的良好之前恢复。获取图像的所有注射​​的胚胎，并进一步培养在28.5℃。 注:在这个协议中，我们使用了荧光体视显微镜用TXR过滤器集，DFC300FX摄像头和相应的应用软件。记下后续图像采集准确采集设置。 </li><li>在24 HPI，如上所述获得的第二轮的图像。一旦所有的图像已被获取，在两个3 HPI和24 HPI，人道地处置胚胎或用于其他的实验手段例如，免疫组织化学。 </li></ol> 5.图像处理<ol><li>量化每个注射胚胎的荧光强度，在3 HPI和24 HPI，通过NIH的ImageJ的软件分析图像。为了测量荧光强度，打开ImageJ的图像，并在1指定的感兴趣区域（ROI）的区域00 PX 2（编辑→选择→指定）。 </li><li>在投资回报率（ 图2C）的中心位置的心脏，集测量，包括平均灰度和面积（分析→设置测量），进行测量（分析→测量）。完成对每个图像组，在3 HPI和24 HPI，每个胚胎和用于进一步处理的平均灰度值传送到电子表格。 注:我们选择100像素2的固定投资回报率的大小，因为这包括胚胎之间的个人的心。的心脏被选择执行测量，由于其大尺寸，使一个方便的位置，以在进入肾脏之前测量血液的荧光内容。 </li><li>使用合适的统计软件进行统计分析。比较使用学生氏t检验组进行统计学意义。 </li></ol>

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bardet-Biedl+syndrome).">Bardet-Biedl syndrome).</a> Eur J Hum Genet. 21 (1), 8-13 (2013).</li><li>Corbetta, S., 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=High+prevalence+of+simple+kidney+cysts+in+patients+with+primary+hyperparathyroidism.">High prevalence of simple kidney cysts in patients with primary hyperparathyroidism.</a> J Endocrinol Invest. 32 (8), 690-694 (2009).</li><li>Veleri, S., 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=Knockdown+of+Bardet-Biedl+syndrome+gene+BBS9/PTHB1+leads+to+cilia+defects.">Knockdown of Bardet-Biedl syndrome gene BBS9/PTHB1 leads to cilia defects.</a> PLoS One. 7 (3), e34389 (2012).</li><li>Vize, P., Woolf, A. S., Bard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Kidney: From Normal Development to Congenital Disease. , (2003).</li><li>Chang, R. L., 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=Permselectivity+of+the+glomerular+capillary+wall+to+macromolecules.+II.+Experimental+studies+in+rats+using+neutral+dextran.">Permselectivity of the glomerular capillary wall to macromolecules. II. Experimental studies in rats using neutral dextran.</a> Biophys J. 15 (9), 887-906 (1975).</li></ol>

作者什么都没有透露。

斑马鱼提供了一个宝贵的工具，人类遗传病模型，他们作为一个科学仪器体内研究使用使许多生物系统，包括肾脏的遗传击穿详细的研究。很多现在被理解如何斑马鱼肾脏发展和功能。惊人的相似，以人类nephrogenesis和同源性致病基因21已示于理解如何斑马鱼已成为基本的基因功能导致肾疾病的病理学缺陷如何。事实上，遗传操作，可以毫不费力地使用反义吗啉击倒技术或更先进的?...

