我们感谢AKI的生物学和斑马鱼技术的有益的讨论，并分享他的甲基纤维素配方C.喋是Wingert实验室的成员。作者还希望圣母院斑马鱼研究中心提供优秀正在进行的畜牧业照顾我们的斑马鱼殖民地的工作人员表示感谢。从美国国立卫生研究院NIDDK补助金DK083512，大方的实验室从圣母大学的启动资金，资金支持这项工作。

1。显微注射过程标签斑马鱼pronephros近端肾小管上皮细胞 此过程中，我们使用显微注射系统（哈佛Appartus，PLI90），由一个空气压缩机（军航，型号6-4），和显微仪器（Narishege部分MN151，IP3，和GJI）提供的加压空气聚集在立体显微镜站每制造商的说明（SMZ - 645变焦尼康，）。葡聚糖结合物很容易吞噬肾近端肾小管，使本上皮细胞人口9优惠标签 。 <ol><li>曝光亚甲蓝加重卵黄囊的自体荧光和形象化的肾管更加困难。 </li><li>提高受精胚胎在28.5 ° C至受精后48-55小时之间（HPF）的一个发展时间点。这是一个理想的的阶段，因为在可以进行肌肉注射显微注射缓解执行幼虫microinjections，因为肾功能此时开始。 </li><li>从任何未孵化的斑马鱼用一双细镊子取出绒毛膜。洗净的培养皿中，然后重新修改E3的解决方案与30 MLS菜的蛋壳碎片。 </li><li>准备注射胚胎托盘。我们首选的注塑模具是1.5％agarose/E3，三角洼地形成的胚胎内注射可定位。为了使注塑模具，塑料模具浮楔形突起（先前11详细描述）在1.5％琼脂糖基地填补了培养皿。 </li><li>准备用针拉马拉毛细管，在斑马鱼书10玻璃微量注射针。总之，拉玻璃毛细管，然后用细的金属钳，切割用锋利的点小费，而观看的立体显微镜的针尖下的显微注射针的尖端。商店切覆盖在培养皿和针头上的橡皮泥定位，以保护切边和防止灰尘堆积。 </li><li>准备注射液标签近端肾小管。在浓度为1 mg / mL蒸馏水溶解，40 kD的葡聚糖荧光共轭（Invitrogen公司，D1845）。 </li><li>麻醉鱼，添加5毫升的0.2％Tricaine，pH值7.0的培养皿中在30日的E3展MLS的斑马鱼幼虫。鱼麻醉时，他们不再表现出触摸响应。至关重要的是，鱼是完全麻醉，或抽搐后，他们将试图注入。 </li><li>麻醉的鱼转移到注塑模具托盘使用的塑料转让吸管。其头部的位置好，并在其一侧主干是沿着井斜角休息三角凹陷最深的部分动物。 </li><li>执行肌肉注射显微注射。 2-3μL葡聚糖荧光负荷削减针，注入约1 NL解决方案与躯干体节的动物。的上半部分的体节的目的，避免卵黄囊扩展。这确保了肾管不是机械显微注射过程中断。 </li><li>转移到一个干净的培养皿中注入斑马鱼，并添加改性E3的解决方案。冲洗动物新鲜的E3 3次，以去除所有的Tricaine痕迹。盖用铝箔培养皿的盖子提供葡聚糖轻保护，并返回的动物，以29 ° C培养箱中过夜。 </li></ol> 2。肾小管上皮细胞的有针对性的激光烧蚀 此过程中，我们使用立体显微镜与epifluorescence得分葡聚糖荧光标记明亮的近端小管的动物，并选择这些激光烧蚀。 然后，我们使用的脉冲micropoint激光系统（光子仪器公司）已连接到一个复合式显微镜（尼康的Eclipse 80I啶附件），并为每个制造商的说明使用校准，进行肾细胞消融 。 