Name: using a whole-mount immunohistochemical method to study the innervation of the biliary tract in suncus murinus
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所有实验程序均由东京都大学体育动物护理与使用委员会（IACUC）批准。根据"实验动物护理和使用 指南"和 "加拿大动物护理委员会实验动物护理和使用指南"，将动物饲养和处理。我们在日本东京都大学前沿健康科学系功能形态学实验室养殖和维护动物。 动物护理和住房<ol><li>从封闭的繁殖群中获得7只小鼠 （3只雌性和4只雄性，重70-90克）。 </li><li>在装有含有纸条的木制巢箱的塑料笼中断奶后分别保存S. murinus （出生后20 d）。将它们保存在常规条件的动物室内:23-27℃，无湿度控制，12:12小时:黑暗循环。供应商业唾液丸和水随意。 </li></ol> 2.组织准备<ol><li>为了制备4％（w / v）多聚甲醛（PFA），将40g PFA溶于1,000mL 0.01M磷酸盐缓冲盐水（PBS，pH7.4）中。在通风柜中，使用涡流进行混合，直到溶液清亮。在4℃下储存4％PFA O / N。 注意:在处理PFA时佩戴适当的个人防护装备（PPE）。 </li><li>灌注并固定动物，用乙醚麻醉，然后腹膜内注射睡眠素（戊巴比妥钠，0.6 mL / kg体重）。 </li><li>动物完全麻醉后，用解剖刀打开腹腔，形成3厘米长的中线腹部切口。在切除下腔静脉用于放血时，将导管逆行插入腹主动脉，在该动脉分叉上方的水平进入髂总动脉。 </li><li>注射嗜睡眠（戊巴比妥）ium，1.0mL / kg体重）。完全安乐死后，用0.01M PBS（pH 7.4）灌注，然后用4％PFA灌装在通风柜中。 </li><li>灌注后，注入2-3mL白色氯丁橡胶胶乳（稀释的白色氯丁橡胶胶乳3:2，用蒸馏水（DW））标记血管。 </li><li>将有关神经和血管的腹部器官，包括肝脏，胆囊，胆总管，十二指肠和胰腺一起提取。 </li><li>将约0.1mL蓝色氯丁橡胶胶乳（向一滴稀释的白色氯丁橡胶胶乳中加入一滴蓝色墨水）注入胆汁以标记胆汁系统。 </li><li>在4℃下用4％PFA固定过夜以进行全套免疫染色。 注意:PFA有毒。避免直接使用PFA。 PFA应用于通风柜。 </li></ol> 3.全身免疫组织化学注意:在整个方案中，包括在洗涤，抗体孵育和着色期间，e样品必须留在钳子上，轻轻地在室温或4℃下摇摆。 <ol><li> 第1天:酶促处理和灭活潜在的内源性过氧化物酶 <ol><li>在适当尺寸的玻璃小瓶中，每个室温下用DW 4x将制备的样品洗涤1小时。将样品轻轻摇匀，以除去PFA。避免在交换溶液时损坏样品。 </li><li>为了制备1％（w / v）的正周期酸，将1g正周期酸溶于100mL的DW中。 </li><li>在室温下将样品在1％正二碘酸中孵育20分钟，以防止任何内在的过氧化物酶反应。 </li><li>通过将0.004g木瓜蛋白酶溶解在100mL 0.025mol / L Tris-HCl缓冲液（pH7.6）中来制备0.004％（w / v）木瓜蛋白酶。 </li><li>在室温下用DW清洗样品10分钟。 </li><li>将样品在新鲜制备的0.004％木瓜蛋白酶中，在37℃下，在温和摇摆的恒温槽中孵育2小时。 </li><li>用D清洗试样W在室温下保持50-60分钟。 </li><li>以4°CO / N将样品储存在4％PFA中。 </li></ol></li><li> 第2天:酶治疗 <ol><li>用DW洗涤存放的样品4次，每次1小时。 </li><li>将样品在新鲜制备的0.004％木瓜蛋白酶中，在37℃下，在温和摇摆的恒温槽中孵育2小时。 </li><li>在室温下用DW洗涤样品50-60分钟。以4°CO / N将样品储存在4％PFA中。 </li></ol></li><li> 第3天:冷冻治疗 <ol><li>按照上述方式，在室温下用DW 4x洗涤储存的样品1小时。 </li><li>通过将2.5g，5g和10g蔗糖分别溶解在100mL的0.01M PBS（0.5g / l）中制备2.5％（w / v），5％（w / v）和10％（w / v） pH 7.4）。 </li><li>将样品浸入2.5％（w / v），5％（w / v）和10％（w / v）蔗糖中30分钟。 </li><li>将样品在-20℃下冷冻30-60分钟，然后在室温下完全解冻;重复周期3x。 </li><li>最佳补充2％Triton X-100（v / v），加入2mL Triton X-100至100mL的0.01M PBS（pH7.4）。 </li><li>将样品以4°CO / N存放在2％Triton X-100中。 </li></ol></li><li> 第4天:初次抗体孵育 <ol><li>通过将0.2g牛血清白蛋白（BSA），0.3mL Triton X-100和0.1g叠氮化钠溶解在100mL 0.01M PBS（pH 7.4）中来制备第一抗体的稀释溶液。 注意:叠氮化钠具有剧毒性。必须避免吸入和接触。穿合适的PPE。 </li><li>在上述稀释缓冲液中稀释第一抗体（NFP）1:600（步骤3.4.1）。在4℃下将标本与NFP抗体孵育3-6天，保持标本轻轻地摇动。较大的标本将需要更长的孵化时间。 </li></ol></li><li> 二次抗体孵育 <ol><li>在PBS中将样品洗涤1次，每次1小时，以除去未结合的一抗。 </l我&gt; <li>通过将0.2g BSA和0.3mL Triton X-100溶解在100mL 0.01M PBS（pH 7.4）中来制备二抗的稀释溶液。 </li><li>在稀释缓冲液中稀释第二抗体（过氧化物酶缀合的，亲和纯化的绵羊抗小鼠IgG-HRP）1:600（步骤3.5.2）。在4℃下将样品与二次抗体孵育3 d，并将样品轻轻晃动在养分器上。 </li></ol></li><li> 着色 <ol><li>通过在通风橱下在100mL 0.05mol / L Tris-HCl缓冲液（pH7.6）中加入0.002g 3,3&#39;-二氨基联苯胺四盐酸盐（DAB）制备DAB着色溶液，将溶液保持在黑暗中。 </li><li>在PBS中4℃洗涤样品1小时。 </li><li>在新制备的DAB着色溶液中加入10μL的30％H 2 O 2 ，并在4°CO / N下孵育3 d，同时在养分器上轻轻晃动。 </li><li>停止pla的反应当达到最佳染色强度时，用0.04％叠氮化钠在PBS中取样。 注意:在此步骤之前，避免使用叠氮化钠，因为它可以抑制辣根过氧化物酶。 </li></ol></li><li> 成像染色的组织 <ol><li>小心地将样品转移到含有PBS的培养皿中（5厘米高，直径10厘米）。使用安装在立体显微镜上的相机拍摄整个安装的图像。 注意:将完全染色的组织成像，完全浸入PBS中的透明玻璃皿上。 </li></ol></li></ol>

