Name: determining bile duct density in the mouse liver
Uploaded: 2019-04-30T14:45:00
Description: Watch this Scientific Journal Video about Determining Bile Duct Density in the Mouse Liver at JoVE.com

作者感谢美国国家卫生研究院(R01 GM084135和R01 DK109982)的支持,来自德克萨斯州医疗中心消化疾病中心的试点/可行性奖,由NIH P30 DK56338颁发,阿拉吉勒综合征加速器奖来自医学基金会

所有动物被安置在贝勒医学院的屏障动物设施,根据机构动物护理和使用委员会的指导方针和批准的动物协议。
1. 小鼠肝脏组织收集
<ol><li>
为肝脏收获准备小鼠
	<ol>
<li>使用胶然化小鼠安乐死。</li>
		<li>执行小鼠的宫颈脱位,以确保死亡。</li>
		<li>在肋骨笼下方约一英寸处进行横向切口。</li>
		<li>露出肝脏的整个腹腔表面。</li>
	</ol>
</li>
	<li>
小鼠肝脏的收?...

<ol>
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小鼠BD发育与修复分析是研究胆囊性疾病发病机制和机理的重要工具。此外,新疗法的发展部分取决于建立可重复的,最好是可量化的表型。小鼠模型中的电流表示通常涉及血清化学、肝脏信息学和细胞类型特定标记物的免疫染色。虽然这些技术产生了关于胆管系统的结构和功能的宝贵信息,但它们不能直接测量给定基因操作对发育系统数量的影响。在解剖病理学中,通过分析活检第9...

胆汁树是哺乳动物肝脏的关键部分,允许胆汁从肝细胞进入肠道。内肝胆管(BDs)是由胆囊形成,通过Notch和TGF+信号1,2与双电位肝细胞区分。正确规范和承诺胆汁及其组装成成熟的BD对肝胆内树的发展至关重要。随着肝脏在发育过程中或器官再生时生长,胆汁系统需要沿着肝脏发展,以确保适当的胆汁排水。此外,一些综合和非合成性疾病导致肝内3的缺乏。此外,一些急性和慢性肝病引起所谓的肝脏导管反应,这些反应被定义为存在大量细胞,这些细胞表示胆道标记,但不一定来自胆道细胞或形式专利BD4.在多系统紊乱阿拉吉勒综合征(ALGS),单体不全的诺奇配体锯齿1(JAG1)导致不良的BD形成和胆汁症5,6。我们的实验室最近证明,以前生成的Jag1杂物小鼠线7是ALGS 8中BD贫乏的动物模型。在这个ALGS的小鼠模型中,胆小板仍然存在。然而,他们未能承诺纳入成熟的专利BD8。因此,在BD缺乏模型中分析肝脏需要的不仅仅是胆汁的明显存在或缺乏。准确评估成熟性BD在肝脏中存在的程度非常重要。
在解剖病理学中,有公认的定量方法来评估BD是否存在9。例如,对人类患者ALGS的研究通常通过分析每个肝脏活检至少10个门户血管9,10来量化BD到门户静脉(PV)比率。分析形状和整体存在或缺乏专利的BD,结合血清化学,可以提供关于小鼠11,12,13的BD发展的宝贵信息。然而,小鼠可能失去大量的BD,只有血清胆红素水平8略有增加。因此,一种定量方法,评估每个PV存在的BD数量,可以提供更直接的测量在小鼠的BD不足程度。在最近的一份报告中,我们量化了所有肝瓣中每PV的BD数,并报告Jag1+/+动物8的BD与PV比显著下降。在分析过程中,我们注意到,尽管炎症反应和管道反应的程度有显著差异,但BD与PV比并没有表现出太大的变异性8。此外,对BD与PV比的量化使我们能够证明,在Jag1+/+动物中去除一个糖基转移酶基因Poglut1副本可以显著改善其BD不足8。在Jag1+/+背景下,血管平滑肌细胞中Poglut1的有条件损失会导致 BD 数量逐渐增加,这是适度的 (20-30%)在P7,但在成人8变得突出。再次,这项技术允许我们证明,即使在P7,这些动物的BD密度的增加是统计显著。值得注意的是,通过树脂铸造分析,也验证了4个月大时这种基因型的BD密度增加。8这些观察和其他报告测量了不同ALGS小鼠模型14、15的BD密度,这促使我们把这种方法纳入我们的整体策略,以分析各种突变体中的胆汁缺陷和转基因小鼠。
在这里,我们详细介绍了一个简单的技术,可用于检查小鼠肝脏疾病模型中的BD缺乏程度(图1)。在此方法中,与胆小球菌标记物 (CK) 8 和 CK19 (下到广谱 CK, wsCK) 共同染色用于可视化小鼠肝脏中的 BD 和未合并的胆汁。对α平滑肌活动蛋白(αSMA)的抗体被添加到染色中,以标记血管。系统分析涵盖所有肝叶的一节中的BD与PV比,确保对每种基因型分析大量的PV。由于我们的方法依赖于在二维图像中量化 BD 和 PV,因此它不适合研究给定突变对胆汁树的 3D 结构的影响或小胆管的完整性。然而,它为调查人员提供了一个简单而客观的策略,以评估小鼠的胆汁发育情况。

