作者承认哈蒙德湾生物站，大湖科学中心，美国地质调查局的贡献。我们也感谢梅林达帧博士在中心高级显微镜在密歇根州立大学在激光扫描共聚焦显微镜她的技术支持。这项研究是由赠款从大湖区渔业委员会YWCD和WML的支持。

1，溶液配制<ol><li>使1升10倍的0.1M磷酸盐缓冲盐水（PBS，pH值为7.4）:称取26.2克磷酸钠（一元），115克磷酸钠（二元），并87.66克NaCl等。溶于约800ml蒸馏水H 2 O，调节pH值，并把音量开到1升，用蒸馏水H 2 O <ol><li>使1升0.1M磷酸缓冲盐水（pH 7.4）:取100毫升10倍的PBS中，并加入900毫升蒸馏H 2 O。 </li><li>使1升0.1M磷酸缓冲液（pH 7.4）中含0.1％的Triton X-100:取百毫升10倍的PBS中，加入1 ml曲通X-100，和使体积达1升，用蒸馏水H 2 O。 </li></ol></li><li>使1微升10x 0.1M磷酸盐缓冲液（PB，pH值为7.4）:称取26.2克磷酸钠（一元）和115克磷酸钠（二元）。溶于约800ml蒸馏水H 2 O，调节pH值，并把音量开到1升，用蒸馏水H 2 O <ol><li>使1升0.1M磷酸缓冲液（pH7.4）:取100毫升10倍的PB，并添加900毫升蒸馏H 2 O </li><li>使1升0.1M磷酸缓冲液（pH7.4），0.1％的Triton X-100:取百毫升10倍的PB，加入1 ml曲通X-100，和使体积达1升，用蒸馏水H 2 O。 </li></ol></li><li>使400毫升水凝胶单体溶液（4％丙烯酰胺，0.25％双丙烯酰胺，4％的多聚甲醛，0.0075％过硫酸铵，在0.1M的PBS 0.0005％皂苷）:称取0.3克过硫酸铵和0.2克皂甙。添加210毫升蒸馏水H 2 O的加入40ml 40％的丙烯酰胺，将10毫升2％双丙烯酰胺加入40ml 10倍的PBS（pH 7.4）中，加入100毫升16％的多聚甲醛，拌匀。 </li><li>使4升清溶液（4％SDS在0.2M硼酸）:称取49.464克硼酸和160g的SDS。溶于约3.5升蒸馏水H 2 O，调节pH值至8.5，用NaOH，并把音量开到4 L。 </li><li>让1升80％的甘油溶液:测量800毫升甘油，加入200毫升蒸馏水H 2 O，拌匀。 </li></ol> 2，组织准备<ol><li>制备水凝胶的单体溶液和等份10毫升各15ml离心管中。 </li><li>解剖整个组织，并在4℃下在水凝胶2天固定确保该水凝胶溶液是至少10倍的组织的体积。 </li><li>使水凝胶固定的组织（在15ml离心管）到通风橱。 </li><li>聚合水凝胶固定的组织，加入5μLTEMED，拌匀，让坐在通风柜在室温下放置2-3小时。 </li><li>彻底去除多余的胶。注:额外的胶会阻碍染色在清算过程中和抗体的渗透。 </li><li>孵育固定的组织在10毫升的培养箱摇床设定在70rpm和50℃下进行数日清溶液直至组织变得透明。改变结算解决方案，至少每天一次，并从组织改变结算解决方案时，去除多余的胶。 </li><li>冲洗组织在10毫升0.1M的PB，0.1％的Triton X-100在70 R下午和37℃过夜。重复此步骤3次，并从该组织改变缓冲溶液时，除去过量的凝胶。 </li><li>孵育在PB中的第一抗体溶液澄清的组织用0.1％的Triton X-100在70rpm和37℃下进行2天。注意:请足够的初级抗体溶液淹没整个组织。 </li><li>漂洗在10毫升PB的组织用0.1％的Triton X-100，在70转和37℃培养过夜。重复此步骤3次，并从该组织改变缓冲溶液时，除去过量的凝胶。 </li><li>执行在一个黑暗的房间或昏暗光线下面的步骤。盖上铝箔样品，以防止照片褪色。 </li><li>孵育澄清组织与在PB中的荧光二抗溶液用0.1％的Triton X-100，并在70转和37℃下进行1天封闭血清。 </li><li>漂洗在10毫升PB的组织用0.1％的Triton X-100，在70转和37℃培养过夜。重复此步骤3次，去除多余的凝胶f改变缓冲溶液时，ROM中的组织。 </li><li>冲洗的组织在10毫升PBS中，在70转和37℃培养过夜。重复此步骤3次，并从该组织改变缓冲溶液时，除去过量的凝胶。 </li><li>孵育澄清组织在80％甘油中，在70转和37℃培养过夜。 </li><li>存储荧光染色的组织在4℃之前，激光共聚焦显微镜。 </li></ol>