人肾起着从血液过滤代谢废物和回收所需溶质以维持细胞稳态的关键作用。有一个数字，引起肾功能障碍的人类遗传疾病。最常见的遗传性肾脏病是常染色体显性多囊肾病（ADPKD）的特征在于肾脏肾小管内的流体填充囊的发展;引起cystogenesis的损害是不利的肾功能10。多囊肾具有1的发生:800 - 1:1000，占8 -的患者在末期肾衰竭（ESRF）11 10％。几个基因有牵连导致多囊肾多囊，包括-1（PKD1）和-2（PKD2），约占85％的病例分别为12,13 15％。此外，对于与PKD1基因产物-2定位于纤毛和是基本的ciliogenesis 14,15。现在有人类遗传性疾病，被称为公认的家庭在ciliopathies，影响纤毛的功能，并导致CKD 16。 越来越多的影响纤毛发育和功能的人类遗传疾病的画在这个曾经被认为是残留的细胞器全球利益。该纤毛，头发般的细胞突起，富含受体和必要的关键细胞信号事件的传导离子通道。该纤毛由一个微管系轴丝，典型地构造成9径向地布置微管双峰具有或不具有中央一对单峰的微管的。该轴丝结构定义的纤毛作用的类型和模式。 9 + 2微管排列赋予蠕动到它被用在跨越上皮表面流体的运动的纤毛。 9 + 0构造为非能动但被认为是主要的功能在细胞信号传递活动17。除了CKD，睫状功能障碍的后果是一组特征ciliopathy功能，包括，肥胖症，视网膜变性，多趾，以及认知缺损16。然而，CKD是其中最不利于患者生活质量，因此后面的适当的体内模型的发展纤毛相关CKD的主要动力。 斑马鱼是一个很好的模型，以了解人类遗传病的病因。他们的快速发展，生产大量的鸡蛋，透明的组织，和前宫内生长使得斑马鱼发育过程进行可视化和生物操纵的事件相当容易。基因可以使用的基因组编辑工具（CRISPR 18和TALENS 19）最近成功进行遗传改变，撞倒使用反义吗啉代技术20，或通过加入化合物以它们的水生环境中的药理学调节。事实上，斑马鱼提供了一个平台，承接éxperiments不在容许在其它动物模型。而斑马鱼都比较简单脊椎动物（相比人类）它们共同与人类有许多共同的功能保守的器官，基因，和信令进程。例如，斑马鱼肾是非常相似的结构和功能相比人类21,22。然而，与哺乳动物肾，通过相的连续发展，各带标记的较发达的肾（前肾，中肾和后肾），胚胎斑马鱼只开发了一个前肾，肾的最不成熟的形式。虽然百万肾单位，可以发现在形成哺乳动物肾的积木，斑马鱼胚胎只具有两个。肾小球，它允许在初始血液滤液，融合在主动脉中线只是腹侧。通过肾小球成运行尾端沿轴线经由泄殖腔退出之前熔合pronephric肾小管血液过滤器。在pronephric TUbules被大量纤毛与运动纤毛是宽容到滤液对尾3,4退出流通。这种简单的pronephric结构经过几个星期的幼虫生长在那里他们最终发展成一个更复杂的结构中肾21斑马鱼保持动态平衡。然而，斑马鱼从未开发出后肾21。尽管斑马鱼特质，斑马鱼肾单位被分段与基因表达谱等于在哺乳动物观察，从而提供了在体内模型无与伦比为nephrogenesis 3,22。 常规的患者通过一系列的血液和尿中的测试测试肾功能。通常将血液溶解的盐，尿素和肌酸酐进行分析。高水平的尿素，肌酸酐和异常盐浓度是指示问题具有肾功能。用比色试纸尿液检测的蛋白质，布卢异常水平D，脓液，细菌和现在的尿液样本中的糖。这样的测试通常需要大约30毫升尿液或5 - 加入10ml的血液。它已经难以在体内模式生物，如斑马鱼翻译这些类型的测定，以小的，主要是由于收集足够的血液或尿液进行测定的不可能性质。在这里，我们解决缺乏相应的斑马鱼肾功能检查，并描述了创新技术的研究。用荧光染料注射入血流中，我们能够通过肾脏，以监测和个别地量化随着时间的推移过滤和荧光活性的排泄从血液。该方法可用于研究疾病引起的肾损害，这是我们提供的一个例子。

<table><tbody><tr><td>P-97 SUTTER Flaming/Brown type micropipette puller</td><td>Intracel</td><td>P-97</td><td></td></tr><tr><td>borosilicate standard wall capillaries</td><td>Harvard Apparatus</td><td>30-0017</td><td></td></tr><tr><td>Glass microscope slides</td><td>VWR International</td><td>631-0109</td><td></td></tr><tr><td>Epoxy Resin Glue</td><td>Evo-Stik</td><td></td><td></td></tr><tr><td>Rhodamine B 10,000 MW labeled Dextran</td><td>Life technologies&amp;nbsp;</td><td>D-1824</td><td></td></tr><tr><td>N-Phenylthiourea&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>P7629</td><td></td></tr><tr><td>Methylene blue&amp;nbsp;</td><td>Sigma-Aldrich</td><td>M9140</td><td></td></tr><tr><td>Ethyl 3-aminobenzoate methanesulfonate salt</td><td>Sigma-Aldrich</td><td>A5040</td><td></td></tr><tr><td>methylcellulose</td><td>Sigma-Aldrich</td><td>M0512</td><td></td></tr><tr><td>air compressor&amp;nbsp;</td><td>Jun-Air</td><td>OF302-15</td><td></td></tr><tr><td>Picospritzer III&amp;nbsp;</td><td>Parker Instruments&amp;nbsp;</td><td>051-0500-900</td><td></td></tr><tr><td>compact 3-axis control micromanipulator&amp;nbsp;</td><td>Marzhauser</td><td>MM33&amp;nbsp;</td><td></td></tr><tr><td>Dissecting stereo microscope</td><td>Nikon</td><td>SMZ1000</td><td></td></tr><tr><td>microloader tips</td><td>Eppendorf</td><td>5242956003</td><td></td></tr><tr><td>Dumont #5 forceps&amp;nbsp;</td><td>Sigma-Aldrich</td><td>F6521</td><td></td></tr><tr><td>stage micrometer&amp;nbsp;</td><td>Pyser- SGI</td><td>02A00404</td><td></td></tr></tbody></table>