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作者声明没有利益冲突。

这项工作描述了使用抗神经丝蛋白的抗体对肝外胆道神经支配的可视化的实验程序。该协议根据Kuratani和Tanaka 1，2，3描述的方案进行了改编。 对于这个实验，计划每个实验的时间表，因为整体染色方法持续2-3周。包含组织的容器必须始终在营养器上旋转，除非在冻融过程中。特别是在与一级和二级抗体一起孵育的同时，将样品置于冷藏室中，同时在养分器上旋转以确保试样均匀地暴露于反应溶液中。几种溶液 - 1％（w / v）正周期酸，0.004％（w / v）木瓜蛋白酶和用于初级/次级抗体的稀释缓冲液死亡 - 必须为每个实验新鲜。在整个协议中，为避免在更换溶液时接触和损坏样品，应将溶液倒出而不是用镊子15去除。 用PFA固定后，应用DW或PBS充分洗涤样品。此外，用木瓜蛋白酶孵育的酶处理是重要的。用于抗原回收的热和酶有助于制备抗体穿透的组织。为了增强溶液的渗透性，组织的外膜应在-20℃冷冻处理30分钟或-80°CO / N 15时被破坏。 将实验样品完全浸没在足够体积的缓冲溶液中以确保溶液能够孵育整个样品是重要的。一次抗体的孵育时间必须经验确定d用于不同的组织和样品量。通常2-3厘米的样品必须是3天。每个抗体必须经验确定最佳稀释度。当使用适当的抗体缓冲液时，推荐使用1:600的稀释度作为主抗体和第二抗体。 这项工作详细描述了一种通用的全蛋白免疫组织化学方法来揭示S. murinus胆道中的神经丝蛋白表达的方案。该方法可用于分析许多物种中所有内脏器官的神经支配。此外，该方案还可以适应于测试其他神经元抗体，但是应该进行抗体的优化。 然而，该技术在标记浅层神经组织和构建三维神经支配模型方面受到限制。此外，它不能确定神经纤维的特征（ 即交感神经或par无症状，运动或感觉等 ）。

房子麝香rew，，，属于食虫纲和家Sor科。这种小型哺乳动物分布在整个东南亚，栖息在靠近人类栖息地或牛栏的房屋和草地上。与其他实验室动物（如小鼠，大鼠和兔5）相比，该物种具有与人类相似的一般形态特征。以前，使用具有S. murinus神经丝蛋白（NFP）的外周神经元标记的全蛋白免疫染色来标记胰腺6 ，主要十二指肠乳头7 ，幽门8 ，胆囊5和肝外胆道9中的外周神经。 NFP是成熟神经元细胞骨架的一部分的大分子复合物。神经丝相关蛋白介导NFP和Zygosome之间的相互作用。酶功能和接头蛋白的结构均受NFP 10调控。 NFP复合物由三种多肽组成:NF-L（70kDa），NF-M（150-160kDa）和NF-H（200kDa）。 NFP可以在中枢神经系统和外周神经系统的神经元轴突中发现。已经显示抗人NFP抗体标记中枢和周围神经系统的轴突。解剖和电生理调查表明，括约肌，胆囊和近端胃肠道连接11,12,13,14。然而，他们的神经元连接的形态特征尚未被定义。 这项工作表明使用全套免疫组织化学染色方法来标记耻骨球菌的神经支配使用NFP抗体。

<table><tbody><tr><td>orthoperiodic acid</td><td>Wako</td><td>162-00732</td><td></td></tr><tr><td>papain</td><td>Wako</td><td>164-00172</td><td></td></tr><tr><td>bovine serum albumin (BSA)</td><td>Wako</td><td>010-15131</td><td></td></tr><tr><td>sodium azide</td><td>Nacalai Tesque</td><td>312-33</td><td></td></tr><tr><td>neurofilament protein (NFP) antibody</td><td>Dako</td><td>M0762, lot 089, clone: 2F11</td><td></td></tr><tr><td>peroxidase-conjugated affinity-purified sheep anti-mouse IgG-HRP</td><td>MBL</td><td>Code 330</td><td></td></tr><tr><td>3,3&#039;-diaminobenzidine tetrahydrochloride (DAB)</td><td>Wako</td><td>349-00903</td><td></td></tr><tr><td>imidazole</td><td>Sigma</td><td>I-0125</td><td></td></tr></tbody></table>

using a whole-mount immunohistochemical method to study the innervation of the biliary tract in suncus murinus

本文详细描述了全基因免疫组织化学染色方法，使用神经丝蛋白抗体标记阳性丝猴（ S. murinus）胆道神经支配 &#160; ）。首先从S. murinus中切下标本并固定在4％多聚甲醛（PFA）中。其次，进行酶处理和潜在的内源性过氧化物酶失活。然后将标本暴露于一抗，抗神经丝蛋白抗体3-6天。然后与与辣根过氧化物酶缀合的二抗孵育。通过使样品与3,3&#39;-二氨基联苯胺（DAB）底物反应来显示显色反应。该方法可用于分析所有内脏器官的神经支配。此外，该方案还可以适应于测试其他神经元抗体，但优化抗体应先做好。这种方法最初由Kuratani和Tanaka 1，2，3引入。