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作者什么都没有透露。

这个协议是由一个称为"净"12新方法，该交联完整的组织与聚丙烯酰胺以形成纳米多孔水凝胶，然后除掉组织的细胞膜，以实现光学透明性及透气性高分子修饰。 "清晰度"允许远程投射和神经系统局部电路接线完好组织成像。这种新方法可用于变态过程中可视化整个胆道系统在海七鳃鳗肝脏。这是生物学中的挑战，获得高分辨率的信息和保持从一个复杂的系统，例如在整个胆道系统或使用常规包埋和切片方法中枢神经系统的全局观点。这个修改​​过的净度方法允许激光束穿透完好海鳃鳗幼体肝最多在常规的激光扫描confoca使用低功率荧光物镜（4倍）的厚度为1至2毫米升显微镜。更深的渗透可以使用更先进的多光子共聚焦显微镜来实现。三维图像可以通过三维重建软件进行重构。实现使用传统方法比较的结果是很费力的，涉及许多路段的零碎的重建，并只限于小组织体积。这种新方法从完整系统12整体结构分析提供了有价值的信息，是劳动密集程度较低，更高效和可重复性。 这个新方法的另一优点是，样品可以与不同的抗体染色12可重复使用多次。这使得在相同的试样不同的分子标记物之间的比较更可靠和可重复的。由水凝胶产生的稳定的框架，可以有效地去除抗体没有精细结构损伤，并保持抗原性的LO纳克时间12。结算解决方案的抗体变性，破坏约束力，因此作为一种抗体，反萃液12。几轮染色的海鳃鳗肝脏样品中已经执行的结构和染色保持最佳的。 有从原来的"清晰度"12协议在本协议中两个主要的修改。 （1）产生自由基的试剂（过硫酸铵和TEMED）被用来发起和加快该水凝胶代替VA044的聚合。 VA044是感光，而不是在美国上市（2）无定制电泳室使用。在净度方法12，由于组织（小鼠脑与七鳃鳗肝脏）的大小，电泳组织清算需要加快质膜的提取。为了避免一个定制的电泳室的初始投资成本，我们首先使用EXI在我们的实验室蜇琼脂糖凝胶电泳设备。这被证明是非常低效的。然而，温育在升高的温度（50℃）的信息交换的解决方案是非常有效的清除质膜。 虽然作者为"净"告诫不要在水凝胶中使用的皂角苷，它提高了在我们的程序的海鳃鳗组织的透明度。一个缺点的共焦成像是海七鳃鳗组织具有较强的自体荧光。荧光团的选择必须由前后免疫荧光染色进行比较的图像进行检测。 在海七鳃鳗变态，对胆囊上皮回归变质阶段2和3之间开始。通过变质阶段4，胆囊完全消失在海七鳃鳗的肝脏14。胆管变性是最DRA岩浆变质阶段3和4 至15。该修改的方法是一个强大的工具来研究整个胆道系统，它可以提供见解的人胆道闭锁的变性的细胞和分子机制。