evaluation of zebrafish kidney function using a fluorescent clearance assay

斑马鱼的胚胎提供了一个听话的模型来研究器官和模拟人类遗传性疾病。尽管其相对简单，斑马鱼肾开发并在几乎相同的方式作为人类的功能。在人类肾脏的结构的主要区别是数以百万计的肾相比，仅具有两个斑马鱼的存在。然而，简化这种复杂的系统进入基本功能单元已资助我们如何肾脏开发和运营的理解。在斑马鱼，位于肾小球中线负责初始血液过滤成发散熔合到彼此的泄殖腔之前两侧向下胚轴运行两个pronephric肾小管。在pronephric肾小管是人口稠密通过运动纤毛有利于滤液的运动沿着分割肾小管，使各种溶质，最后才通过泄殖腔2-4退出交流。负责CKD，INC许多基因泸那些与ciliogenesis，已经研究了在斑马鱼5。然而，一个主要的平局背面已在遗传操作之后评估斑马鱼肾功能的困难。传统的检测来衡量肾功能不全的人证明非平移，以斑马鱼，主要是由于他们的水生环境和小尺寸。例如，它不是物理上能够提取血液从对于尿素，肌酐含量分析胚胎上演鱼，因为它们太小。此外，斑马鱼不产生足够的尿液用于测试在一个简单的蛋白尿&#39;试纸&#39;，这是经常在初始病人检查执行。我们描述了一种荧光测定法，利用了斑马鱼的光学透明度，定量监测荧光染料的间隙，随着时间的推移，从脉管并通过肾脏，得到读出肾功能1,6-9的。

巴比二氏综合征（BBS）是一种罕见的异类ciliopathy影响约为1:全球16 16万人。患者目前有许多相关的问题，包括多囊性肾脏，随后患者经常需要肾透析或移植24。 ESRF是死亡BBS的最常见的原因，大约有30％的患者发展CKD 16。目前，20个非相关的基因有牵连的BBS，没有发表基因型 - 表型的关联。在BBS蛋白质共享纤毛和基体，随着患者的特性特征，推断ciliopathy诊断中常见蛋白质定?...

Watch this Scientific Journal Video about Evaluation of Zebrafish Kidney Function Using a Fluorescent Clearance Assay at JoVE.com

Evaluation of Zebrafish Kidney Function Using a Fluorescent Clearance Assay

斑马鱼是一种流行的工具来模拟慢性肾脏病（CKD）。然而，它们的小尺寸使得它不可能用传统方法评估肾功能。我们描述一个荧光染料肾清除率法1，允许在CKD斑马鱼肾功能定量分析。

斑马鱼肾功能评价采用荧光测定间隙

The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish ...

evaluation-zebrafish-kidney-function-using-fluorescent-clearance

Research

JoVE Journal

Biology

13.1K 次观看.  University College London. 斑马鱼是一种流行的工具来模拟慢性肾脏病（CKD）。然而，它们的小尺寸使得它不可能用传统方法评估肾功能。我们描述一个荧光染料肾清除率法1，允许在CKD斑马鱼肾功能定量分析。 

视频: Evaluation of Zebrafish Kidney Function Using a Fluorescent Clearance Assay

Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is the nephron. Mammals are able to repair existing nephrons after injury, but lose the ability to form new nephrons soon after birth. In contrast to mammals, adult fish produce new nephrons (neonephrogenesis) throughout their lives in response to growth requirements or injury. Recently, lhx1a has been shown to mark nephron progenitor cells in the adult zebrafish kidney, however mechanisms controlling the formation of new nephrons after injury remain unknown. Here we show our method for robust and reproducible injury in the adult zebrafish kidney by intraperitoneal (i.p.) injection of gentamicin, which uses a noninvasive visual screening process to select for fish with strong but nonlethal injury. Using this method, we can determine optimal gentamicin dosages for injury and go on to demonstrate the effect of higher temperatures on kidney regeneration in zebrafish.