我们不得不使用FITC标记的过滤器，使研究者查看荧光灯照明下明肾小管地区最成功的细胞消融 。 在此过程之前，准备消融tricaine/E3 0.02％，1.5％甲基纤维素溶解在固定媒体。甲基纤维素在室温下直接使用的商店分装，或在4 ° C下长期贮存 。 <ol><li>加入0.2％Tricaine pH值7.0的5毫升含有培养皿在30日的E3展MLS的斑马鱼幼虫麻醉72 HPF斑马鱼。鱼麻醉时，他们不再表现出触摸响应。至关重要的是，鱼是完全麻醉，或抽搐后，他们将试图执行的激光烧蚀。 </李&gt; <li>检查注射的动物，在适当的荧光设置，并选择在其中您可以轻松地可视化近端小管的动物。 </li><li>选定的胚胎转移到一个单独的菜含有tricaine。动物可长达30分钟的麻醉，然后再执行细胞消融。如果你的得分&gt; 15的动物，退回到一个单独的菜，含有新鲜修改E3的每个细胞消融需要几分钟的等待消融。 </li><li>要执行消融，转让一个麻醉斑马鱼的小培养皿中含有1.5％methylcellulose/0.02％tricaine洗2分钟的胚胎在固定媒体。 </li><li>下一步，将胚胎玻璃抑郁症的幻灯片，其中包含的1.5％methylcellulose/0.02％tricaine下降。轻轻的位置用细探针，其腹侧面的幻灯片和背侧朝上的动物。 </li><li>将复方显微镜阶段的幻灯片，并专注于动物的背侧。执行踩下脚踏板下的焦点肾细胞消融。你会看到散布的荧光，肾小管上皮细胞是烧蚀（图1A）。 </li><li>轻轻转移消融动物以及12以及随后的培养盘。冲洗2-3次的E3洗去甲基纤维素。 </li><li>注射后，提高动物所需的时间点，并执行所需的分析，为年轻的斑马鱼的胚胎（图2）成立的组织学和分子技术。 </li></ol> 3。代表性的成果： 如图1所示的数据表明消融timecourse。肌注右旋糖酐偶联后，肾小管上皮优先标记。注射右旋糖酐FITC标签激光烧蚀肾，并重新注入在一个额外的时间点以下消融消融动物右旋糖酐罗丹明重新映像近端肾小管人口。表达原位杂交分析技术一样，可以用来分析在固定样本的变化，在单一的时间点细胞消融后，如在图2所示。 <img src="/files/ftp_upload/2845/2845fig1.jpg" alt="图1"/> 图1：激光消融术是其次的肾小管上皮细胞快速再生 （AC）Timecourse细胞的变化过程中和后的斑马鱼幼虫近端小管的激光消融消融的时间（第3天，A组），或一个或三个。天消融术（B组第4天，第7天，C组）。 （上）原理图显示肾的位置，与右旋糖酐- FITC（绿色）和右旋糖酐罗丹明（红色），和（底部）控制和激光烧蚀（LA）的肾单位的现场图像。 <img src="/files/ftp_upload/2845/2845fig2.jpg" alt="图2"/> 图2：分析基因的表达，激光烧蚀激光消融在第3天，胚胎的固定和处理整装原位杂交检测 slc20a1a，这标志着近端的肾曲小管段的成绩单。 ：（A）控制胚胎没有受到激光烧蚀，（二）胚胎中的肾小管上皮细胞的大片消融，（三）胚胎中的肾小管上皮细胞的间隔时间短是烧蚀。黑线表示在近端小管的程度提供参考，蓝线显示面板公元前激光烧蚀程度，*表示的控制（非烧蚀）面板公元前对侧肾。