海七鳃鳗通过三个不同的人生阶段发展1,2。幼虫海七鳃鳗（L）的花费大部分时间在洞穴作为底栖滤食性动物。在经历的外部形态和重组内脏3剧变7变质阶段，由此产生的少年（合资）输入期间，他们吸食血液和组织液从宿主鱼寄生阶段，增加体重的100倍以上。经过1.0〜1.5年在海洋或大湖主机喂鱼，大人在早春停止喂养和迁移到河流产卵，然后死亡1,2。 变态期间，海七鳃鳗肝脏失去胆囊和整个胆管树，进化的突变表型，模仿人类婴儿疾病胆道闭锁。婴儿胆道闭锁是一种罕见的小儿肝脏疾病严重的并发症4,5,6,7，8,9,10，但是胆道闭锁的发病机制和病因在很大程度上是未知4。出生后的两年内病人胆道闭锁死去，除非手术治疗（葛西程序）执行5。随后，这些患者需要丰富的临床管理及常肝移植6。胆道闭锁的发病机理许多理论被提出，如病毒感染，先天性畸形，自身免疫性疾病，并且毒性损伤。然而，每到胆道闭锁的发展所作的贡献仍未有定论7,8,9,10。 不像遭受病理胆道闭锁的婴儿，海七鳃鳗经受发育编程胆道闭锁没有广泛的坏死性炎症，纤维化或肝硬化10。在此过程中10，动物可能会受到短暂的胆汁淤积，但适应这种发展的条件通过从头合成和胆汁盐在发育胆道闭锁，除了已知的机制，如在肝11减少胆汁盐合成后肠的分泌。这在海七鳃鳗的发展过程提供了唯一的已知的机会检查胆道闭锁的进展。 一个名为"清晰度"新开发的方法，通过将完整的组织成一个光学透明的纳米多孔水凝胶12可实现高清晰度成像在复杂的哺乳动物神经系统。使用海七鳃鳗的肝脏和修改CLARITY协议，胆道病变的完好组织成像可以记录整个肝脏变态。

<table><tbody><tr><td>40% acrylamide&amp;nbsp;</td><td>Bio-Rad</td><td>161-0140</td><td></td></tr><tr><td>2% bis-acrylamide&amp;nbsp;</td><td>Bio-Rad</td><td>161-0142</td><td></td></tr><tr><td>TEMED</td><td>Bio-Rad</td><td>161-0800</td><td></td></tr><tr><td>ammonium persulfate&amp;nbsp;</td><td>Sigma</td><td>A3678-25G</td><td></td></tr><tr><td>boric acid</td><td>Sigma</td><td>B7901-1KG</td><td></td></tr><tr><td>saponin&amp;nbsp;</td><td>Sigma</td><td>47036</td><td></td></tr><tr><td>sodium dodecyl sulfate&amp;nbsp;</td><td>Sigma</td><td>L337-500G</td><td></td></tr><tr><td>sodium phosphate (monobasic)</td><td>Sigma</td><td>04269-1KG</td><td></td></tr><tr><td>sodium phosphate (dibasic)</td><td>Sigma</td><td>S5136-1KG</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>X100-500ML</td><td></td></tr><tr><td>glycerol&amp;nbsp;</td><td>Sigma</td><td>G9012-500ML</td><td></td></tr><tr><td>16% paraformaldehyde&amp;nbsp;</td><td>Electron Microscopy Sciences</td><td>15710-S</td><td></td></tr><tr><td>NaOH pellets&amp;nbsp;</td><td>EMD</td><td>SX0590-3</td><td></td></tr><tr><td>15 ml centrifuge tubes</td><td>Any brand</td><td></td><td></td></tr><tr><td>dissecting tools&amp;nbsp;</td><td>Any brand</td><td></td><td></td></tr></tbody></table>

a new clarification method to visualize biliary degeneration during liver metamorphosis in sea lamprey (petromyzon marinus)

胆道闭锁是婴儿期一种罕见的疾病，估计有1 15,000频率在美国东南部，但在东亚国家较为常见，与1台5000报频率。虽然知道关于胆道闭锁的管理，其发病机制仍不清楚。海七鳃鳗（Petromyzon马里努斯 ）提供了一个独特的机会来审视胆管变性的机制和进展。海七鳃鳗发展经历三个不同的人生阶段:幼虫，寄生，和成人。在从幼虫寄生少年的过渡，海七鳃鳗经历蜕变与戏剧性的重组和重构的外部形态和内部器官。在肝脏中，整个胆道系统丢失，包括胆囊和胆管树。一个名为"清晰度"新开发的方法进行了修改，以澄清整个肝脏和结与变质海七鳃鳗肠道。该胆管变性过程是通过使用激光扫描共聚焦显微镜观察和海七鳃鳗变态过程中看出端倪。这种方法提供了一个强大的工具来研究胆道闭锁在一个独特的动物模型。