Here we present a reliable method to study adult kidney regeneration by inducing acute kidney injury by gentamicin injection. We show that injury is dependent on gentamicin dosage and environmental temperature using in situ hybridization to label lhx1a+ developing new nephrons.

肾再生的成年斑马鱼庆大霉素性损伤

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is ...

科研

发育生物学

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 ...

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method described here allows time-lapse analysis of normal and impaired kidney development in zebrafish embryos by using a fluorescence dissecting microscope equipped for structured illumination and z-stack acquisition. To visualize nephrogenesis, transgenic zebrafish (Tg(wt1b:GFP)) with fluorescently labeled kidney structures were used. Renal defects were triggered by injection of an antisense morpholino oligonucleotide against the Wilms tumor gene wt1a, a factor known to be crucial for kidney development.
The advantage of the experimental setup is the combination of a zoom microscope with simple strategies for re-adjusting movements in x, y or z direction without additional equipment. To circumvent focal drift that is induced by temperature variations and mechanical vibrations, an autofocus strategy was applied instead of utilizing a usually required environmental chamber. In order to re-adjust the positional changes due to a xy-drift, imaging chambers with imprinted relocation grids were employed.
In comparison to more complex setups for time-lapse recording with optical sectioning such as confocal laser scanning&#160;or light sheet microscopes, a zoom microscope is easy to handle. Besides, it offers dissecting microscope-specific benefits such as high depth of field and an extended working distance.
The method to study organogenesis presented here can also be used with fluorescence stereo microscopes not capable of optical sectioning. Although limited for high-throughput, this technique offers an alternative to more complex equipment that is normally used for time-lapse recording of developing tissues and organ dynamics.

The method described here allows time-lapse analysis of organ development in zebrafish embryos by using a fluorescence dissecting microscope capable of performing optical sectioning and simple strategies of readjustment to correct focal and planar drift.

配备了光学切片与成像定时使用解剖显微镜斑马鱼肾脏发展分析

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method ...

Precise Cellular Ablation Approach for Modeling Acute Kidney Injury in Developing Zebrafish

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore adequate kidney function after supportive treatment. However, a better understanding of how nephron cell death and repair occur on the cellular level is required to minimize cell death and to enhance the regenerative process. The zebrafish pronephros is a good model system to accomplish this goal because it contains anatomical segments that are similar to the mammalian nephron. Previously, the most common model used to study kidney injury in fish was the pharmacological gentamicin model. However, this model does not allow for precise spatiotemporal control of injury, and hence it is difficult to study cellular and molecular processes involved in kidney repair. To overcome this limitation, this work presents a method through which, in contrast to the gentamicin approach, a specific Green Fuorescent Protein (GFP)-expressing nephron segment can be photoablated using a violet laser light (405 nm). This novel model of AKI provides many advantages that other methods of epithelial injury lack. Its main advantages are the ability to &#34;dial&#34; the level of injury and the precise spatiotemporal control in the robust in vivo animal model. This new method has the potential to significantly advance the level of understanding of kidney injury and repair mechanisms.

This work presents a new in vivo model of segmental kidney injury using kidney GFP transgenic zebrafish. The model allows for the induction of the targeted ablation of kidney epithelial cells to show the cellular mechanisms of nephron injury and repair.

精确的细胞消融方法建模发展中的斑马鱼急性肾损伤

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore ...

Intravenous Microinjections of Zebrafish Larvae to Study Acute Kidney Injury

In this video article we describe a zebrafish model of AKI using gentamicin as the nephrotoxicant.  The technique consists of intravenous microinjections on 2 dpf zebrafish. This technique represents an efficient and rapid method to deliver soluble substances into the bloodstream of zebrafish larvae, allowing for the injection of 15-20 fish per hour. In addition to AKI studies, this microinjection technique can also be used for other types of experimental studies such as angiography.  We provide a detailed protocol of the technique from equipment required to visual measures of decreased kidney function. In addition, we also demonstrate the process of fixation, whole mount immunohistochemistry with a kidney tubule marker, plastic embedding and sectioning of the larval zebrafish. We demonstrate that zebrafish larvae injected with gentamicin show morphological features consistent with AKI: edema, loss of cell polarity in proximal tubular epithelial cells, and morphological disruption of the tubule.