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Q., Ma, D., Holm, T., Naylor, R., Arora, N., Wingert, R., Bollig, F., Djordjevic, G., Lichman, B., Zhu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+adult+nephron+progenitors+capable+of+kidney+regeneration+in+zebrafish.">Identification of adult nephron progenitors capable of kidney regeneration in zebrafish.</a> Nature. 470, 95-100 (2011).</li></ol>

没有利益冲突的声明。

斑马鱼是一种广泛使用的模式生物，在整个科学的许多方面，应用增长 6 。喜欢AKI的肾脏疾病的研究斑马鱼模型系统的就业导致了重要的意见，并将继续是一个很好的模型来研究肾损伤7,8。随着一个被测序的基因组和分子协议中发生的许多，它已变得越来越容易，执行复杂的斑马鱼的研究，利用转基因模型，基因敲除，错误表达研究。斑马鱼有一个庞大的大小和定义良好的发展阶段的生命周期短。此外，在很大程度上是透明的斑马鱼的胚胎和幼体阶段，使影像学研究成为可能，而剥离的有机体。整个胚胎和幼体阶段，斑马鱼有两个肾，仍然在这些发展的时间点中可以看到，组成一个pronephric肾脏。斑马鱼pronephric肾的结构和功能，以他们的哺乳动物相似，使得它为急性肾功能衰竭的研究4,5的良好模型。 肾脏是人类和其他脊椎动物的生存至关重要，作为净化体内的液体代谢废物的器官。此清洗功能取决于肾肾过滤血液，然后修改滤液收集分泌含氮废物，同时维持体液和电解质平衡的能力。肾是由血液过滤，肾小管上皮细胞，并成为一个管道漏。所有三个部分的结构和功能的完整是不可分割的肾功能。各种侮辱肾脏，包括曝光nephrotoxins，缺血，或尿流出道阻塞，可导致急性肾损伤 1 。 AKI的病理往往伴有急性肾小管坏死1,2。临床研究表明，随后的肾功能衰竭的范围可以从7-80％取决于肾功能衰竭的原因和背景下，任何地方的死亡率。然而，快速诊断和逆转的AKI的根本原因是越来越多地与通过回收再生坏死的肾单位的肾小管。最近的命运映射的研究表明，主要细胞群，重新填充受损的肾单位的肾小管上皮细胞的起源，从相邻未受伤的地区，12 。这些研究结果并没有被淘汰的可能性，肾干/祖细胞可能参与的过程中，并有独立的报告表明，骨髓细胞可以促进肾再生13。很明显，需要继续调查，以更好地解决肾小管上皮再生的细胞来源。 两个肾的再生和发展的份额之间的间质细胞和上皮细胞表型的细胞状态的开关的现象。在肾开发，骨髓间充质祖细胞分化成一个固定的表型，附加基底膜形成的肾小管上皮3。推测急性肾功能衰竭后的上皮细胞的迁徙现象，有必要去分化成间充质表型。有趣的是，最近的研究表明，在损坏肾脏的间质细胞呈现类似到 14在开发过程中的间质细胞的表达谱。各种报告检测细胞粘附分子，基质蛋白，细胞因子和趋化因子的变化。以下建议的间质上皮过渡，细胞重组基底膜和肾小管上皮细胞的活动再次启动。确定在这个过程中重要的基因，仍然是我们的实验室和其他人的研究主题。的工作，以澄清在这个过程中的关键因素，仍然有许多工作要做，但庆大霉素的影响的研究预示着在该领域已取得重大进展。 庆大霉素引起急性肾损伤和肾功能衰竭是考虑药15中使用的肾毒性的化合物患病率有关。氨基糖甙类抗生素的管理作为一种用于治疗革兰氏阴性细菌感染，导致在10-25％的情况下急性损伤。这种药物会导致过多的负面细胞的影响，导致许多肾小管上皮细胞凋亡或坏死。有研究发现绑定优先megalin和cubulin涉及内吞作用形成了一个复杂的药物。这些分子被发现在近端肾小管上皮细胞的丰度，虽然他们不是只限于在这些细胞中的表达。通过诱导的氧化应激，血管收缩剂的释放，细胞内ATP，缺氧的耗竭，磷脂酶抑制造成的细胞信号，庆大霉素和类似抗生素导致上皮细胞的凋亡和坏死。此外，抗生素引发的肾血液过滤器（肾小球）系膜收缩，导致肾小球滤过率下降和负面的改变肾脏过滤血液的能力。生产的活性氧，免疫刺激分子，和磷脂酶的激活在肾弥漫性的影响，创造了灾难性的，导致急性肾功能衰竭的病理。 虽然庆大霉素和类似抗生素的研究，已经并将继续肾脏再生研究的重要工具，他们缺乏一个精确的，可以在解决某些问题有用的。我们使用在本议定书中所描述的方法用激光烧蚀一起的斑马鱼系统地回答了这样一个精确的研究工具的需要。激光烧蚀，使研究人员能够诱导细胞破坏肾小管的重点领域，从一个小的（2-3）大（50-100）人口与无与伦比的精度。在这个协议中，我们利用以前开发的曝光右旋糖酐偶联优先标签近端肾小管上皮人口的方法。此外，一个或多个地区的肾小管表达绿色荧光蛋白的转基因斑马鱼线都可以使用。例如，cadherin17：EGFP转基因斑马鱼表现出较强的荧光在远端肾小管地区（虽然在近端人口弱），和可再生研究在其他小管段中的16宝贵的。肾的知名度，使时间推移细胞随着时间的推移变化的视频录制。当如整装原位杂交或免疫组织化学基因表达研究中，研究人员可以随着时间的推移肾检测的分子变化。两者合计，这种战略可以开始制定一个更动态的了解，蒸发时，肾小管上皮细胞被破坏的事件。 我们的激光烧蚀模型的主要需要注意的是，它可能重述的，在人类AKI的蒸腾的生理条件下的局部方面。通过瞬时物理损坏引起的细胞死亡是从不同的级联事件，导致细胞坏死，并在这些不同的微环境体液因素可能不完全相同。此外，存在诱导细胞凋亡在肾损伤的证据，它不知道这样的后果是否蒸腾用激光以下侮辱。然而，仅仅是一个工具，一些更好的答案的问题，需要高度控制的环境，激光烧蚀将作为一个高度控制体内AKI的模型，细胞培养。这样的见解，得到了肾上皮消融在斑马鱼的研究，将有可能促进发现可应用于其他肾损伤的调查，并有可能使用在人类创造更好的诊断的基本见解。