发生于肝胆系统的几个重要发展事件海七鳃鳗变态过程。胆管和胆囊发生凋亡和退化（ 图1）。与肝细胞标志物细胞角蛋白19（CK19，存在于两个胆管上皮细胞和肝细胞前和变态13之后），并使用共聚焦显微镜的抗凋亡BCL2基因标记物相结合的改性澄清法和染色法，在整个胆道系统沿Z轴（捕获图2）和3 -维结构被重建（ 图3）。激光扫描共聚焦显微镜通过完整的肝脏，因为大多数胆道系统的嵌入在肝脏内，除非胆管进入肠道的交界处进行光学部分。退变过程中发生变质阶段2-4，从周边小胆小管向中央大intrahep开始ATIC胆总管（ 图2），与先前的研究一致10。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51648/51648fig1highres.jpg" width="500" /> 变态期间海七鳃鳗变态过程如图1所示。胆囊病变。海七鳃鳗失去了胆（黑色箭头）。试样取自动物的变质阶段2和3之间的外部形态， 即轮眼无（阶段2）或（3阶段），虹膜和瞳孔的急剧分化，对口腔的内背侧表面突出的乳头状突起罩（两个阶段），增稠的口服罩（阶段2）的形成矩形入口入口腔（3阶段），或"哈巴狗状"口鼻部（阶段3）3的嘴唇。需要注意的是胆也变了颜色指示胆汁分泌的差异。我:肠。 L:肝脏<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51648/51648fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51648/51648fig2highres.jpg" width="500" /> 图2。共聚焦图像显示在蜕变过程中海鳃鳗肝脏的胆管树。标本取自动物的变质阶段2和3之间的外部形态。顶面板被从前方拍摄，并从后端的下面板。的肝细胞标志物细胞角蛋白19的图像（1:100小鼠抗-CK19;染色的2微克/毫升的Alexa Fluor 350山羊抗小鼠IgG，蓝色）和抗凋亡BCL2基因标记物（1:100兔抗BCL2;用2微克/毫升的Alexa Fluor 488驴抗兔IgG抗体染色;绿色）被叠加，以产生合并后的图像。白色箭头指示的Biliary树。黑色箭头表示在黑暗树突寻找凋亡​​细胞和小管周围和小管在肝脏中。我:肠。 L:肝。比例尺:500微米<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51648/51648fig2highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51648/51648fig3highres.jpg" width="500" /> 图3。从合并后的共焦的z序列重构三维图像。三维图像从共焦的z系列的变质阶段2和3之间3肝脏进行重建。的肝细胞标志物细胞角蛋白19合并图像的光学部分（1:100鼠标抗CK19;染色的2微克/毫升的Alexa Fluor 350山羊抗小鼠IgG，蓝色）和抗凋亡BCL2基因标记物（1:100兔抗Bcl2的;染色的2微克/毫升的Alexa Fluor 488驴抗兔IgG;绿色）进行重构制造商的软件来生成三维图像。顶面板被从前方拍摄，并从后端的下面板。白色箭头指示的胆管树。黑色箭头表示在黑暗树突寻找凋亡​​细胞和小管周围和小管在肝脏中。我:肠。 L:肝。比例尺:500微米<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51648/51648fig3highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" fo:content-width="5in" src="/files/ftp_upload/51648/51648fig4highres.jpg" width="500" /> 。从3荧光通道图4显示，在管腔内胆汁滴胆管共聚焦光学部分合并后的图像;蓝色:鼠抗c-Myc基因（1:100）/ 2微克/毫升的Alexa Fluor 350羊抗鼠IgG抗体;绿色:2微克/毫升尼氏绿色;红:兔抗-TGFβRII（1:100）/ 2微克/毫升的Alexa Fluor 594驴抗兔。比例尺:500微米。胆汁小滴用黑箭头表示。肝脏样本被送往变质阶段2和3之间。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51648/51648fig4highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about A New Clarification Method to Visualize Biliary Degeneration During Liver Metamorphosis in Sea Lamprey Petromyzon marinus at JoVE.com

A New Clarification Method to Visualize Biliary Degeneration During Liver Metamorphosis in Sea Lamprey Petromyzon marinus

海七鳃鳗失去变态在胆囊和胆管，类似于人类的胆道闭锁的过程。一种新的固定和澄清方法（CLARITY）进行了修改，使用可视化激光扫描共聚焦显微镜在整个胆道系统。这种方法提供了一个强大的工具来研究胆管变性。

一种新的澄清方法，以可视化胆道变性在肝癌变态海七鳃鳗（ Petromyzon马里努斯）

Biliary atresia is a rare disease of infancy, with an estimated 1 in 15,000 frequency in the southeast United States, but more common in East Asian ...