We describe a technique of microinjecting the aminoglycoside, gentamicin, into 2 days post-fetilization (dpf) zebrafish larvae to induce acute kidney injury (AKI). We also describe a method for whole mount immunohistochemistry, plastic embedding and sectioning of zebrafish larvae to visualize the AKI mediated damage.

斑马鱼幼虫的研究急性肾损伤的静脉Microinjections

In this video article we describe a zebrafish model of AKI using gentamicin as the nephrotoxicant.  The technique consists of intravenous microinjections ...

生物学

Transplantation of Cells Directly into the Kidney of Adult Zebrafish

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic diseases1. However, most organs are not easily accessible, necessitating the need to develop surgical methods to gain access to these structures. In this video article, we describe a method for transplanting cells directly into the kidney of adult zebrafish, a popular model to study regeneration and disease2. Recipient fish are pre-conditioned by irradiation to suppress the immune rejection of the injected cells3. We demonstrate how the head kidney can be exposed by a lateral incision in the flank of the fish, followed by the injection of cells directly in to the organ. Using fluorescently labeled whole kidney marrow cells comprising a mixed population of renal and hematopoietic precursors, we show that nephron progenitors can engraft and differentiate into new renal tissue - the gold standard of any cell-based regenerative therapy. This technique can be adapted to deliver purified stem or progenitor cells and/or small molecules to the kidney as well as other internal organs and further enhances the zebrafish as a versatile model to study regenerative medicine.

Cell transplantation is an essential technique for studying tissue regeneration and for developing cell-based therapies of disease. We demonstrate here a microsurgical technique that permits the transplantation of genetically labeled cells directly into the kidney of adult zebrafish fish.

直接进入成年斑马鱼的肾细胞移植

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic ...

Dissection of the Adult Zebrafish Kidney

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem cells during normal homeostasis and disease states. The understanding of adult stem cells has broad reaching implications for the future of regenerative medicine1. For example, better knowledge about adult stem cell biology can facilitate the design of therapeutic strategies in which organs are triggered to heal themselves or even the creation of methods for growing organs in vitro that can be transplanted into humans1. The zebrafish has become a powerful animal model for the study of vertebrate cell biology2. There has been extensive documentation and analysis of embryonic development in the zebrafish3. Only recently have scientists sought to document adult anatomy and surgical dissection techniques4, as there has been a progressive movement within the zebrafish community to broaden the applications of this research organism to adult studies. For example, there are expanding interests in using zebrafish to investigate the biology of adult stem cell populations and make sophisticated adult models of diseases such as cancer5. Historically, isolation of the zebrafish adult kidney has been instrumental for studying hematopoiesis, as the kidney is the anatomical location of blood cell production in fish6,7. The kidney is composed of nephron functional units found in arborized arrangements, surrounded by hematopoietic tissue that is dispersed throughout the intervening spaces. The hematopoietic component consists of hematopoietic stem cells (HSCs) and their progeny that inhabit the kidney until they terminally differentiate8. In addition, it is now appreciated that a group of renal stem/progenitor cells (RPCs) also inhabit the zebrafish kidney organ and enable both kidney regeneration and growth, as observed in other fish species9-11. In light of this new discovery, the zebrafish kidney is one organ that houses the location of two exciting opportunities for adult stem cell biology studies. It is clear that many outstanding questions could be well served with this experimental system. To encourage expansion of this field, it is beneficial to document detailed methods of visualizing and then isolating the adult zebrafish kidney organ. This protocol details our procedure for dissection of the adult kidney from both unfixed and fixed animals.
Dissection of the kidney organ can be used to isolate and characterize hematopoietic and renal stem cells and their offspring using established techniques such as histology, fluorescence activated cell sorting (FACS)11,12, expression profiling13,14, and transplantation11,15. 
We hope that dissemination of this protocol will provide researchers with the knowledge to implement broader use of zebrafish studies that ultimately can be translated for human application.

