作者希望到她的协助下，在实验室消耗品的管理感谢Esmaili陈怡蓉。 AA是收件人的卓越中心阿尔茨海默氏病的研究和护理学院医学科学院，埃迪斯科文大学的博士奖学金。 MC接收博士，扶轮社，珀斯的资金支持。 AM，GV，KT，RNM由麦国琛阿尔茨海默氏症的研究基础。

1。系统维护<ol><li>斑马鱼被保存在一个循环体系，不断过滤器和充气系统的水，以保持水质，需要一个健康的水生环境。循环的系统还有助于去除过量的食物和鱼的排泄物。不同的公司提供了斑马鱼系统，但水生栖息地，美国在我们的实验室，我们使用的系统。室温或油箱温度一般保持在26-28.5℃，并在光照条件之间是十四时一十小时（光照：黑暗）。斑马鱼系统由水生栖息地（ 例如 ，台式系统）的费用约9,000美元。这种台式系统具有两个货架可以容纳6个10升，12 3升，或20个1.5升罐每个货架上。鱼的多行（ 例如 ，转基因，突变体，野生型）也可以被容纳在同一系统上。 </li><li>不同种的一组过滤器，用于在系统中。在我们的系统中，所有的坦克每年的水小规模企业，通过一个120微米的过滤垫，50微米罐过滤，生物过滤，活性炭吸附过滤器和紫外线消毒器，然后分发流回油箱。 de-chlorinated/aged水斑马鱼系统中使用。水可以氯化老化至少48小时。在理想的条件下，水应保持在水库泵循环水，以保持温暖，并加快除氯。 </li><li>应每天检查系统的水的pH值，并保持在6.8和7.5之间。在必要时，应该使用碳酸氢钠以提高pH值。 </li><li>应定期清洗鱼缸。要清洁鱼缸，关闭水流的该型坦克，沥干多余的水向后倾斜罐的罐小心地从系统中删除。将是显而易见的，在槽的底部和侧面的污垢和藻类生长。 </li><li>将挡板到一个干净的罐，并填写与氯化水（又名系统娃之三）。小心渔网的鱼转移到该型坦克。盖上盖子，转让的标签名称的坦克。小心地放置在干净的坦克进入了系统和交换机的供水。 </li><li>净化鱼网，喷以70％的乙醇，在水冲洗，并让它干，然后再重新使用。拆下挡板从污槽和喷洒的两个部分用70％的乙醇。用自来水彻底冲洗，并允许坦克和挡板要重新使用前充分干燥。 </li><li>循环系统的过滤器必须定期进行检查和修改，以确保其正常功能。这些过滤器应定期更换，以确保适当和清洁水供应的所有鱼缸。 </li><li>的120微米过滤器垫通常是重新定位或者每天更换;朝向水流重新定位，以用它完全与一个新的更换前。 </li><li>筒式过滤器应每周更换一次。要更改罐过滤器，去除过滤器单元的扭曲蚂蚁扳手或手iclockwise与。将塑料片或毛巾以下，以防止水或吸收溢漏。用一个新的过滤器更换碳罐和适合的过滤器单元回系统中，小心地拧紧用的手，如有必要的扳手。 </li><li>活性炭过滤器应改为半月刊（每两个星期）。要更改的碳过滤器，仔细去除碳过滤器单元，用扳手。丢弃用过的活性炭，取而代之的是新的活性炭。重新装上碳的持有人，并把它放回过滤器单元。适合的过滤器单元的背面进入系统。切换系统，并检查，水流入到过滤器。如果有必要，冲洗一些水通过管道清洁之前嵌合到系统中的过滤器管道中的污垢。 </li><li>生物过滤器应每半年清洗。通常位于之间的碳罐和碳过滤器，在循环系统中的生物过滤器。要改变生物过滤器，REM奥雅纳从系统中的过滤器单元。释放压力从过滤器按压力释放按钮。用扳手拧开过滤器。一般情况下，两个人都需要这一步。卸下的过滤器单元的盖体。清空的内容的过滤器成筛分的的容器分开siporax从水中。注：Siporax是一个很好的孔隙生物过滤介质，有能力进行硝化和反硝化。 </li><li> ，如果siporax非常脏更换新的。填写过滤器单元与系统的水（含氯的水），盖上盖子，返回过滤装置的供水系统和开关。注意：在一个适应的水族馆，siporax将几个硝化细菌。这些微生物是保持水族箱内的氮循环，并消除一手住房的生物过滤器（脏siporax），其次是亚硝酸盐峰值，可能会导致严重的氨尖峰而n：EW生物过滤器（新siporax）重新建立。这些中间状态的氮循环对水生生物有毒，可能会杀死如果没有回应，适当的斑马鱼。因此，重要的是有继发其他生物过滤器中的斑马鱼的住房制度（ 例如 ，在集水箱水生生境系统），让这些重要微生物的新siporax上的快速复育。 </li><li> UV过滤器是用来控制系统的生物污染物（如细菌），应改为每九十二个月。应当指出，过滤紫外线的消毒剂量率是〜110毫焦/厘米2在灯的寿命的开始和剂量率的降低超过时间的过程中，因此即使当它出现仍然是必要的，以取代的EUROPAGES功能。 </li></ol> 2。馈送<ol><li>斑马鱼可喂干（食物从100微米大小的幼虫吨O 300/400微米的成鱼）或活的食物（丰年虾）。丰年虾（ 卤虫 ）卵可从当地的宠物商店，可以在实验室中孵化以下简单的步骤如下所述。 <ol class="lalpha"><li>溶解红海盐老年水置于烧杯中，与更好的溶解度在磁力搅拌器上的盐。