图 1-4显示了S. murinus肝外胆道中NFP阳性神经纤维的典型结果。抗NFP的抗体可重复标记肝外胆管（ 图1 ），胆囊（ 图2 ），上胆管（ 图3 ）和十二指肠乳头区域（ 图4 ）的整个图像中的神经支配，具有高特异性和最小背景。 对于样本的所有组织，无论形状和大小如何，可以同时染色被盖的神经。除了肝外胆道的神经支配外，食管和胃的迷走神经的运行和分布密度明确地展现（ 图1 ）。 图2 ）。 在上胆管中，标记了两种类型的神经束。精神神经束形成不规则且密集的神经网络，粘连，并居住在胆道上/胆道;较粗的神经束平行于胆道表面分布（ 图3 ）。 胆总管用蓝色氯丁橡胶胶乳和含有氯丁橡胶胶乳的容器进行标记。在胆总管末端和十二指肠乳头区域的细小神经纤维明显表现为高c<html> ontrast（ 图4 ）。 <img alt="图1" class="xfigimg" src="/files/ftp_upload/55483/55483fig1.jpg" /> 图1:胆道，食道和胃中的NFP阳性神经纤维。箭头显示迷走神经沿食道行进到胃。 CBD，常见胆管;食道，食道;圣，胃。刻度棒=1,300μm。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55483/55483fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" class="xfigimg" src="/files/ftp_upload/55483/55483fig2.jpg" /> 图2:胆囊中的NFP阳性神经纤维。胆囊的血管用白色胶乳标记。在胆囊颈部发现丰富的神经支配（箭头）。刻度棒=1,000μm。ref ="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55483/55483fig2large.jpg"target ="_ blank"&gt;请点击此处查看此图的较大版本。 <img alt="图3" class="xfigimg" src="/files/ftp_upload/55483/55483fig3.jpg" /> 图3:上胆管中NFP阳性神经纤维。观察到两种类型的神经束。一种类型是粘连地存在于肝外胆道（箭头）上的细神经束;另一种类型是较厚的神经束，其平行于肝外胆道表面分布并在胆囊和十二指肠（三角形）之间运行。 PV，门静脉。刻度尺=650μm。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55483/55483fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图4" class="xfigimg"src ="/ files / ftp_upload / 55483 / 55483fig4.jpg"/&gt; 图4:十二指肠乳头区域中的NFP阳性神经纤维。胆总管标有蓝色胶乳和白色胶乳（*）。箭头显示胆总管末端的神经支配，三角形显示来自胆总管和血管的十二指肠乳头区域（P）的神经支配。刻度棒=1,000μm。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55483/55483fig4large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about Using a Whole-mount Immunohistochemical Method to Study the Innervation of the Biliary Tract in Suncus murinus at JoVE.com

Using a Whole-mount Immunohistochemical Method to Study the Innervation of the Biliary Tract in Suncus murinus

一种全身免疫组织化学方法，可以显示Suncus murinus肝外胆道中的神经丝蛋白表达。在这里介绍。该方案可用于分析小鼠或其他物种中所有内脏器官的神经支配。

使用全身免疫组织化学方法研究胆道的神经支配<em&gt; Suncus murinus</em

This work describes the whole-mount immunohistochemistry staining method in detail, using neurofilament protein antibody to label the innervation of the ...

using-whole-mount-immunohistochemical-method-to-study-innervation

Universidad San Sebastian

Research

JoVE Journal

Biology

Using a Whole-mount Immunohistochemical Method to Study the Innervation of the Biliary Tract in Suncus murinus

6.7K 次观看.  Tokyo Metropolitan University. 一种全身免疫组织化学方法，可以显示Suncus murinus肝外胆道中的神经丝蛋白表达。在这里介绍。该方案可用于分析小鼠或其他物种中所有内脏器官的神经支配。 