<table>
	<tbody>
		<tr>
			<td>Isothesia (Isoflurane)</td>
			<td>Henry Schein</td>
			<td>11695-6776-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Desiccator</td>
			<td>Bel-Art</td>
			<td>16-800-552</td>
			<td></td>
		</tr>
		<tr>
			<td>10% PFA</td>
			<td>Electron Microscopy Sciences</td>
			<td>15712</td>
			<td></td>
		</tr>
		<tr>
			<td>50mL tube</td>
			<td>ThermoScientific</td>
			<td>339653</td>
			<td></td>
		</tr>
		<tr>
			<td>70% Ethanol</td>
			<td>Decon Laboratories</td>
			<td>2401</td>
			<td></td>
		</tr>
		<tr>
			<td>95% Ethanol</td>
			<td>Decon Laboratories</td>
			<td>2801</td>
			<td></td>
		</tr>
		<tr>
			<td>100% Ethanol</td>
			<td>Decon Laboratories</td>
			<td>2701</td>
			<td></td>
		</tr>
		<tr>
			<td>HistoChoice</td>
			<td>VWR Life Sciences</td>
			<td>H103-4L</td>
			<td>clearing agent</td>
		</tr>
		<tr>
			<td>Omnisette Tissue Cassette</td>
			<td>Fisher HealthCare</td>
			<td>15-197-710E</td>
			<td></td>
		</tr>
		<tr>
			<td>Macrosette</td>
			<td>Simport</td>
			<td>M512</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraplast X-TRA</td>
			<td>McCormick Scientific</td>
			<td>39503002</td>
			<td>Parrafin</td>
		</tr>
		<tr>
			<td>Tissue Mold</td>
			<td>Fisher Scientific</td>
			<td>62528-32</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Microm</td>
			<td>HM 325</td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost Plus Microscope Slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Fisher Scientific</td>
			<td>C8H10</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-Based Antigen Retrieval</td>
			<td>Vector Laboratories</td>
			<td>H-3301</td>
			<td></td>
		</tr>
		<tr>
			<td>Pressure Cooker</td>
			<td>Instant Pot</td>
			<td>Lux Mini</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini Pap Pen</td>
			<td>Life Technologies</td>
			<td>8877</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyoxyethylene 20 Sorbitan Monolaurate (Tween-20)</td>
			<td>J.T. Baker</td>
			<td>X251-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Octyl Phenol Ethoxylate (Triton-X-100)</td>
			<td>J.T. Baker</td>
			<td>X198-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Goat Serum</td>
			<td>Jackson Immunoresearch</td>
			<td>005-000-121</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CK8</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>TROMA-I</td>
			<td>Antibody Registry ID AB531826</td>
		</tr>
		<tr>
			<td>anti-CK19</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>TROMA-III</td>
			<td>Antibody Registry ID AB2133570</td>
		</tr>
		<tr>
			<td>anti-&#945;SMA</td>
			<td>Sigma Aldrich</td>
			<td>A2547, Clone 1A4</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-rat-Alexa488</td>
			<td>ThermoFisher</td>
			<td>A21208</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse-Cy5</td>
			<td>Jackson Immunoresearch</td>
			<td>715-175-151</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>22x50 micro cover glass</td>
			<td>VWR Life Sciences</td>
			<td>48393 059</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence Microscope</td>
			<td>Leica</td>
			<td>DMI6000 B</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Kimtech Science</td>
			<td>05511</td>
			<td></td>
		</tr>
		<tr>
			<td>VECTASHIELD</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td>Antifade Mounting Medium</td>
		</tr>
	</tbody>
</table>