a-new-clarification-method-to-visualize-biliary-degeneration-during

Nanyang Technological University

Research

JoVE Journal

Biology

A New Clarification Method to Visualize Biliary Degeneration During Liver Metamorphosis in Sea Lamprey (Petromyzon marinus)

13.7K 次观看.  Michigan State University. 海七鳃鳗失去变态在胆囊和胆管，类似于人类的胆道闭锁的过程。一种新的固定和澄清方法（CLARITY）进行了修改，使用可视化激光扫描共聚焦显微镜在整个胆道系统。这种方法提供了一个强大的工具来研究胆管变性。 

视频: A New Clarification Method to Visualize Biliary Degeneration During Liver Metamorphosis in Sea Lamprey Petromyzon marinus

Hepatocyte-specific Ablation in Zebrafish to Study Biliary-driven Liver Regeneration

The liver has a great capacity to regenerate. Hepatocytes, the parenchymal cells of the liver, can regenerate in one of two ways: hepatocyte- or biliary-driven liver regeneration. In hepatocyte-driven liver regeneration, regenerating hepatocytes are derived from preexisting hepatocytes, whereas, in biliary-driven regeneration, regenerating hepatocytes are derived from biliary epithelial cells (BECs). For hepatocyte-driven liver regeneration, there are excellent rodent models that have significantly contributed to the current understanding of liver regeneration. However, no such rodent model exists for biliary-driven liver regeneration. We recently reported on a zebrafish liver injury model in which BECs extensively give rise to hepatocytes upon severe hepatocyte loss. In this model, hepatocytes are specifically ablated by a pharmacogenetic means. Here we present in detail the methods to ablate hepatocytes and to analyze the BEC-driven liver regeneration process. This hepatocyte-specific ablation model can be further used to discover the underlying molecular and cellular mechanisms of biliary-driven liver regeneration. Moreover, these methods can be applied to chemical screens to identify small molecules that augment or suppress liver regeneration.

To help assess the molecular mechanisms underlying zebrafish biliary-driven liver regeneration, we established a liver injury model in which the nitroreductase-expressing hepatocytes are genetically ablated upon metronidazole treatment. In this protocol, we describe how to adeptly manipulate, monitor and analyze hepatocyte ablation and biliary-driven liver regeneration.

肝细胞特异性消融在斑马鱼研究胆驱动肝再生

The liver has a great capacity to regenerate. Hepatocytes, the parenchymal cells of the liver, can regenerate in one of two ways: hepatocyte- or ...

科研

发育生物学

The Identification of Sea Lamprey Pheromones Using Bioassay-Guided Fractionation

Bioassay-guided fractionation is an iterative approach that uses the results of physiological and behavioral bioassays to guide the isolation and identification of an active pheromone compound. This method has resulted in the successful characterization of the chemical signals that function as pheromones in a wide range of animal species. Sea lampreys rely on olfaction to detect pheromones that mediate behavioral or physiological responses. We use this knowledge of fish biology to posit functions of putative pheromones and to guide the isolation and identification of active pheromone components. Chromatography is used to extract, concentrate, and separate compounds from the conditioned water. Electro-olfactogram (EOG) recordings are conducted to determine which fractions elicit olfactory responses. Two-choice maze behavioral assays are then used to determine if any of the odorous fractions are also behaviorally active and induce a preference. Spectrometric and spectroscopic methods provide the molecular weight and structural information to assist with the structure elucidation. The bioactivity of the pure compounds is confirmed with EOG and behavioral assays. The behavioral responses observed in the maze should ultimately be validated in a field setting to confirm their function in a natural stream setting. These bioassays play a dual role to 1) guide the fractionation process and 2) confirm and further define the bioactivity of isolated components. Here, we report the representative results of a sea lamprey pheromone identification that exemplify the utility of the bioassay-guided fractionation approach. The identification of sea lamprey pheromones is particularly important because a modulation of its pheromone communication system is among the options considered to control the invasive sea lamprey in the Laurentian Great Lakes. This method can be readily adapted to characterize the chemical communication in a broad array of taxa and shed light on waterborne chemical ecology.

Here, we present a protocol to isolate and characterize the structure, olfactory potency, and behavioral response of putative pheromone compounds of sea lampreys.

利用生物测定引导分馏法鉴定海洋鳗信息素

Bioassay-guided fractionation is an iterative approach that uses the results of physiological and behavioral bioassays to guide the isolation and ...