另外，盐可以溶解在水中，通过与曝气管曝气。即时海洋盐也可使用作为孵化的解决方案，如果不可用食用盐红色的海洋。丰年虾可以容忍宽范围的盐度，但是，我们在30-35克/ L红色的海洋咸水孵化的丰年虾（10-15克）。注：如果没有现成的丰年虾卵，然后再封装盐水虾“的去”前分批将它们添加到虾孵化场，也可用于后在当地的宠物商店。这也可能避免频繁旅行的宠物商店。 </li><li>填写丰年虾孵化器与一个公升水和1.2汤匙/升的浓度添加的虾卵。大力通气的孵化器，空气泵和离开丰年虾卵孵化〜48小时。 注意：应该注意的是，未增浓盐水虾失去营养价值每隔一小时孵化后。因此，它可能是适合要么舱口超过24小时或丰富超过48小时，以便获得最大的营养价值从盐水虾。 </li><li>从孵化系统的废水中加入含漂白剂的消毒和设置稍后检疫废物。 </li></ol></li><li>要收集丰年虾，清除管道中的空气，让文化为4-5分钟解决，但不超过10分钟。孵化丰年虾聚集在底部的孵化器。 <ol class="lalpha"><li>收集丰年虾的孵化器的底部使用自来水。其中包括未孵化的丰年虾卵的初始流量丢弃。 </li><l我后丢弃的未孵化的丰年虾，收集孵化丰年虾将被用来作为斑马鱼的食物。 </li><li>分开的盐水虾，咸的水，用盐水虾收藏网（〜350微米的尼龙网）。冲洗盐水虾从网系统的水容器使用。 </li></ol></li><li>将收集的盐水洗涤虾一般存在于高浓度的底部的容器，给它一个相当橙色。 </li><li>饲料的盐水洗涤虾到斑马鱼使用吸移管或滴管/ squeezy的瓶。分配的食物的量取决于个人坦克的人口规模。斑马鱼是普遍接受的比率在每天的食物中获得4％的体重。斑马鱼千万不要吃得太多，因为这可能会增加在水中的硝酸盐含量，可能会影响其繁殖，活力，有些鱼可能是由于饮食过量而死亡。 <ol class="lalpha"><li>当注入的食物进入水，饥饿的鱼游，赶上丰年虾。 </li></ol></li><li>干喂养可利用的水生生态系统的/水生生境简单的基于Spring的鱼食分配器。另外，干燥的进料也可以使用一个简单的勺子来执行，或通过切割塑料滴管斜用剪刀给它的外观的一小汤匙。 </li></ol> 3。选育<ol><li>斑马鱼开始繁育开始时的光。受精卵可通过在水箱养殖或成对繁殖。虽然在水箱养殖是更多的劳动效率，并定期的胚胎收集在我们的实验室实施基因或突变时，最好是从鱼个体进行筛选，配对繁殖。 </li><li>在水箱养殖，聚集在坦克饲养员和缓慢下降到鱼缸发病后的光。此外，在水箱养殖设置可以在鱼缸放置过夜。 </li><li> ł屋檐坦克种鸡约15分钟，让鱼交配，然后删除，饲养员从水箱和收集鸡蛋。 </li><li>配对繁殖通常在下午晚些时候喂奶后。 </li><li>组装养殖池，并填写账龄系统的水。 </li><li>一名女性和一名男性转移到养殖池的两侧。女性可以区别于男性，因为其更大的下腹部。男性也可以从女性的区别，因为他们更修长，颜色较深高于女性。此外，男性在臀鳍有黄色着色相比，女性（参见图1）。当疑问，寻找产卵的雌性斑马鱼（ 见图4）。 </li><li>卸下分频器第二天早晨在发病后不久的光。允许交配发生静置20分钟或直到足够数量的胚胎在水箱底部铺设。 </li><li>繁殖季结束后，鱼返回到他们的坦克。使用过滤器收集的鸡蛋。 </li><li>系统的水彻底洗净胚胎。 </li><li>将胚胎转移到陪替氏培养皿KH 2漂洗过滤器与胚胎介质;又名EM3氯化钠（NaCl，13.7毫摩尔;氯化钾，0.54毫摩尔，用MgSO 4，1.0mM的的CaCl 2，1.3毫摩尔的Na 2 HPO 4，0.025毫摩尔; 0.044毫米; PO 4，碳酸氢钠 ，4.2毫米）。 </li><li>胚胎可以在显微镜下观察。受精卵，然后分离从用一个针头和一个移液管（参见图3）的未受精的卵。 </li></ol> 4。提高幼虫<ol><li>受精卵保持的培养箱中（〜28.5℃）下72小时，直至孵出幼虫。 </li><li>现在的幼虫是的绒毛膜和游泳的自由，他们准备转移到一个主要鱼缸。幼虫需要从5天受精后（dpf）的馈送，并保持在胚胎培养基（组合物中所述第3部分，10）或系统的水。幼虫可保持在第二轮的菜肴〜50％或更多的水其中每天改变。水改应包括去除死亡或患病的幼虫和任何其他碎片。 </li><li>轻轻的幼虫转移到坦克，包含一个小型挡板（300-400微米）。应除去死亡和患病的幼虫，每天几毫升的水应缓慢加入。 </li><li> 14天之后，幼虫坦克可以被搁置到系统中，并附带一条小溪的循环水（每秒1-2滴）。在幼虫生长，水流量可以增加。不同尺寸的挡板，可以根据使用上的幼虫的大小（ 例如，挡板尺寸300-400，500，700-750，和1000微米）和一个正常的塑料挡板应用于成人鱼类。 </li><li>它通常需要3个月的胚胎发育到性成熟的成年。 </li></ol>