determining bile duct density in the mouse liver

老鼠被广泛用作研究胆汁疾病的模型。为了评估胆汁系统的发展和功能,使用了各种技术,包括血清化学、组织学分析和特定标记物的免疫染色。虽然这些技术可以提供有关胆汁系统的重要信息,但它们通常不能显示整个肝脏胆管 (BD) 发育缺陷的完整情况。部分原因在于小鼠肝脏能够强大的能力排出胆汁,即使在胆汁发育有显著损害的动物中也是如此。在这里,我们提出了一个简单的方法,以计算与每个门户静脉 (PV) 相关的 D 的平均数量,这些部分涵盖突变/转基因小鼠的所有叶。在这种方法中,肝脏以立体的方式安装和分割,以方便各种基因型和实验条件之间的比较。BD通过细胞角素染色胆汁的光学显微镜进行识别,然后除以肝脏部分的PV总数。例如,我们展示了这种方法如何能够清楚地区分野生型小鼠和Alagille综合征的小鼠模型。这里介绍的方法不能替代可视化胆汁树三维结构的技术。然而,它提供了一种简单而直接的方法,定量评估BD的发展和小鼠的导管反应形成程度。

我们之前记录了Jag1+/+动物的胆汁缺陷,这是ALGS8的小鼠模型。为了确定BD与PV比,我们对P30小鼠肝脏进行切片,并将其与血管标记αSMA一起用于CK8和CK19(wsCK)。共染色。然后,我们拍摄了每个肝叶中的所有 PV。如图2A所示,我们将 PV 定义为具有相邻 wsCK 染色(箭头)的 _SMA 染色容器。没有 wsCK 的 _SMA 染色结构是中央静脉,不应包含在分析中(...

Watch this Scientific Journal Video about Determining Bile Duct Density in the Mouse Liver at JoVE.com

Determining Bile Duct Density in the Mouse Liver

我们提出了一种相当简单和敏感的方法,用于准确定量小鼠肝脏中的胆管密度。该方法有助于确定遗传和环境修饰剂的影响,以及潜在疗法在胆汁疾病小鼠模型中的有效性。

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 ...

该协议使研究人员能够量化小鼠肝脏疾病模型中的胆管发育。它还允许评估遗传修饰剂对胆管发育的影响。该技术的优点是，它是直接评估胆管发育的直接、敏感和定量的方法。首先，用钳子紧绷的皮肤，用小剪刀做一个横向切口约一英寸的排骨下，安乐死鼠标的肋骨笼。暴露肝脏的整个腹体表面。使用小剪刀小心地切开连接肝脏和腹部其他器官的韧带。然后，切开普通胆管，将肝脏从肠道中分离出来。抓住胆囊，小心切除肝脏。立即将它放在一个50毫升的管子中，用4%的甲醛填充四分之三。要修复肝脏组织，在4摄氏度下孵育4%PFA，48小时。一系列乙醇洗涤后，在室温下用清除剂清洗肝脏三次，每次洗涤30分钟。要开始嵌入，在组织模具中清洗组织盒三次，用预热至60摄氏度的石蜡30分钟。然后，用石蜡填充组织模具至四分之三高，并在60摄氏度的加热块上保持。将肝脏放在模具中，腹侧朝上。小心地从加热块中取出模具后，将盒式磁带的顶部放在模具上。用热液体石蜡顶上，让其冷却到室温过夜。从模具中取出肝脏块后，使用微切子开始切分肝脏的表层侧，进行五微米的切块。在解剖显微镜下检查表面部分，确保截面不会剪切或折叠。要处理免疫组化学的幻灯片，选择每个基因型的幻灯片进行分析，用二甲苯、100%乙醇、95%乙醇和最后70%乙醇洗涤15分钟。在电离水中清洗幻灯片后，将其浸入基于 Tris 的高 pH 抗原检索溶液中。然后，在压力锅中在压力下加热三分钟，每平方英寸 10 磅。使幻灯片冷却到室温后，使用 PAP 笔勾勒出幻灯片上的各部分。使用 PBS Tween 洗涤后，通过添加 100 微升的阻滞溶液，每节和孵育覆盖在 4 摄氏度一个小时， 堵塞组织部分。将含有所有三种原抗体的稀释抗体溶液的100微升应用到每个部分，并在4摄氏度下孵育。洗涤幻灯片后，加入100微升的二次抗体溶液，同时含有二次抗体，并在室温下孵育一小时。最后，在幻灯片上应用安装介质和盖玻片。使用荧光显微镜以每个部分的 1 倍变焦拍摄 20 倍图像。确保肝脏的每个入口静脉都成像。创建包含以下列的电子表格：动物/样本编号、图像编号、门户静脉数和胆管数量。识别并记录每个图像的门户静脉数。然后，通过围绕可定义的流明的胆血管细胞的存在，识别每个图像中的专利胆汁导管。这些结构应该不同，由与其他广谱细胞素阳性细胞分离。这项技术的一个主要挑战是识别专利胆汁导管。确定什么是专利管道需要分析管道的形状和流明周围的细胞。计算每个专利胆管，并放在与图像编号相同的列中，并重复每个门户静脉图像。最后，计算肝脏样本中所有入口静脉和所有胆管的总和，并计算肝脏样本的胆管与门户静脉比率。P30小鼠肝脏被分段并共同染色的CK8和CK19以及血管标记，α-SMA，以确定BD到PV的比例。当每个肝叶的所有入口静脉被成像时，入口静脉被定义为具有相邻广谱细胞角蛋白染色的α-SMA染色血管。没有广谱细胞素的α-SMA染色结构是被排除在分析之外的中心静脉。专利管道具有明确定义的流明，周围环绕着广谱细胞素阳性胆管，通常通过中游分离出附近的导管或胆管细胞。没有可定义流明的广谱细胞角蛋白阳性细胞不计入胆管总数。野生动物的肝脏部分显示了与完全专利管道以及几个未合并细胞相关的入口静脉。来自JAG1异质动物的代表性肝脏部分显示，三个入口静脉周围不存在专利胆管。这里所有的广谱细胞角素阳性细胞都是未合并的，因此，不应计算在内。对三种野生型动物和三种JAG1异型动物的BD与PV比率的分析表明，如何根据胆管计数可以方便地区分这两种基因型。评估胆管的所有入口容器非常重要。如果肝脏存在表型变异性，它对于确定胆管密度尤为重要。该技术用于鉴定赫尔加森小鼠模型中胆道表型的遗传抑制器。因此，它可能有助于评估胆道疾病动物模型的治疗模式。