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th></tr><tr><td>胚胎菜</td><td>鹘</td><td> 35-1005 </td></tr><tr><td>解剖钳</td><td> Roboz </td><td> RS - 5010 </td></tr><tr><td>注射针头</td><td>萨特Intruments </td><td> BF100 - 50 - 10 </td></tr><tr><td>超纯琼脂糖</td><td> Invitrogen公司</td><td> 16500-500 </td></tr><tr><td> 40 kilodalton（KD）葡聚糖荧光</td><td> Invitrogen公司</td><td> D1845 </td></tr><tr><td>塑料转移吸管</td><td>萨姆肯科学，费舍尔</td><td> 204，13-711-23 </td></tr><tr><td> Tricaine </td><td>西格玛</td><td> E10521 </td></tr><tr><td>甲基纤维素</td><td>西格玛</td><td> M0262 </td></tr><tr><td>萧条幻灯片</td><td>费舍尔</td><td> S175201 </td></tr><tr><td> 12以及菜</td><td>康宁</td><td> 3512 </td></tr></tbody></table>

laser ablation of the zebrafish pronephros to study renal epithelial regeneration

急性肾损伤（AKI）的特点是高死亡率，肾功能恶化，过几个小时或几天内，在肾功能衰竭1的高潮。 AKI的可能造成的一些因素，包括缺血，药物的毒性，或阻塞性损伤1。在无力维持体液和电解质的平衡的结果。虽然几十年来一直观察AKI的，有效的临床治疗，还有待开发。有趣的是，一些与AKI的患者恢复肾功能，随着时间的推移，一个神秘的现象已rudimentally 特点 1,2 。使用AKI的哺乳动物模型的研究表明，缺血性或nephrotoxin受伤的肾脏经验上皮细胞死亡在肾管1,2，肾脏的功能单位，作出了一系列专门的区域（段）的上皮细胞类型3 。在肾上皮细胞的死亡是最高的肾小管上皮细胞。有证据表明，细胞的破坏是去分化，增殖和迁移周围的上皮细胞，可再生的肾完全 1,2 。不过，也有许多悬而未决的问题，对肾小管上皮再生的机制，从信号调节这些事件的能力在人与人之间的差异很大的原因，再生受伤的肾脏。 幼虫斑马鱼提供了一个极好的模型来研究pronephric肾肾上皮再生与高等脊椎动物包括哺乳动物 4,5保守的肾单位组成。斑马鱼幼虫的肾可与荧光技术，因为相对年轻的斑马鱼 6的透明度可视化。这提供了一个独特的机会，相反，肾都无法访问的哺乳动物模型，形象的细胞和分子实时变化，因为肾脏是内在结构复杂的系统内的动物。最近的研究已受聘为一种有毒的病原体Aki和随后的肾功能衰竭的研究氨基糖苷类庆大霉素：庆大霉素等抗生素已经导致人类AKI的，研究人员已经制定的方法来使用这个代理，引发肾损害在斑马鱼7 8。然而，在斑马鱼的幼虫毒性氨基糖甙类的影响是灾难性的，致命的，它提出了一个研究上皮再生和功能随着时间的推移，当困难。我们的方法提出了一个新的工具在斑马鱼的上皮损伤的研究使用的靶细胞消融。激光烧蚀，使研究人员能够诱导细胞死亡的细胞有限的人口。基于形态学的位置，功能，甚至特定的细胞表型的表达，细胞的不同地区可以有针对性。因此，激光烧蚀会增加研究人员可以研究的特殊性，可以成为一个强大的新方法，阐明肾小管上皮再生的机制。该协议可以广泛应用到其他器官中的细胞群，在斑马鱼胚胎研究任何利益的上下文中的损伤和再生目标。

