Name: a new clarification method to visualize biliary degeneration during liver metamorphosis in sea lamprey (petromyzon marinus)
Uploaded: 2014-06-06T14:15:00.000+00:00
Duration: 7 min 3 s
Description: 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

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?...

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?...

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

Universidad de Cartagena UDEC

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

Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development

Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM compared to confocal systems are its low phototoxicity, gentle mounting strategies, fast acquisition with high signal to noise ratio and the possibility of imaging samples from various angles (views) for long periods of time. Imaging from multiple views unleashes the full potential of LSFM, but at the same time it can create terabyte-sized datasets. Processing such datasets is the biggest challenge of using LSFM. In this protocol we outline some solutions to this problem. Until recently, LSFM was mostly performed in laboratories that had the expertise to build and operate their own light sheet microscopes. However, in the last three years several commercial implementations of LSFM became available, which are multipurpose and easy to use for any developmental biologist. This article is primarily directed to those researchers, who are not LSFM technology developers, but want to employ LSFM as a tool to answer specific developmental biology questions. 
Here, we use imaging of zebrafish eye development as an example to introduce the reader to LSFM technology and we demonstrate applications of LSFM across multiple spatial and temporal scales. This article describes a complete experimental protocol starting with the mounting of zebrafish embryos for LSFM. We then outline the options for imaging using the commercially available light sheet microscope. Importantly, we also explain a pipeline for subsequent registration and fusion of multiview datasets using an open source solution implemented as a Fiji plugin. While this protocol focuses on imaging the developing zebrafish eye and processing data from a particular imaging setup, most of the insights and troubleshooting suggestions presented here are of general use and the protocol can be adapted to a variety of light sheet microscopy experiments.

Light sheet fluorescence microscopy is an excellent tool for imaging embryonic development. It allows recording of long time-lapse movies of live embryos in near physiological conditions. We demonstrate its application for imaging zebrafish eye development across wide spatio-temporal scales and present a pipeline for fusion and deconvolution of multiview datasets.

采用轻质板材荧光显微镜到图像斑马鱼眼睛发育

Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM ...

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

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling processes of excitable neuronal and muscular cells and many other biological processes, such as embryonic developmental patterning. However, there is a need for in vivo electrical activity monitoring in vertebrate embryogenesis. The advances of genetically encoded fluorescent voltage indicators (GEVIs) have made it possible to provide a solution for this challenge. Here, we describe how to create a transgenic voltage indicator zebrafish using the established voltage indicator, ASAP1 (Accelerated Sensor of Action Potentials 1), as an example. The Tol2 kit and a ubiquitous zebrafish promoter, ubi, were chosen in this study. We also explain&#160;the processes of Gateway site-specific cloning, Tol2 transposon-based zebrafish transgenesis, and the imaging process for early-stage fish embryos and fish tumors using regular epifluorescent microscopes. Using this fish line, we found that there are cellular electric voltage changes during zebrafish embryogenesis, and fish larval movement. Furthermore, it was observed that in a few zebrafish malignant peripheral nerve sheath tumors, the tumor cells were generally polarized compared to the surrounding normal tissues.

Here, we show the process of creating a cellular electric voltage reporter zebrafish line to visualize embryonic development, movement, and fish tumor cells in vivo.

斑马鱼早期胚胎和肿瘤细胞电活动的可视化研究

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

用扫描电镜和全贴免疫荧光技术可视化小鼠胚胎中的节点和外型板

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

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 ...

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

Partial Hepatectomy in Adult Zebrafish

Liver failure is one of the leading causes of death worldwide, and mortality from chronic liver disease is rising sharply in the United States. Healthy livers are capable of regenerating from toxic damage, but in advanced liver disease, the natural ability of the liver to regenerate is impaired. Zebrafish have emerged as a powerful experimental system for studying regeneration. They are an ideal model for studying liver regeneration from partial hepatectomy, a procedure with direct clinical relevance in which part of the liver is surgically removed, leaving the rest intact. There is no standard protocol for partial hepatectomy; previous studies using this model have used slightly different protocols and reported disparate results. Described here is an efficient, reproducible protocol for performing a partial hepatectomy in adult zebrafish. We use this technique to demonstrate that zebrafish are capable of epimorphic regeneration of the resected lobe. This protocol can be used to further interrogate the mechanisms required for liver regeneration in zebrafish.

This protocol describes the procedure for removing the ventral lobe of the liver in adult zebrafish to enable the study of liver regeneration.

成人斑马鱼的部分肝切除术

Liver failure is one of the leading causes of death worldwide, and mortality from chronic liver disease is rising sharply in the United States. Healthy ...