The zebrafish kidney is home to both renal and hematopoietic adult stem/progenitor cells, and represents an outstanding opportunity to study these cell types and their progeny in a vertebrate model organism. Here, we demonstrate a detailed dissection procedure that enables the researcher to identify and surgically remove the adult zebrafish kidney, which can be used for applications such as cell isolation, transplantation, and expression studies of kidney and/or blood cell populations.

成年斑马鱼肾脏的解剖

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem ...

Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys are composed of functional units known as nephrons, which are highly conserved with other vertebrates, including mammals. Research in zebrafish has recently demonstrated that two distinctive phenomena transpire after adult nephrons incur damage: first, there is robust regeneration within existing nephrons that replaces the destroyed tubule epithelial cells; second, entirely new nephrons are produced from renal progenitors in a process known as neonephrogenesis. In contrast, humans and other mammals seem to have only a limited ability for nephron epithelial regeneration. To date, the mechanisms responsible for these kidney regeneration phenomena remain poorly understood. Since adult zebrafish kidneys undergo both nephron epithelial regeneration and neonephrogenesis, they provide an outstanding experimental paradigm to study these events. Further, there is a wide range of genetic and pharmacological tools available in the zebrafish model that can be used to delineate the cellular and molecular mechanisms that regulate renal regeneration. One essential aspect of such research is the evaluation of nephron structure and function. This protocol describes a set of labeling techniques that can be used to gauge renal composition and test nephron functionality in the adult zebrafish kidney. Thus, these methods are widely applicable to the future phenotypic characterization of adult zebrafish kidney injury paradigms, which include but are not limited to, nephrotoxicant exposure regimes or genetic methods of targeted cell death such as the nitroreductase mediated cell ablation technique. Further, these methods could be used to study genetic perturbations in adult kidney formation and could also be applied to assess renal status during chronic disease modeling.

The zebrafish adult kidney is an excellent system for renal regeneration and disease studies. An essential aspect of such research is the assessment of nephron structure and function. This protocol describes several methodologies that can be implemented to assess nephron tubule composition and to evaluate renal reabsorption.

肾单位的组成与功能的成年斑马鱼肾脏分析

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys ...

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.

The zebrafish is an excellent experimental organism to study vertebrate developmental processes and model human disease. Here, we describe a protocol on how to perform a manual high-throughput chemical screen in zebrafish embryos with a whole-mount in situ hybridization (WISH) read-out.

手册小分子屏幕接近高通量使用斑马鱼胚胎

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology ...

Acute Kidney Injury Model Induced by Cisplatin in Adult Zebrafish

Cisplatin is commonly used as chemotherapy. Although it has positive effects in cancer-treated individuals, cisplatin can easily accumulate in the kidney due to its low molecular weight. Such accumulation causes the death of tubular cells and can induce the development of Acute Kidney Injury (AKI), which is characterized by a quick decrease in kidney function, tissue damage, and immune cells infiltration. If administered in specific doses cisplatin can be a useful tool as an AKI inducer in animal models. The zebrafish has appeared as an interesting model to study renal function, kidney regeneration, and injury, as renal structures conserve functional similarities with mammals. Adult zebrafish injected with cisplatin shows decreased survival, kidney cell death, and increased inflammation markers after 24 h post-injection (hpi). In this model, immune cells infiltration and cell death can be assessed by flow cytometry and TUNEL assay. This protocol describes the procedures to induce AKI in adult zebrafish by intraperitoneal cisplatin injection and subsequently demonstrates how to collect the renal tissue for flow cytometry processing and cell death TUNEL assay. These techniques will be useful to understand the effects of cisplatin as a nephrotoxic agent and will contribute to the expansion of AKI models in adult zebrafish. This model can also be used to study kidney regeneration, in the search for compounds that treat or prevent kidney damage and to study inflammation in AKI. Moreover, the methods used in this protocol will improve the characterization of tissue damage and inflammation, which are therapeutic targets in kidney-associated comorbidities.

This protocol describes the procedures to induce Acute Kidney Injury (AKI) in adult zebrafish using cisplatin as a nephrotoxic agent. We detailed the steps to evaluate the reproducibility of the technique and two techniques to analyze inflammation and cell death in the renal tissue, flow cytometry and TUNEL, respectively.

成人斑马鱼中西斯铂诱导的急性肾损伤模型

Cisplatin is commonly used as chemotherapy. Although it has positive effects in cancer-treated individuals, cisplatin can easily accumulate in the kidney ...