视频: Using a Whole-mount Immunohistochemical Method to Study the Innervation of the Biliary Tract in Suncus murinus

Immunostaining to Visualize Murine Enteric Nervous System Development

The enteric nervous system is formed by neural crest cells that proliferate, migrate and colonize the gut. Following colonization, neural crest cells must then differentiate into neurons with markers specific for their neurotransmitter phenotype. Cholinergic neurons, a major neurotransmitter phenotype in the enteric nervous system, are identified by staining for choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine. Historical efforts to visualize cholinergic neurons have been hampered by antibodies with differing specificities to central nervous system versus peripheral nervous system ChAT. We and others have overcome this limitation by using an antibody against placental ChAT, which recognizes both central and peripheral ChAT, to successfully visualize embryonic enteric cholinergic neurons. Additionally, we have compared this antibody to genetic reporters for ChAT and shown that the antibody is more reliable during embryogenesis. This protocol describes a technique for dissecting, fixing and immunostaining of the murine embryonic gastrointestinal tract to visualize enteric nervous system neurotransmitter expression.

The enteric nervous system is formed by neural crest cells that proliferate, migrate and colonize the gut. Neural crest cells differentiate into neurons with markers specific for their neurotransmitter phenotype. This protocol describes a technique for dissecting, fixing and immunostaining of the murine embryonic gastrointestinal tract to visualize enteric nervous system neurotransmitter expression.

免疫组化可视化小鼠肠神经系统的发展

The enteric nervous system is formed by neural crest cells that proliferate, migrate and colonize the gut. Following colonization, neural crest cells must ...

科研

发育生物学

In Vivo Immunofluorescence Localization for Assessment of Therapeutic and Diagnostic Antibody Biodistribution in Cancer Research

Monoclonal antibodies (mAbs) are important tools in cancer detection, diagnosis, and treatment. They are used to unravel the role of proteins in tumorigenesis, can be directed to cancer biomarkers enabling tumor detection and characterization, and can be used for cancer therapy as mAbs or antibody-drug conjugates to activate immune effector cells, to inhibit signaling pathways, or directly kill cells carrying the specific antigen. Despite clinical advancements in the development and production of novel and highly specific mAbs, diagnostic and therapeutic applications can be impaired by the complexity and heterogeneity of the tumor microenvironment. Thus, for the development of efficient antibody-based therapies and diagnostics, it is crucial to assess the biodistribution and interaction of the antibody-based conjugate with the living tumor microenvironment. Here, we describe In Vivo Immunofluorescence Localization (IVIL) as a new approach to study interactions of antibody-based therapeutics and diagnostics in the in vivo physiological and pathological conditions. In this technique, a therapeutic or diagnostic antigen-specific antibody is intravenously injected in&#160;vivo and localized ex vivo with a secondary antibody in isolated tumors. IVIL, therefore, reflects the in vivo biodistribution of antibody-based drugs and targeting agents. Two IVIL applications are described assessing the biodistribution and accessibility of antibody-based contrast agents for molecular imaging of breast cancer. This protocol will allow future users to adapt the IVIL method for their own antibody-based research applications.

The in vivo immunofluorescence localization (IVIL) method can be used to examine in vivo biodistribution of antibodies and antibody conjugates for oncological purposes in living organisms using a combination of in vivo tumor targeting and ex vivo immunostaining methods.

在Vivo免疫荧光定位中用于评估癌症研究中的治疗和诊断抗体生物分布

Monoclonal antibodies (mAbs) are important tools in cancer detection, diagnosis, and treatment. They are used to unravel the role of proteins in ...

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 ...