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没有利益冲突的声明。

斑马鱼起源在印度北部的恒河，并正在成为流行在研究中的成虫及幼虫阶段2，斯宾塞等人的审查。3。斑马鱼比其他的动物模型，如繁殖力高，便于维护，光学间隙的胚胎，胚胎发育迅速，维护成本低，具有几个优点。它们是适于遗传操作6，适合于高通量药物筛选4,5。他们的施肥是外部的，这是有利的，他们使用的发育生物学家。由于这些有利的特点，斑马鱼的日益普及，8，7，药理遗传学和行为学研究9,10。有许多挑战，保持斑马鱼设施和斑马鱼的饲养获得胚胎。在这里，我们描述我们的经验和建议，在应对这些挑战和outline一个协议，用于系统维护，饲养，繁殖和饲养的幼虫。 系统维护为了保持在健康状态下的斑马鱼，重要的是为他们提供一个清洁的环境中正常工作的水族馆系统。这方面的一个重要组成部分，是定期改变系统的过滤器，使所有的坦克获得适当的水流量和干净的水。至关重要的是要避免失败的循环供水系统管道堵塞，由于每罐。管道可以使用高于正常的水的压力和流量，如果待清洁堵塞确实发生。在理想的情况下，10％左右的系统的水应每天更换，以保持良好的水质。另外，水可以被替换，而改变密封罐或碳过滤器。这可以确保除去沉积在配管连接这些滤波器的脏物。应定期检查水的质量。碱度，pH，TEMPERAT参数，如余吕杏茜，硬 ​​度，氨氮，溶解氧，盐度，电导率应考虑的重要因素，在系统的水的质量（有关详细信息，请参阅表2）。最起码的硝酸盐，pH值和温度应定期监控，以确保良好水质住房斑马鱼。理想的硝酸盐（NO 3 - ）的水平&lt;50 mg / L的11;如果高，这些水平，可以减少更换的循环系统中的水与新鲜的系统水。有时，过滤器不适合和泄漏，因此，建议检查是否有泄漏后更换过滤器。如果水流从该主贮存器被阻塞后，无论是改变水泵或改变一个过滤器，水流量可以恢复由任何松动或为几秒钟，以除去过滤器释放任何在管道中所产生的真空。更换过滤器所需的时间可能有所不同，这取决于各种因素，如总生物负荷Øn的系统，其他的过滤器清洁，和污垢沉积在管道中。因此，过滤器，应立即改变，如果他们出现脏或如果所有的坦克都没有接收到正确的供水。此外，还建议鱼网，用70％乙醇清洗，并在水中漂洗，进行除污，并让其干燥之前被重新利用。干燥确保蒸发乙醇否则是有毒的鱼。 大多数的斑马鱼系统使用氯化消毒的自来水，但是，有些系统使用去离子水。重要的是保持系统的水的电导率在300〜1,500μS之间，因为这会减少的能量的鱼需要保持身体的盐。因此，斑马鱼不能保持在去离子水中，除非的盐已加入，以保持最佳的电导率水平。存在这样的风险的系统中的水的浓度尽可能高的铜，如果自来水的碳过滤器的使用，因为不除去铜。因此，U表面增强拉曼光谱检查，并在可能的情况下避免铜管铜含量。 馈送斑马鱼绝不能吃得太多，因为这可能会增加在水中的硝酸盐含量，可能会影响它们的繁殖，或生存能力，因为有些鱼可能是由于饮食过量而死亡。我们建议在任何一个超过一缸鱼可以在10分钟内完成进料没有提供更多的食物。这是非常重要的，，去除盐分前的丰年虾喂养它们的斑马鱼作为多余的盐分浓度会导致死亡。如果需要更多的斑马鱼鸡蛋，鱼可以喂一天三次。每天清洁，饲养员鱼缸也可以提高产蛋量的水平。 对水生生物的栖息地系统喂奶时，我们通常会关闭水泵，气泵让鱼所吃的食物10分钟。这将减少食物量，洗净，放入过滤器。然而，用户必须小心，以REM烬后再次打开这些泵。 选育斑马鱼是在3个月和18个月的年龄通常在最佳饲养条件。不应该进行配对繁殖连续两天11然而，在水箱养殖可以作为一个坦克每天可以容纳很多的鱼，从而降低了对鱼饲养两天，在一排的机会。育种应定期进行，即使蛋不是必需的。此过程将确保维持鱼的繁殖周期。建议，有更多的女性比男性在繁殖的设置。雄性斑马鱼改变他们的女性伴侣，每天12，进一步支持这一建议。此外，在我们的实验室中，我们最初遇到的问题与繁殖，然而，女性多于男性在繁殖的建立有助于解决这个问题。此外，喂食高蛋白质的小故事NT饮食和盐水虾，每天两三次，从不同的坦克（从不同的父母），维持滋生的温度设置在27和28之间°C，挤压腹部的女性，卵巢管阻塞的混合鱼轻柔的按摩，进一步提高产蛋量。我们建议保持创纪录的鱼线/来源，以避免在兄弟姐妹之间滋生。这进一步提高了胚胎生产。保持的记录的数量从每罐鱼的胚胎奠定还建议。这有助于保持最佳的养殖鱼缸的轨道，并采取措施，提高养殖的鱼不产卵。 提高幼虫饲喂幼虫开始从5 DPF（天受精后）。低龄幼虫可以喂干粮〜100微米大小（ 例如 ，ZM100）或活的食物，如草履虫和轮虫（刺激经济增长）。食物的大小可以慢慢增加至200微米（ 例如 ZM200）或300/400微米（ 例如 ZM300）。人口的成鱼应该是6-7每升水的鱼。这种做法被推荐为更好地维护BOD（生物需氧量）的坦克。