生物化学

Analysis of Liver Microenvironment During Early Progression of Non-Alcoholic Fatty Liver Disease-Associated Hepatocellular Carcinoma in Zebrafish

Liver cancer is currently the third&#160;leading cause of cancer related death worldwide, and Hepatocellular Carcinoma (HCC) accounts for 75-90% of all liver cancer cases. With the introduction of effective treatments to prevent and treat hepatitis B/C, non-alcoholic fatty liver disease (NAFLD), and the more aggressive form know as non-alcoholic steatohepatitis (NASH), are quickly becoming the number one risk factors to develop HCC in modern societies. To better understand the role NASH has on the development of HCC we designed a NASH-associated HCC zebrafish. The optical clarity and genetic tractability of the zebrafish larvae make them an appealing and powerful model to study the liver microenvironment and immune cell composition using non-invasive fluorescent live imaging. This protocol describes how to use a NASH-associated HCC zebrafish model to investigate the effect of cholesterol surplus in the liver microenvironment and its impact on immune cell composition at early stages of the disease. First, we feed HCC larvae (s704Tg), which express hepatocyte-specific activated beta-catenin, with a 10% high cholesterol diet for 8 days to develop a NASH-associated HCC model. Here we describe how to make use of different transgenic lines to evaluate several early malignancy features in the liver by non-invasive confocal microscopy, such as liver area, cell, and nuclear morphology (hepatocytes area, nuclear area, nuclear:cytoplasmic ratio (N:C ratio), nuclear circularity, micronuclei/nuclear herniation scoring) and angiogenesis. Then, using transgenic lines with tagged immune cells (neutrophils, macrophages, and T cells) we show how to analyze liver immune cell composition in NASH-associated HCC larvae. The described techniques are useful to evaluate liver microenvironment and immune cell composition at early hepatocarcinogenesis stages, but they can also be modified to study such features in other liver disease models.

Here, we present how to generate a non-alcoholic fatty liver disease (NAFLD)-associated Hepatocellular Carcinoma (HCC) zebrafish model to study the impact of cholesterol surplus on liver microenvironment and immune cell landscape.

斑马鱼非酒精性脂肪肝相关肝细胞癌早期进展期间的肝脏微环境分析

Liver cancer is currently the third&#160;leading cause of cancer related death worldwide, and Hepatocellular Carcinoma (HCC) accounts for 75-90% of all ...

癌症研究

Determining Bile Duct Density in the Mouse Liver

Mouse is broadly used as a model organism to study biliary diseases. To evaluate the development and function of the biliary system, various techniques are used, including serum chemistry, histological analysis, and immunostaining for specific markers. Although these techniques can provide important information about the biliary system, they often do not present a full picture of bile duct (BD) developmental defects across the whole liver. This is in part due to the robust ability of the mouse liver to drain the bile even in animals with significant impairment in biliary development. Here we present a simple method to calculate the average number of BDs associated with each portal vein (PV) in sections covering all lobes of mutant/transgenic mice. In this method, livers are mounted and sectioned in a stereotypic manner to facilitate comparison among various genotypes and experimental conditions. BDs are identified via light microscopy of cytokeratin-stained cholangiocytes, and then counted and divided by the total number of PVs present in liver section. As an example, we show how this method can clearly distinguish between wild-type mice and a mouse model of Alagille syndrome. The method presented here cannot substitute for techniques that visualize the three-dimensional structure of the biliary tree. However, it offers an easy and direct way to quantitatively assess BD development and the degree of ductular reaction formation in mice.

We present a rather simple and sensitive method for accurate quantification of bile duct density in the mouse liver. This method can aid in determining the effects of genetic and environmental modifiers and the effectiveness of potential therapies in mouse models of biliary diseases.

确定小鼠肝脏中的胆汁密度

Mouse is broadly used as a model organism to study biliary diseases. To evaluate the development and function of the biliary system, various techniques ...

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several weeks, and this is accompanied by short distance regeneration (a few mm) of propriospinal axons and spinal-projecting axons from the brainstem. Among the 36 large identifiable spinal-projecting neurons, some are good regenerators and others are bad regenerators. These neurons can most easily be identified in wholemount CNS preparations. In order to understand the neuron-intrinsic mechanisms that favor or inhibit axon regeneration after injury in the vertebrates CNS, we determine differences in gene expression between the good and bad regenerators, and how expression is influenced by spinal cord transection. This paper illustrates the techniques for housing larval and recently transformed adult sea lampreys in fresh water tanks, producing complete spinal cord transections under microscopic vision, and preparing brain and spinal cord wholemounts for in situ hybridization. Briefly, animals are kept at 16&#160;&#176;C and anesthetized in 1% Benzocaine in lamprey Ringer. The spinal cord is transected with iridectomy scissors via a dorsal approach and the animal is allowed to recover in fresh water tanks at 23 &#176;C. For in situ hybridization, animals are reanesthetized and the brain and cord removed via a dorsal approach.