Watch this Scientific Journal Video about Laser Ablation of the Zebrafish Pronephros to Study Renal Epithelial Regeneration at JoVE.com

Laser Ablation of the Zebrafish Pronephros to Study Renal Epithelial Regeneration

在人类急性肾损伤（AKI）是临床常见的问题，造成损害的上皮细胞，包括肾肾，AKI的是与高死亡率的50-70％1。上皮细胞破坏，肾有再生能力有限，但指导这一现象的机制和限制，仍然知之甚少。在这个视频文章中，我们描述了我们的技术在斑马鱼胚胎的肾脏，肾肾细胞有针对性的激光烧蚀或pronephros。我们的新方法，可以用来补充AKI的肾毒性引起的模型和肾肾上皮再生的细胞和分子变化，获得高分辨率的认识。

激光烧蚀斑马鱼Pronephros研究肾小管上皮再生

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in ...

laser-ablation-zebrafish-pronephros-to-study-renal-epithelial

Research

JoVE Journal

Biology

15.2K 次观看.  University of Notre Dame. 在人类急性肾损伤（AKI）是临床常见的问题，造成损害的上皮细胞，包括肾肾，AKI的是与高死亡率的50-70％1。上皮细胞破坏，肾有再生能力有限，但指导这一现象的机制和限制，仍然知之甚少。在这个视频文章中，我们描述了我们的技术在斑马鱼胚胎的肾脏，肾肾细胞有针对性的激光烧蚀或pronephros。我们的新方法，可以用来补充AKI的肾毒性引起的模型和肾肾?...

视频: Laser Ablation of the Zebrafish Pronephros to Study Renal Epithelial Regeneration

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

测试的速度和运动方向的视觉灵敏度在蜥蜴

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

科研

生物学

Murine Renal Transplantation Procedure

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell et al over 30 years ago (1) and refined by Zhang et al years later (2), few people in the world have mastered this procedure. In our laboratory we have successfully performed 1200 orthotopic kidney transplantations with &gt; 90% survival rate. The key points for success include stringent control of reperfusion injury, bleeding and thrombosis, both during the procedure and post-transplantation, and use of 10-0 instead of 11-0 suture for anastomoses.
Post-operative care and treatment of the recipient is extremely important to transplant success and evaluation. All renal graft recipients receive antibiotics in the form of an injection of penicillin immediately post-transplant and sulfatrim in the drinking water continually. Overall animal health is evaluated daily and whole blood creatinine analyses are performed routinely with a portable I-STAT machine to assess graft function.

Renal transplantation in mice is a technically challenging procedure that requires careful post-operative care and treatment for success.

小鼠肾移植程序

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell ...

Methods for the Study of the Zebrafish Maxillary Barbel

Barbels are skin sensory appendages found in fishes, reptiles and amphibians. The zebrafish, Danio rerio, develops two pairs of barbels- a short nasal pair and a longer maxillary pair. Barbel tissue contains cells of ectodermal, mesodermal and neural crest origin, including skin cells, glands, taste buds, melanocytes, circulatory vessels and sensory nerves. Unlike most adult tissue, the maxillary barbel is optically clear, allowing us to visualize the development and maintenance of these tissue types throughout the life cycle. 
This video shows early development of the maxillary barbel (beginning approximately one month post-fertilization) and demonstrates a surgical protocol to induce regeneration in the adult appendage (&gt;3 months post-fertilization). Briefly, the left maxillary barbel of an anesthetized fish is elevated with sterile forceps just distal to the caudal edge of the maxilla. A fine, sterile spring scissors is positioned against the forceps to cut the barbel shaft at this level, establishing an anatomical landmark for the amputation plane. Regenerative growth can be measured with respect to this plane, and in comparison to the contralateral barbel. Barbel tissue regenerates rapidly, reaching maximal regrowth within 2 weeks of injury.
Techniques for analyzing the regenerated barbel include dissecting and embedding matched pairs of barbels (regenerate and control) in the wells of a standard DNA electrophoresis gel. Embedded specimens are conveniently photographed under a stereomicroscope for gross morphology and morphometry, and can be stored for weeks prior to downstream applications such as paraffin histology, cryosectioning, and/or whole mount immunohistochemistry. These methods establish the maxillary barbel as a novel in vivo tissue system for studying the regenerative capacity of multiple cell types within the genetic context of zebrafish.