<table border="1"><tbody><tr><td> 试剂/设备名称 </td><td> 公司 </td><td> 目录编号 </td><td></td></tr><tr><td>斑马鱼的循环系统</td><td>水生生物栖息地，美国</td><td> AHAB独立，台式系统</td><td></td></tr><tr><td>碳酸氢钠</td><td> Sigma Aldrich公司</td><td> S6297 </td><td></td></tr><tr><td>鱼的食物机/干喂食器</td><td>水生生物栖息地，美国</td><td> AH19 </td><td></td></tr><tr><td>显微镜</td><td>奥林巴斯</td><td> SZX12 </td><td></td></tr><tr><td>干粮</td><td> ZM鱼的食物，英国</td><td> ZM100，ZM200，ZM300 </td><td></td></tr><tr><td>丰年虾卵</td><td>盐河公司，美国</td><td>优等品</td><td></td></tr><tr><td>在坦克饲养者</td><td>水生生物栖息地，美国</td><td> ITSTS-A </td><td></td></tr><tr><td>活性炭</td><td>宾州贝齿亲蟹，美国</td><td> PBC3MF </td><td></td></tr><tr><td>育种坦克</td><td>水生生物栖息地，美国</td><td> SBTANK（1L） 的饲养员坦克2（2L） </td><td></td></tr><tr><td>红海盐</td><td>美国红海盐， </td><td>当地的宠物商店/ <a href="http://www.redseafish.com" target="_blank">www.redseafish.com</a> </td><td></td></tr><tr><td>过滤器（罐，过滤垫，UV） </td><td>水生生物栖息地，美国</td><td> <a href="http://www.aquatichabitats.com" target="_blank">aquatichabitats.com</a> </td><td></td></tr><tr><td> Siporax媒体</td><td>水生生物栖息地，美国</td><td> BF 820 </td><td></td></tr><tr><td>盐水虾网</td><td>水生生物栖息地，美国</td><td> BSN1 </td><td></td></tr><tr><td>丰年虾孵化器</td><td>水生生物栖息地，美国</td><td> BS252 </td><td></td></tr><tr><td>挡板</td><td>水生生物栖息地，美国</td><td> <a href="http://www.aquatichabitats.com" target="_blank">aquatichabitats.com</a> </td><td></td></tr><tr><td></td><td></td><td></td><td> 表1，表在本协议中所使用的特定的试剂和设备。 </td></tr></tbody></table>

regular care and maintenance of a zebrafish (danio rerio) laboratory: an introduction