Bile Duct Ligation in Mice: Induction of Inflammatory Liver Injury and Fibrosis by Obstructive Cholestasis

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well as to excrete bilirubin and other metabolic products. In the liver, the experimental obstruction of the extrahepatic biliary system initiates a complex cascade of pathological events that leads to cholestasis and inflammation resulting in a strong fibrotic reaction originating from the periportal fields. Therefore, surgical ligation of the common bile duct has become the most commonly used model to induce obstructive cholestatic injury in rodents and to study the molecular and cellular events that underlie these pathophysiological mechanisms induced by inappropriate bile flow. In recent years, different surgical techniques have been described that either allow reconnection or reanastomosis after bile duct ligation (BDL), e.g., partial BDL, or other microsurgical methods for specific research questions. However, the most frequently used model is the complete obstruction of the common bile duct that induces a strong fibrotic response after 21 to 28 days. The mortality rate can be high due to infectious complications or technical inaccuracies. Here we provide a detailed surgical procedure for the BDL model in mice that induce a highly reproducible fibrotic response in accordance to the 3R rule for animal welfare postulated by Russel and Burch in 1959.

Disruption of bile flow results in severe inflammatory cholestatic liver injury with a characteristic time-dependent sequence of morphological alterations. Here we present a protocol for the surgical ligation of the common bile duct in mice that allows to induce a strong fibrotic response after 21 to 28 days.

胆管结扎小鼠:感应炎性肝损伤和纤维化的阻塞性胆汁淤积

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well ...

医学

In Situ Ca2+ Imaging of the Enteric Nervous System

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ganglionated plexuses housed in the gut wall. Enteric neurons and enteric glia are the only cell types within the enteric ganglia. The activity of enteric neurons and glia is responsible for coordinating intestinal functions. This protocol describes methods for observing the activity of neurons and glia within the intact ENS by imaging intracellular calcium (Ca2+) transients with fluorescent indicator dyes. Our technical discussion focuses on methods for Ca2+ imaging in whole-mount preparations of the myenteric plexus from the rodent bowel. Bulk loading of ENS whole-mounts with a high-affinity Ca2+ indicator such as Fluo-4 permits measurements of Ca2+ responses in individual neurons or glial cells. These responses can be evoked repeatedly and reliably, which permits quantitative studies using pharmacological tools. Ca2+ responses in cells of the ENS are recorded using a fluorescence microscope equipped with a cooled charge-coupled device (CCD) camera. Fluorescence measurements obtained using Ca2+ imaging in whole-mount preparations offer a straightforward means of characterizing the mechanisms and potential functional consequences of Ca2+ responses in enteric neurons and glial cells.

In Situ Ca2+ Imaging of the Enteric Nervous System

The enteric nervous system (ENS) is a network of neurons and glia located in the gut wall that controls intestinal reflexes. This protocol describes methods for recording the activity of enteric neurons and glia in live preparations of ENS using Ca2+ imaging.

在肠神经系统的原位 钙成像

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ...

神经科学

Visualization of the Interstitial Cells of Cajal (ICC) Network in Mice

The interstitial cells of Cajal (ICC) are mesenchymal derived "pacemaker cells" of the gastrointestinal (GI) tract that generate spontaneous slow waves required for peristalsis and mediate neuronal input from the enteric nervous system1. Different subtypes of ICC form distinct networks in the muscularis of the GI tract 2,3. Loss or injury to these networks is associated with a number of motility disorders4. ICC cells express the KIT receptor tyrosine kinase on the plasma membrane and KIT immunostaining has been used for the past 15 years to label the ICC network5,6. Importantly, normal KIT activity is required for ICC development5,6. Neoplastic transformation of ICC cells results in gastrointestinal stromal tumor (GIST), that frequently harbor gain-of-function KIT mutations7,8. We recently showed that ETV1 is a lineage-specific survival factor expressed in the ICC/GIST lineage and is a master transcriptional regulator required for both normal ICC network formation and for of GIST tumorigenesis9. We further demonstrate that it cooperates with activating KIT mutations in tumorigenesis. Here, we describe methods for visualization of ICC networks in mice, largely based on previously published protocols10,11. More recently, the chloride channel anoctamin 1 (ANO1) has also been characterized as a specific membrane marker of ICC11,12. Because of their plasma membrane localization, immunofluorescence of both proteins can be used to visualize the ICC networks. Here, we describe visualization of the ICC networks by fixed-frozen cyrosections and whole mount preparations.