The zebrafish maxillary barbel is an integumentary sense organ containing ectodermal, mesodermal and neural crest derivatives. Importantly, the adult barbel can regenerate after proximal amputation. This video introduces maxillary barbel development and demonstrates a surgical protocol to induce regeneration, followed by collection, embedding and downstream imaging of barbel specimens.

上颌Barbel斑马鱼的研究方法

Barbels are skin sensory appendages found in fishes, reptiles and amphibians.  The zebrafish, Danio rerio, develops two pairs of barbels- a short nasal ...

Using the optokinetic response to study visual function of zebrafish

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the visual functions of larval zebrafish1-5. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adult zabrafish. Here, we introduce how to measure the OKR of adult zebrafish with our simple custom-built apparatus using a new protocol which is established in our lab. Both our apparatus and step-by-step procedure of OKR in adult zebrafish are illustrated in this video. In addition, the measurements of the larval OKR, as well as the optomotor response (OMR) test of adult zebrafish, are also demonstrated in this video. This OKR assay of adult zebrafish in our experiment may last for up to 4 hours. Such OKR test applied in adult fish will benefit to visual function investigation more efficiently when the adult fish vision system is manipulated.
Su-Qi Zou and Wu Yin contributed equally to this paper.

Optokinetic response has been widely used to assess the visual functions of larval zebrafish. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adults1-5. Here, we introduce how to measure the OKR of adult zebrafish using a new protocol which is established in our lab.

使用的视动反应研究斑马鱼的视觉功能

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the ...

A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, heart, the jaw 1. Specifically, newts are unique for its lens regeneration capability. Upon lens removal, IPE cells of the dorsal iris transdifferentiate to lens cells and eventually form a new lens in about a month 2,3. This property of regeneration is never exhibited by the ventral iris cells. The regeneration potential of the iris cells can be studied by making transplants of the in vitro cultured IPE cells. For the culture, the dorsal and ventral iris cells are first isolated from the eye and cultured separately for a time period of 2 weeks (Figure 1). These cultured cells are reaggregated and implanted back to the newt eye. Past studies have shown that the dorsal reaggregate maintains its lens forming capacity whereas the ventral aggregate does not form a lens, recapitulating, thus the in vivo process (Figure 2) 4,5. This system of determining regeneration potential of dorsal and ventral iris cells is very useful in studying the role of genes and proteins involved in lens regeneration.

In newt, the lens regenerates always from the dorsal iris by transdifferentiation of the iris pigmented epithelial cells (IPEs). Here we describe a procedure to culture dorsal and ventral newt IPE cells and their implantation to the newt eye. The implanted cells are then studied by tissue sectioning and immunohistochemistry.

一个系统为培养虹膜色素上皮细胞，研究蝾螈镜头再生

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

在毛发再生的上皮 - 间充质信号的快速遗传分析

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Application of Retinoic Acid to Obtain Osteocytes Cultures from Primary Mouse Osteoblasts

The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell numbers. Therefore, the most widely used cellular system is the osteocyte-like MLO-Y4 cell line.
The method here described refers to the use of retinoic acid to generate a homogeneous population of ramified cells with morphological and molecular osteocyte features.
After isolation of osteoblasts from mouse calvaria, all-trans retinoic acid (ATRA) is added to cell medium, and cell monitoring is conducted daily under an inverted microscope. First morphological changes are detectable after 2 days of treatment and differentiation is generally complete in 5 days, with progressive development of dendrites, loss of the ability to produce extracellular matrix, down-regulation of osteoblast markers and up-regulation of osteocyte-specific molecules.
Daily cell monitoring is needed because of the inherent variability of primary cells, and the protocol can be adapted with minimal variation to cells obtained from different mouse strains and applied to transgenic models. 
The method is easy to perform and does not require special instrumentation, it is highly reproducible, and rapidly generates a mature osteocyte population in complete absence of extracellular matrix, allowing the use of these cells for unlimited biological applications.