determining-bile-duct-density-in-the-mouse-liver

Research

JoVE Journal

Developmental Biology

Stimulation of Notch Signaling in Mouse Osteoclast Precursors

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro investigation of its varying and sometimes contradictory roles a challenge. As a component of direct cell-cell communication with both receptors and ligands bound to the plasma membrane, Notch signaling cannot be activated in vitro by simple addition of ligands to culture media, as is possible with many other signaling pathways. Instead, Notch ligands must be presented to cells in an immobilized state. 
Variations in methods of Notch signaling activation can lead to different outcomes in cultured cells. In osteoclast precursors, in particular, differences in methods of Notch stimulation and osteoclast precursor culture and differentiation have led to disagreement over whether Notch signaling is a positive or negative regulator of osteoclast differentiation. While closer comparisons of osteoclast differentiation under different Notch stimulation conditions in vitro and genetic models have largely resolved the controversy regarding Notch signaling and osteoclasts, standardized methods of continuous and temporary stimulation of Notch signaling in cultured cells could prevent such discrepancies in the future.
This protocol describes two methods for stimulating Notch signaling specifically in cultured mouse osteoclast precursors, though these methods should be applicable to any adherent cell type with minor adjustments. The first method produces continuous stimulation of Notch signaling and involves immobilizing Notch ligand to a tissue culture surface prior to the seeding of cells. The second, which uses Notch ligand bound to agarose beads allows for temporary stimulation of Notch signaling in cells that are already adhered to a culture surface. This protocol also includes methods for detecting Notch activation in osteoclast precursors as well as representative transcriptional markers of Notch signaling activation.

Notch signaling is a form of cellular communication that relies upon direct contact between cells. To properly induce Notch signaling in vitro, Notch ligands must be presented to cells in an immobilized state. This protocol describes methods for in vitro stimulation of Notch signaling in mouse osteoclast precursors.

在鼠标破骨细胞前体Notch信号的刺激

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro ...

科研

发育生物学

Histological Analyses of Acute Alcoholic Liver Injury in Zebrafish

Alcoholic Liver Disease (ALD) refers to damage to the liver due to acute or chronic alcohol abuse. It is among the leading causes of alcohol-related morbidity and mortality and affects more than 2 million people in the United States. A better understanding of the cellular and molecular mechanisms underlying alcohol-induced liver injury is crucial for developing effective treatment for ALD. Zebrafish larvae exhibit hepatic steatosis and fibrogenesis after just 24 h of exposure to 2% ethanol, making them useful for the study of acute alcoholic liver injury. This work describes the procedure for acute ethanol treatment in zebrafish larvae and shows that it causes steatosis and swelling of the hepatic blood vessels. A detailed protocol for Hematoxylin and Eosin (H&#38;E) staining that is optimized for the histological analysis of the zebrafish larval liver, is also described. H&#38;E staining has several unique advantages over immunofluorescence, as it marks all liver cells and extracellular components simultaneously and can readily detect hepatic injury, such as steatosis and fibrosis. Given the increasing usage of zebrafish in modeling toxin and virus-induced liver injury, as well as inherited liver diseases, this protocol serves as a reference for the histological analyses performed in all these studies.