本协议描述了斑马鱼实验室的定期保养和维护。斑马鱼是现在日益普及，遗传学，药理学和行为学研究。斑马鱼的遗传序列相似性与人类和脊椎动物，被用作各种人类疾病的动物模型条件。斑马鱼相比其他常见的脊椎动物模型的优点包括繁殖力高，维护成本低，胚胎透明，快速的发展。由于在斑马鱼研究的兴趣，需要建立和维持一个高效的斑马鱼的住房设施的刺激也越来越多。虽然文学是为维护斑马鱼实验室，一个简洁的视频协议是缺乏的。该视频演示了普通住房的协议，斑马鱼幼虫的饲养，繁殖和饲养。这个过程将有助于研究人员理解的自然行为和最佳工艺条件为zebrafisĤ牧，因此，解决实验问题源于鱼的饲养条件。该协议将规划，建立了斑马鱼的实验室，同时也为毕业的学生正打算利用斑马鱼作为动物模型的研究人员有很大帮助。

斑马鱼的住房和维护更容易，更便宜比传统的啮齿动物模型。数千名斑马鱼可以被安置在一个小型实验室。作为此协议的结果，研究人员将能够管理斑马鱼设施，将提供健康的条件下的斑马鱼。此外，下面的插图将有助于识别受精卵，成年斑马鱼，和他们的食物。说明的雄性斑马鱼（ 图1A）和雌性斑马鱼（ 图1B和1C），以帮助研究人员之间的雄性和雌性斑马鱼养殖的区别。 图2描述了微观角度，盐水虾12倍（ 图2A），90X（图2B），一个单一的盐水虾和90X（ 图2C）的未受精的盐水虾蛋。这将有助于了解受精，受精卵的丰年虾为支柱的区别呃喂养的目的。受精和未受精的卵的是在图3中所示， 图3A示出了微观图受精和未受精的胚胎。未受精胚胎通常是不透明和/或破裂的单元格内的绒毛膜（黑色箭头），而受精胚胎，完整和不断增长的下一个细胞分裂的状态（详细阅读不同阶段的斑马鱼胚胎参见图1），会出现。放大视图的一个未受精的卵子受精，显示在图3B和3C。图4说明了一个雌性斑马鱼的产卵器，以帮助研究人员之间的雄性和雌性斑马鱼区分开来。 中可能出现的斑马鱼实验室的几个关键问题，包括个人/所有的坦克在住房制度，水质较差，管道泄漏或水库的循环系统堵塞的供水。另外，在获得胚胎繁殖的问题可能是另外一个问题。故障排除这些问题将在下面讨论。 <img src="/files/ftp_upload/4196/4196fig1.jpg" alt="图1" /> 图1是示的阳斑马鱼（A）和女性斑马鱼（B，C）。 <img src="/files/ftp_upload/4196/4196fig2.jpg" alt="图2" /> 图2：微观上看，盐水虾在12X（A），一个盐水虾在90X（B），未受精的丰年虾卵在90X（C）。 <img src="/files/ftp_upload/4196/4196fig3.jpg" alt="图3" /> 图3。微观角度（16X）的未受精的卵子受精和未受精的卵，其中只有两个是未受精的鸡蛋，是用黑色箭头表示（A）。更高的放大倍率视图（90X）的受精（B）和（C）未受精卵。 <img src="/files/ftp_upload/4196/4196fig4.jpg" alt="图4" /> 图4。一只雌性斑马鱼的产卵器（用黑色箭头表示）的说明。 <table border="1"><tbody><tr><td fo:width="2in"> 参数 </td><td fo:width="3in"> 最佳范围 </td></tr><tr><td>碱度</td><td> 50-150 mg / L的CaCO 3的 </td></tr><tr><td> pH值</td><td> 6.8-7.5（6.0-8.5耐受性） </td></tr><tr><td>温度</td><td> 26-28.5°C </td></tr><tr><td>硬度</td><td> 50-100 mg / L的CaCO 3的 </td></tr><tr><td>联合国离子氨</td><td> &lt;0.02 mg / L的</td></tr><tr><td>硝酸盐（NO 3 - ） </td><td> &lt;50毫克/升</td></tr><tr><td>亚硝酸盐（NO 2 - ） </td><td> &lt;0.1 ​​mg / L的</td></tr><tr><td>溶解氧</td><td> &gt; 6.0 mg / L的</td></tr><tr><td>盐度</td><td> 0.5-1克/ L </td></tr><tr><td>电导率</td><td> 300 -1,500μS </td></tr></tbody></table> 表2中。水质参数水质参数。在斑马鱼的水族馆11环境参数的最佳范围。

Watch this Scientific Journal Video about Regular Care and Maintenance of a Zebrafish Danio rerio Laboratory: An Introduction at JoVE.com

Regular Care and Maintenance of a Zebrafish Danio rerio Laboratory: An Introduction

该协议概述了定期的维修和保养，以保持最佳斑马鱼的饲养条件。视频显示系统维护，普通住房，饲养，繁殖，提高斑马鱼幼虫的协议。

定期保养和维护的斑马鱼（<em&gt;斑马鱼</em&gt;）实验室简介

This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and ...