Visualization of the Interstitial Cells of Cajal ICC Network in Mice

The interstitial cells of Cajal (ICC) are the pacemaker cells of the gastrointestinal (GI) tract. They form complex networks between smooth muscle cells and post-ganglionic neuronal fibers to regulate GI contractility. Here, we present immunofluorescence methods cross-sectional and whole-mount visualization of murine ICC networks.

卡哈尔（ICC）的网络的间质细胞在小鼠体内的可视化

The interstitial cells of Cajal (ICC) are mesenchymal derived "pacemaker cells" of the gastrointestinal (GI) tract that generate spontaneous slow waves ...

生物学

Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal epithelium. Due in part to the lack of a definitive ligand for the &#947;&#948; T cell receptor, our understanding of the regulation of &#947;&#948; IEL activation and their function in vivo remains limited. This necessitates the development of alternative strategies to interrogate signaling pathways involved in regulating &#947;&#948; IEL function and the responsiveness of these cells to the local microenvironment. Although &#947;&#948; IELs are widely understood to limit pathogen translocation, the use of intravital imaging has been critical to understanding the spatiotemporal dynamics of IEL/epithelial interactions at steady-state and in response to invasive pathogens. Herein, we present a protocol for visualizing IEL migratory behavior in the small intestinal mucosa of a GFP &#947;&#948; T cell reporter mouse using inverted spinning disk confocal laser microscopy. Although the maximum imaging depth of this approach is limited relative to the use of two-photon laser-scanning microscopy, spinning disk confocal laser microscopy provides the advantage of high speed image acquisition with reduced photobleaching and photodamage. Using 4D image analysis software, T cell surveillance behavior and their interactions with neighboring cells can be analyzed following experimental manipulation to provide additional insight into IEL activation and function within the intestinal mucosa.

We describe a method to visualize GFP-labeled &#947;&#948; IELs using intravital imaging of murine small intestine by inverted spinning disk confocal microscopy. This technique enables the tracking of live cells within the mucosa for up to 4 h and can be used to investigate a variety of intestinal immune-epithelial interactions. &#160;

毛里小肠内皮淋巴细胞的内质成像

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal ...

免疫与感染

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

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 countries, with a reported frequency of 1 in 5,000 in Taiwan. Although much is known about the management of biliary atresia, its pathogenesis is still elusive. The sea lamprey (Petromyzon marinus) provides a unique opportunity to examine the mechanism and progression of biliary degeneration. Sea lamprey develop through three distinct life stages: larval, parasitic, and adult. During the transition from larvae to parasitic juvenile, sea lamprey undergo metamorphosis with dramatic reorganization and remodeling in external morphology and internal organs. In the liver, the entire biliary system is lost, including the gall bladder and the biliary tree. A newly-developed method called &#8220;CLARITY&#8221; was modified to clarify the entire liver and the junction with the intestine in metamorphic sea lamprey. The process of biliary degeneration was visualized and discerned during sea lamprey metamorphosis by using laser scanning confocal microscopy. This method provides a powerful tool to study biliary atresia in a unique animal model.

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

Sea lamprey lose the gall bladder and bile ducts during metamorphosis, a process similar to human biliary atresia. A new fixation and clarification method (CLARITY) was modified to visualize the entire biliary tree using laser scanning confocal microscopy. This method provides a powerful tool to study biliary degeneration.

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 ...

Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic fatty liver disease, metabolic liver disease, and liver cancer) requires experimental manipulation of animal models and in vitro cell cultures. Both techniques have limitations, such as the requirement of large numbers of animals for in vivo manipulation. However, in vitro cell cultures do not reproduce the structure and function of the multicellular hepatic environment. The use of precision-cut liver slices is a technique in which uniform slices of viable mouse liver are maintained in laboratory tissue culture for experimental manipulation. This technique occupies an experimental niche that exists between animal studies and in vitro cell culture methods. The presented protocol describes a straightforward and reliable method to isolate and culture precision-cut liver slices from mice. As an application of this technique, ex vivo liver slices are treated with bile acids to simulate cholestatic liver injury and ultimately assess the mechanisms of hepatic fibrogenesis.