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

Lampreys recover locomotion after a complete spinal cord injury. However, some spinal-projecting neurons are good regenerators and others are not. This paper illustrates the techniques for housing sea lamprey larvae (and recently transformed adults), producing complete spinal cord transections and preparing wholemount brains and spinal cords for in situ hybridization.

完全性脊髓损伤和脑解剖协议后续Wholemount<em&gt;原位</em&gt;杂交苗种海七鳃鳗

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several ...

神经科学

Quantifying Liver Size in Larval Zebrafish Using Brightfield Microscopy

In several transgenic zebrafish models of hepatocellular carcinoma (HCC), hepatomegaly can be observed during early larval stages. Quantifying larval liver size in zebrafish HCC models provides a means to rapidly assess the effects of drugs and other manipulations on an oncogene-related phenotype. Here we show how to fix zebrafish larvae, dissect the tissues surrounding the liver, photograph livers using bright-field microscopy, measure liver area, and analyze results. This protocol enables rapid, precise quantification of liver size. As this method involves measuring liver area, it may underestimate differences in liver volume, and complementary methodologies are required to differentiate between changes in cell size and changes in cell number. The dissection technique described herein is an excellent tool to visualize the liver, gut, and pancreas in their natural positions for myriad downstream applications including immunofluorescence staining and in situ hybridization. The described strategy for quantifying larval liver size is applicable to many aspects of liver development and regeneration.

Here we demonstrate a method for quantifying liver size in larval zebrafish, providing a way to assess the effects of genetic and pharmacologic manipulations on liver growth and development.

使用光明场显微镜定量拉瓦尔斑马鱼的肝脏大小

In several transgenic zebrafish models of hepatocellular carcinoma (HCC), hepatomegaly can be observed during early larval stages. Quantifying larval ...

Examining Muscle Regeneration in Zebrafish Models of Muscle Disease

Skeletal muscle has a remarkable ability to regenerate following injury, which is driven by obligate tissue resident muscle stem cells. Following injury, the muscle stem cell is activated and undergoes cell proliferation to generate a pool of myoblasts, which subsequently differentiate to form new muscle fibers. In many muscle wasting conditions, including muscular dystrophy and ageing, this process is impaired resulting in the inability of muscle to regenerate. The process of muscle regeneration in zebrafish is highly conserved with mammalian systems providing an excellent system to study muscle stem cell function and regeneration, in muscle wasting conditions such as muscular dystrophy. Here, we present a method to examine muscle regeneration in zebrafish models of muscle disease. The first step involves the use of a genotyping platform that allows the determination of the genotype of the larvae prior to eliciting an injury. Having determined the genotype, the muscle is injured using a needle stab, following which polarizing light microscopy is used to determine the extent of muscle regeneration. We therefore provide a high throughput pipeline which allows the examination of muscle regeneration in zebrafish models of muscle disease.

Skeletal muscle regeneration is driven by tissue resident muscle stem cells, which are impaired in many muscle diseases such as muscular dystrophy, and this results in the inability of muscle to regenerate. Here, we describe a protocol that allows the examination of muscle regeneration in zebrafish models of muscle disease.

检查斑马鱼肌肉疾病模型中的肌肉再生

Skeletal muscle has a remarkable ability to regenerate following injury, which is driven by obligate tissue resident muscle stem cells. Following injury, ...

Eye Removal in Living Zebrafish Larvae to Examine Innervation-dependent Growth and Development of the Visual System

Zebrafish exhibit remarkable life-long growth and regenerative abilities. For example, specialized stem cell niches established during embryogenesis support continuous growth of the entire visual system, both in the eye and the brain. Coordinated growth between the retinae and the optic tectum ensures accurate retinotopic mapping as new neurons are added in the eyes and brain. To address whether retinal axons provide crucial information for regulating tectal stem and progenitor cell behaviors such as survival, proliferation, and/or differentiation, it is necessary to be able to compare innervated and denervated tectal lobes within the same animal and across animals. 
Surgical removal of one eye from living larval zebrafish followed by observation of the optic tectum achieves this goal. The accompanying video demonstrates how to anesthetize larvae, electrolytically sharpen tungsten needles, and use them to remove one eye. It next shows how to dissect brains from fixed zebrafish larvae. Finally, the video provides an overview of the protocol for immunohistochemistry and a demonstration of how to mount stained embryos in low-melting-point agarose for microscopy.