Treatment of primary mouse osteoblasts with retinoic acid produces a homogeneous population of ramified cells bearing morphological and molecular features of osteocytes. The method overcomes the difficulty of obtaining and maintaining primary osteocytes in culture, and can be advantageous to study cells derived from transgenic models.&#160;

维甲酸的应用获取从初级小鼠成骨细胞骨细胞培养

The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

在肾小管上皮细胞胞浆钙的测量流式细胞仪

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

Laser Micro-Irradiation to Study DNA Recruitment During S Phase

DNA damage repair maintains the genetic integrity of cells in a highly reactive environment. Cells may accumulate various types of DNA damage due to both endogenous and exogenous sources such as metabolic activities or UV radiation. Without DNA repair, the cell's genetic code becomes compromised, undermining the structures and functions of proteins and potentially causing disease. 
Understanding the spatiotemporal dynamics of the different DNA repair pathways in various cell cycle phases is crucial in the field of DNA damage repair. Current fluorescent microscopy techniques provide great tools to measure the recruitment kinetics of different repair proteins after DNA damage induction. DNA synthesis during the S phase of the cell cycle is a peculiar point in cell fate regarding DNA repair. It provides a unique window to screen the entire genome for mistakes. At the same time, DNA synthesis errors also pose a threat to DNA integrity that is not encountered in non-dividing cells. Therefore, DNA repair processes differ significantly in S phase as compared to other phases of the cell cycle, and those differences are poorly understood. 
The following protocol describes the preparation of cell lines and the measurement of dynamics of DNA repair proteins in S phase at locally induced DNA damage sites, using a laser-scanning confocal microscope equipped with a 405 nm laser line. Tagged PCNA (with mPlum) is used as a cell cycle marker combined with an AcGFP-labeled repair protein of interest (i.e., EXO1b) to measure the DNA damage recruitment in S phase.

This protocol describes a non-invasive method to efficiently identify S-phase cells for downstream microscopy studies, such as measuring DNA repair protein recruitment by laser micro-irradiation.

激光微辐射研究S阶段DNA招募

DNA damage repair maintains the genetic integrity of cells in a highly reactive environment. Cells may accumulate various types of DNA damage due to both ...

Nitroreductase/Metronidazole-Mediated Ablation and a MATLAB Platform (RpEGEN) for Studying Regeneration of the Zebrafish Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) resides at the back of the eye and performs functions essential for maintaining the health and integrity of adjacent retinal and vascular tissues. At present, the limited reparative capacity of mammalian RPE, which is restricted to small injuries, has hindered progress to understanding in vivo RPE regenerative processes. Here, a detailed methodology is provided to facilitate the study of in vivo RPE repair utilizing the zebrafish, a&#160;vertebrate model capable of robust tissue regeneration. This protocol describes a transgenic nitroreductase/metronidazole (NTR/MTZ)-mediated injury paradigm (rpe65a:nfsB-eGFP), which results in ablation of the central two-thirds of the RPE after 24 h treatment with MTZ, with subsequent tissue recovery. Focus is placed on RPE ablations in larval zebrafish and methods for testing the effects of pharmacological compounds on RPE regeneration are also outlined. Generation and validation of RpEGEN, a MATLAB script created to automate quantification of RPE regeneration based on pigmentation, is also discussed. Beyond active RPE repair mechanisms, this protocol can be expanded to studies of RPE degeneration and injury responses as well as the effects of RPE damage on adjacent retinal and vascular tissues, among other cellular and molecular processes. This zebrafish system holds significant promise in identifying genes, networks, and processes that drive RPE regeneration and RPE disease-related mechanisms, with the long-term goal of applying this knowledge to mammalian systems and, ultimately, toward therapeutic development.

Nitroreductase/Metronidazole-Mediated Ablation and a MATLAB Platform RpEGEN for Studying Regeneration of the Zebrafish Retinal Pigment Epithelium

This protocol describes the methodology to genetically ablate the retinal pigment epithelium (RPE) using a transgenic zebrafish model. Adapting the protocol to incorporate signaling pathway modulation using pharmacological compounds is extensively detailed. A MATLAB platform for quantifying RPE regeneration based on pigmentation was developed and is presented and discussed.

硝基还原酶/甲硝唑介导的消融和MATLAB平台（RpEGEN）研究斑马鱼视网膜色素上皮的再生

The retinal pigment epithelium (RPE) resides at the back of the eye and performs functions essential for maintaining the health and integrity of adjacent ...