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Research

JoVE Journal

Biology

Regular Care and Maintenance of a Zebrafish (Danio rerio) Laboratory: An Introduction

88.4K 次观看.  Edith Cowan University. 该协议概述了定期的维修和保养，以保持最佳斑马鱼的饲养条件。视频显示系统维护，普通住房，饲养，繁殖，提高斑马鱼幼虫的协议。

视频: Regular Care and Maintenance of a Zebrafish Danio rerio Laboratory: An Introduction

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish (Danio rerio)

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the cellular and molecular features of the epithelia, which are mainly composed of two types of cells: hair cells (HCs) and supporting cells (SCs). Here we choose to study the inner ear sensory epithelia in adult zebrafish not only because the epithelial structures are highly conserved in all vertebrates studied, but also because the adult zebrafish is able to regenerate HCs, an ability that mammals lose shortly after birth. We use the inner ear of adult zebrafish as a model system to study the mechanisms of inner ear HC regeneration in adult vertebrates that could be helpful for clinical therapy of hearing/balance deficits in human as a result of HC loss.
Here we demonstrate how to do gross and fine dissections of inner ear sensory epithelia in adult zebrafish. The gross dissection removes the tissues surrounding the inner ear and is helpful for preparing tissue sections, which allows us to examine the detailed structure of the sensory epithelia. The fine dissection cleans up the non-sensory-epithelial tissues of each individual epithelium and enables us to examine the heterogeneity of the whole epithelium easily in whole-mount epithelial samples.

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish Danio rerio

The inner ear sensory epithelium of adult zebrafish is a good model system for understanding the mechanisms of hair cell regeneration in adult vertebrates. This protocol demonstrates the fine dissection of the epithelia, through which we can get tissue samples for studying the regenerative events at cellular and subcellular levels.

格罗斯和精细解剖内耳感觉上皮细胞在成年斑马鱼（斑马鱼）

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the ...

科研

生物学

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work examining post-embryonic through adult cardiac development has been limited7-10. Examining the changing morphology of the maturing and aging heart are restricted by the lack of techniques available for staging and isolating juvenile and adult hearts. In order to analyze heart development over the fish's lifespan, we dissect zebrafish hearts at numerous stages and photograph them for further analysis11. The morphological features of the heart can easily be quantified and individual hearts can be further analyzed by a host of standard methods. Zebrafish grow at variable rates and maturation correlates better with fish size than age, thus, post-fixation, we photograph and measure fish length as a gauge of fish maturation. This protocol explains two distinct, size dependent dissection techniques for zebrafish, ranging from larvae 3.5mm standard length (SL) with hearts of 100&mu;m ventricle length (VL), to adults, with SL of 30mm and VL 1mm or larger. Larval and adult fish have quite distinct body and organ morphology. Larvae are not only significantly smaller, they have less pigment and each organ is visually very difficult to identify. For this reason, we use distinct dissection techniques.
We used pre-dissection fixation procedures, as we discovered that hearts dissected directly after euthanization have a more variable morphology, with very loose and balloon like atria compared with hearts removed following fixation. The fish fixed prior to dissection, retain in vivo morphology and chamber position (data not shown). In addition, for demonstration purposes, we take advantage of the heart (myocardial) specific GFP transgenic Tg(myl7:GFP)twu34 (12), which allows us to visualize the entire heart and is particularly useful at early stages in development when the cardiac morphology is less distinct from surrounding tissues. Dissection of the heart makes further analysis of the cell and molecular biology underlying heart development and maturation using in situ hybridization, immunohistochemistry, RNA extraction or other analytical methods easier in post-embryonic zebrafish. This protocol will provide a valuable technique for the study of cardiac development maturation and aging.

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

A clear, standardized method for dissection and isolation of the zebrafish heart at multiple developmental stages are described. Annotation and quantification techniques are also discussed.

幼虫，少年和成年斑马鱼的心脏解剖，斑马鱼</em

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work ...

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish. Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated.1 These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present a systematic approach for developing physiologically relevant, sensitive and specific in vivo assays for interpreting variation in human pathology. Transient genetic manipulation via microinjection of WT and mutant human mRNA and morpholino (MO) antisense oligonucleotides harness the tractability of the developing zebrafish embryo to rapidly assay pathogenic mutations, especially, but not exclusively, in the context of human developmental disorders.

在体内病态的人类基因组建模使用斑马鱼

Here,  we present methods for the development of assays to query potentially clinically  significant nonsynonymous changes using in  vivo complementation ...