The article explains how to surgically remove eyes from living zebrafish larvae as the first step toward investigating how retinal input influences optic tectum growth and development. In addition, the article provides information about larval anesthetization, fixation, and brain dissection, followed by immunohistochemistry and confocal imaging.

活斑马鱼幼虫的眼睛去除以检查视觉系统的神经支配依赖性生长和发育

Zebrafish exhibit remarkable life-long growth and regenerative abilities. For example, specialized stem cell niches established during embryogenesis ...

DUCT: Double Resin Casting followed by Micro-Computed Tomography for 3D Liver Analysis

The liver is the biggest internal organ in humans and mice, and high auto-fluorescence presents a significant challenge for assessing the three-dimensional (3D) architecture of the organ at the whole-organ level. Liver architecture is characterized by multiple branching lumenized structures, which can be filled with resin, including vascular and biliary trees, establishing a highly stereotyped pattern in the otherwise hepatocyte-rich parenchyma. This protocol describes the pipeline for performing double resin casting micro-computed tomography, or &#34;DUCT&#34;. DUCT entails injecting the portal vein and common bile duct with two different radiopaque synthetic resins, followed by tissue fixation. Quality control by clearing one lobe, or the entire liver, with an optical clearing agent, allows for pre-screening of suitably injected samples. In the second part of the DUCT pipeline, a lobe or the whole liver can be used for micro-computed tomography (microCT) scanning, (semi-)automated segmentation, and 3D rendering of the portal venous and biliary networks. MicroCT results in 3D coordinate data for the two resins allowing for qualitative as well as quantitative analysis of the two systems and their spatial relationship. DUCT can be applied to postnatal and adult mouse liver and can be further extended to other tubular networks, for example, vascular networks and airways in the lungs.

Double resin casting micro-computed tomography, or DUCT, enables visualization, digitalization, and segmentation of two tubular systems simultaneously to facilitate 3D analysis of organ architecture. DUCT combines ex vivo injection of two radiopaque resins followed by micro-computed tomography scanning and segmentation of the tomographic data.

DUCT:双树脂铸造，然后进行微型计算机断层扫描，用于3D肝脏分析

The liver is the biggest internal organ in humans and mice, and high auto-fluorescence presents a significant challenge for assessing the ...

医学

Dissection and Flat-mounting of the Threespine Stickleback Branchial Skeleton

The posterior pharyngeal segments of the vertebrate head give rise to the branchial skeleton, the primary site of food processing in fish. The morphology of the fish branchial skeleton is matched to a species' diet. Threespine stickleback fish (Gasterosteus aculeatus) have emerged as a model system to study the genetic and developmental basis of evolved differences in a variety of traits. Marine populations of sticklebacks have repeatedly colonized countless new freshwater lakes and creeks. Adaptation to the new diet in these freshwater environments likely underlies a series of craniofacial changes that have evolved repeatedly in independently derived freshwater populations. These include three major patterning changes to the branchial skeleton: reductions in the number and length of gill raker bones, increases in pharyngeal tooth number, and increased branchial bone lengths. Here we describe a detailed protocol to dissect and flat-mount the internal branchial skeleton in threespine stickleback fish. Dissection of the entire three-dimensional branchial skeleton and mounting it flat into a largely two-dimensional prep allows for the easy visualization and quantification of branchial skeleton morphology. This dissection method is inexpensive, fast, relatively easy, and applicable to a wide variety of fish species. In sticklebacks, this efficient method allows the quantification of skeletal morphology in genetic crosses to map genomic regions controlling craniofacial patterning.

The branchial skeleton, including gill rakers, pharyngeal teeth, and branchial bones, serves as the primary site of food processing in most fish. Here we describe a protocol to dissect and flat-mount this internal skeleton in threespine sticklebacks. This method is also applicable to a variety of other fish species.

解剖和三刺鳃骨架的平面安装

The posterior pharyngeal segments of the vertebrate head give rise to the branchial skeleton, the primary site of food processing in fish. The morphology ...