This protocol provides a simple and reliable method for the production of viable precision-cut liver slices from mice. The ex vivo tissue samples can be maintained under laboratory tissue culture conditions for multiple days, providing a flexible model to examine liver pathobiology.

毛利精密切肝片作为肝脏生物学的Ex Vivo模型

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic ...

Location, Dissection, and Analysis of the Murine Stellate Ganglion

The autonomic nervous system is a substantial driver of cardiac electrophysiology. Especially&#160;the role of its sympathetic branch is an ongoing matter of investigation in the pathophysiology of ventricular arrhythmias (VA). Neurons in the stellate ganglia (SG)&#160;&#8211;&#160;bilateral star-shaped structures of the sympathetic chain &#8211;&#160;are an important component of the sympathetic infrastructure. The SG are a recognized target for&#160;treatment via cardiac sympathetic denervation in patients with therapy-refractory VA. While neuronal remodeling and glial activation in the SG have been described in patients with VA, the underlying cellular and molecular processes that potentially precede the onset of arrhythmia are only insufficiently understood and should be elucidated to improve autonomic modulation. Mouse models allow us to study sympathetic neuronal remodeling, but identification of the murine SG is challenging for the inexperienced investigator. Thus, in-depth cellular and molecular biological studies of the murine SG are lacking for many common cardiac diseases. Here, we describe a basic repertoire for dissecting and studying the SG in adult mice for analyses at RNA level (RNA isolation for gene expression analyses, in situ hybridization), protein level (immunofluorescent whole mount staining), and cellular level (basic morphology, cell size measurement). We present potential solutions to overcome challenges in the preparation technique, and how to improve staining via quenching of autofluorescence. This allows for the visualization of neurons as well as glial cells via established markers in order to determine cell composition and remodeling processes. The methods presented here allow characterizing the SG to gain further information on autonomic dysfunction in mice prone to VA and can be complemented by additional techniques investigating neuronal and glial components of the autonomic nervous system in the heart.

Pathophysiological changes in the cardiac autonomic nervous system, especially in its sympathetic branch, contribute to the onset and maintenance of ventricular arrhythmias. In the present protocol, we show how to characterize murine stellate ganglia to improve the understanding of the underlying molecular and cellular processes.

穆林·斯特拉特·甘利翁的位置、解剖和分析

The autonomic nervous system is a substantial driver of cardiac electrophysiology. Especially&#160;the role of its sympathetic branch is an ongoing matter ...

使用全身免疫组织化学方法研究胆道的神经支配

摘要

探索更多视频

此视频中的章节

免疫组化可视化小鼠肠神经系统的发展

在Vivo免疫荧光定位中用于评估癌症研究中的治疗和诊断抗体生物分布

确定小鼠肝脏中的胆汁密度

胆管结扎小鼠:感应炎性肝损伤和纤维化的阻塞性胆汁淤积

在肠神经系统的原位 ^钙成像

卡哈尔（ICC）的网络的间质细胞在小鼠体内的可视化

毛里小肠内皮淋巴细胞的内质成像

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）

毛利精密切肝片作为肝脏生物学的Ex Vivo模型

穆林·斯特拉特·甘利翁的位置、解剖和分析

使用全身免疫组织化学方法研究胆道的神经支配

摘要

探索更多视频

此视频中的章节

免疫组化可视化小鼠肠神经系统的发展

在Vivo免疫荧光定位中用于评估癌症研究中的治疗和诊断抗体生物分布

确定小鼠肝脏中的胆汁密度

胆管结扎小鼠:感应炎性肝损伤和纤维化的阻塞性胆汁淤积

在肠神经系统的原位 钙成像

卡哈尔（ICC）的网络的间质细胞在小鼠体内的可视化

毛里小肠内皮淋巴细胞的内质成像

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）

毛利精密切肝片作为肝脏生物学的Ex Vivo模型

穆林·斯特拉特·甘利翁的位置、解剖和分析

在肠神经系统的原位 ^钙成像