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of energy homeostasis and obesity. The small size of zebrafish allows for assays on embryos to be conducted in a 96- or 384-well plate format, Morpholino and CRISPR based technologies promote ease of genetic manipulation, and drug treatment by bath application is viable. Moreover, zebrafish are ideal for forward genetic screens allowing for novel gene discovery. Given the relative novelty of zebrafish as a model for obesity, it is necessary to develop tools that fully exploit these benefits. Herein, we describe a method to measure energy expenditure in thousands of embryonic zebrafish simultaneously. We have developed a whole animal microplate platform in which we use 96-well plates to isolate individual fish and we assess cumulative NADH2 production using the commercially available cell culture viability reagent alamarBlue. In poikilotherms the relationship between NADH2 production and energy expenditure is tightly linked. This energy expenditure assay creates the potential to rapidly screen pharmacological or genetic manipulations that directly alter energy expenditure or alter the response to an applied drug (e.g. insulin sensitizers).

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism for the study of energy homeostasis. By utilizing a NADH2 sensitive redox indicator, alamar Blue, we have developed an assay that measures the metabolic rate of zebrafish larvae in a 96 well plate format and can be applied to drug or gene discovery.

高通量<em&gt;斑马鱼</em&gt;能量消耗分析

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of ...

Maintenance of a Drosophila melanogaster Population Cage

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life cycle of Drosophila enables collection of large quantities of material from embryos, larvae, pupae and adult flies, in a synchronized way, at a low economic cost. A major strategy for propagating large numbers of flies is the use of a fly population cage. This useful and common tool in the Drososphila community is an efficient way to regularly produce milligrams to tens of grams of embryos, depending on uniformity of developmental stage desired. While a population cage can be time consuming to set up, maintaining a cage over months takes much less time and enables rapid collection of biological material in a short period. This paper describes a detailed and flexible protocol for the maintenance of a Drosophila melanogaster population cage, starting with 1.5 g of harvested material from the previous cycle.

Maintenance of a Drosophila melanogaster Population Cage

This manuscript reports a detailed protocol for culturing, on a regular basis, a population of Drosophila melanogaster using a fly population cage.

的维持<em&gt;果蝇</em&gt;人口笼

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

肾毒素显微注射斑马鱼到模型急性肾损伤

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

Detecting Estrogenic Ligands in Personal Care Products using a Yeast Estrogen Screen Optimized for the Undergraduate Teaching Laboratory

The Yeast Estrogen Screen (YES) is used to detect estrogenic ligands in environmental samples and has been broadly applied in studies of endocrine disruption. Estrogenic ligands include both natural and manmade &#34;Environmental Estrogens&#34; (EEs) found in many consumer goods including Personal Care Products (PCPs), plastics, pesticides, and foods. EEs disrupt hormone signaling in humans and other animals, potentially reducing fertility and increasing disease risk. Despite the importance of EEs and other Endocrine Disrupting Chemicals (EDCs) to public health, endocrine disruption is not typically included in undergraduate curricula. This shortcoming is partly due to a lack of relevant laboratory activities that illustrate the principles involved while also being accessible to undergraduate students. This article presents an optimized YES for quantifying ligands in personal care products that bind estrogen receptors alpha (ER&#945;) and/or beta (ER&#946;). The method incorporates one of the two colorimetric substrates (ortho-nitrophenyl-&#946;-D-galactopyranoside (ONPG) or chlorophenol red-&#946;-D-galactopyranoside (CPRG)) that are cleaved by &#946;-galactosidase, a 6-day refrigerated incubation step to facilitate use in undergraduate laboratory courses, an automated application for LacZ calculations, and R code for the associated 4-parameter logistic regression analysis. The protocol has been designed to allow undergraduate students to develop and conduct experiments in which they screen products of their choosing for estrogen mimics. In the process, they learn about endocrine disruption, cell culture, receptor binding, enzyme activity, genetic engineering, statistics, and experimental design. Simultaneously, they also practice fundamental and broadly applicable laboratory skills, such as: calculating concentrations; making solutions; demonstrating sterile technique; serially diluting standards; constructing and interpolating standard curves; identifying variables and controls; collecting, organizing, and analyzing data; constructing and interpreting graphs; and using common laboratory equipment such as micropipettors and spectrophotometers. Thus, implementing this assay encourages students to engage in inquiry-based learning while exploring emerging issues in environmental science and health.

This article presents an optimized yeast estrogen screen for quantifying ligands in Personal Care Products (PCPs) that bind estrogen receptors alpha (ER&#945;) and/or beta (ER&#946;). The method incorporates two colorimetric substrate options, a six-day refrigerated incubation for use in undergraduate courses, and statistical tools for data analysis.

应用酵母雌激素筛检优选本科教学实验室的个人护理产品中的雌激素配体

The Yeast Estrogen Screen (YES) is used to detect estrogenic ligands in environmental samples and has been broadly applied in studies of endocrine ...

Anaerobic Growth and Maintenance of Mammalian Cell Lines

Most mucosal surfaces along with the midpoints in tumors and stem cell niches are geographic areas of the body that are anoxic. Previous studies show that the incubation in normoxic (5% CO2 in air) or hypoxic (low oxygen levels) conditions followed by an anoxic incubation (an absence of free oxygen) results in limited viability (2&#8211;3 days). A novel methodology was developed that enables an anoxic cell cultivation (for at least 17 days; the maximum time tested). The most critical aspect of this methodology is to ensure that no oxygen is introduced into the system. This is obtained by the degassing of media, and by flushing tubes, dishes, flasks, and pipettes with an anaerobic gas mixture (H2, CO2, N2) followed by permitting the materials to equilibrate to the anoxic (non-oxygen) environment prior to usage.&#160;Additional care must be exercised when acquiring photomicrographs to ensure that the micrographs obtained do not contain artifacts. In the absence of oxygen, cell morphology is significantly altered. Two distinct morphotypes are noted for all anaerobically-grown cells. The ability to grow and maintain mammalian cells in the absence of oxygen can be applied to the analysis of cell physiology, polymicrobial interactions, and the characterization of biosynthetic pathways for novel cancer drug development.

Here, we describe a new method that enables the anaerobic long-term cultivation of established cell lines. The maximum survival time that was tested is 17 days. This method is suitable for the testing of cytotoxic agents and exploring the physiology of anoxically replicating cells.

哺乳动物细胞系的厌氧生长与维持

Most mucosal surfaces along with the midpoints in tumors and stem cell niches are geographic areas of the body that are anoxic. Previous studies show that ...

Establishment and Maintenance of Gnotobiotic American Cockroaches (Periplaneta americana)

Gnotobiotic animals are a powerful tool for the study of controls on microbiome structure and function. Presented here is a protocol for the establishment and maintenance of gnotobiotic American cockroaches (Periplaneta americana). This approach includes built-in sterility checks for ongoing quality control. Gnotobiotic insects are defined here as cockroaches that still contain their vertically transmitted endosymbiont (Blattabacterium) but lack other microbes that normally reside on their surface and in their digestive tract. For this protocol, egg cases (oothecae) are removed from a (nonsterile) stock colony and surface sterilized. Once collected and sterilized, the oothecae are incubated at 30 &#176;C for approximately 4&#8722;6 weeks on brain-heart infusion (BHI) agar until they hatch or are removed due to contamination. Hatched nymphs are transferred to an Erlenmeyer flask containing a BHI floor, sterile water, and sterile rat food. To ensure that the nymphs are not housing microbes that are unable to grow on BHI in the given conditions, an additional quality control measure uses restriction fragment-length polymorphism (RFLP) to test for nonendosymbiotic microbes. Gnotobiotic nymphs generated using this approach can be inoculated with simple or complex microbial communities and used as a tool in gut microbiome studies.

Establishment and Maintenance of Gnotobiotic American Cockroaches Periplaneta americana

This protocol is used in establishing and maintaining gnotobiotic American cockroaches (Periplaneta americana) by surface sterilizing the egg cases (oothecae) prior to hatching. These gnotobiotic insects contain their vertically transmitted Blattabacterium endosymbionts but have axenic guts.

建立和维护侏罗育美国蟑螂 （佩里普拉内塔美洲）

Gnotobiotic animals are a powerful tool for the study of controls on microbiome structure and function. Presented here is a protocol for the establishment ...

Fluorescence In Situ Hybridization on DNA Halo Preparations to Reveal Whole Chromosomes, Telomeres and Gene Loci

The genome is associated with several structures inside cell nuclei, in order to regulate its activity and anchor it in specific locations. These structures are collectively known as the nucleoskeleton and include the nuclear lamina, the nucleoli, and nuclear bodies. Although many variants of fluorescence in situ hybridization (FISH) exist to study the genome and its organization, these are often limited by resolution and provide insufficient information on the genome&#39;s association with nuclear structures. The DNA halo method uses high salt concentrations and nonionic detergents to generate DNA loops that remain anchored to structures within nuclei through attachment regions within the genome. Here, soluble nuclear proteins, such as histones, lipids, and DNA not tightly bound to the nuclear matrix, are extracted. This leads to the formation of a halo of unattached DNA surrounding a residual nucleus which itself contains DNA closely associated with internal nuclear structures and extraction-resistant proteins. These extended DNA strands enable increased resolution and can facilitate physical mapping. In combination with FISH, this method has the added advantage of studying genomic interactions with all the structures that the genome is anchored by. This technique, termed HALO-FISH, is highly versatile whereby DNA halos can be coupled with nucleic acid probes to reveal gene loci, whole chromosomes, alpha satellite, telomeres and even RNA. This technique provides an insight into nuclear organization and function in normal&#160;cells and in disease progression such as with cancer.

Combining DNA halo preparations with fluorescence in situ hybridization enables high-resolution analysis of genomic interactions with the nucleoskeleton. Attached genome leads to hybridized fluorescent signals located within the residual extracted nuclei, whereas non-attached genome is in the halo of DNA surrounding the residual nuclei.