This protocol describes histological analyses of the livers from zebrafish larvae that have been treated with 2% ethanol for 24 h. Such acute ethanol treatment results in hepatic steatosis and swelling of the hepatic vasculature.

斑马鱼急性酒精性肝损伤的组织学分析

Alcoholic Liver Disease (ALD) refers to damage to the liver due to acute or chronic alcohol abuse. It is among the leading causes of alcohol-related ...

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We quantitatively assessed follicle survival, measured follicle and oocyte diameters, and examined ultrastructure of the oocytes produced from the system. We observed decreased follicle survival, downregulation of expressions of proliferating cell nuclear antigen and growth differentiation factor 9, as indicators for the development of granulosa cells and oocytes, respectively, and oocyte ultrastructural abnormalities under the simulated microgravity condition. The simulated microgravity experimental setup needs to be optimized to provide a model for investigation of the mechanisms involved in the oocyte/follicle in vitro development.

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

A highly promising technique to generate tissue constructs without using matrix is to culture cells in a simulated microgravity condition. Using a rotary culture system, we examined ovarian follicle growth and oocyte maturation in terms of follicle survival, morphology, growth, and oocyte function under the simulated microgravity condition.

体外模拟微重力下小鼠腔卵泡生长的研究

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We ...

Quantifying Branching Density in Rat Mammary Gland Whole-mounts Using the Sholl Analysis Method

An increasing number of studies are utilizing the rodent mammary gland as an endpoint for assessing the developmental toxicity of a chemical exposure. The effects these exposures have on mammary gland development are typically evaluated using either basic dimensional measurements or by scoring morphological characteristics. However, the broad range of methods for interpreting developmental changes could lead to inconsistent translations across laboratories. A common method of assessment is needed so that proper interpretations can be formed from data being compared across studies. The present study describes the application of the Sholl analysis method to quantify mammary gland branching characteristics. The Sholl method was originally developed for use in quantifying neuronal dendritic patterns. By using ImageJ, an open-source image analysis software package, and a plugin developed for this analysis, the mammary gland branching density and the complexity of a mammary gland from a peripubertal female rat were determined. The methods described here will enable the use of the Sholl analysis as an effective tool for quantifying an important characteristic of mammary gland development.

Mammary gland development in the rodent has typically been evaluated using descriptive assessments or by measuring basic physical attributes. Branching density is an indicator of mammary development that is difficult to quantify objectively. This protocol describes a reliable method for the quantitative assessment of mammary gland branching characteristics.

使用Sholl分析方法量化大鼠乳腺组织中的分支密度

An increasing number of studies are utilizing the rodent mammary gland as an endpoint for assessing the developmental toxicity of a chemical exposure. The ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

小鼠第二腭融合的实时成像

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is routinely used as an in vivo model to study embryo implantation and pregnancy progression. Unfortunately, such murine studies require pregnancy interruption to enable follow-up phenotypic analysis. To address this issue, we used three-dimensional (3-D) reconstruction of HFUS imaging data for early detection and characterization of murine embryo implantation sites and their individual developmental progression in utero. Combining HFUS imaging with 3-D reconstruction and modeling, we were able to accurately quantify embryo implantation site number as well as monitor developmental progression in pregnant C57BL6J/129S mice from 5.5 days post coitus (d.p.c.) through to 9.5 d.p.c. with the use of a transducer. Measurements included: number, location, and volume of implantation sites as well as inter-implantation site spacing; embryo viability was assessed by cardiac activity monitoring. In the immediate post-implantation period (5.5 to 8.5 d.p.c.), 3-D reconstruction of the gravid uterus in both mesh and solid overlay format enabled visual representation of the developing pregnancies within each uterine horn. As genetically engineered mice continue to be used to characterize female reproductive phenotypes derived from uterine dysfunction, this method offers a new approach to detect, quantify, and characterize early implantation events in vivo. This novel use of 3-D HFUS imaging demonstrates the ability to successfully detect, visualize, and characterize embryo-implantation sites during early murine pregnancy in a non-invasive manner. The technology offers a significant improvement over current methods, which rely on the interruption of pregnancies for gross tissue and histopathologic characterization. Here we use a video and text format to describe how to successfully perform ultrasounds of early murine pregnancy to generate reliable and reproducible data with reconstruction of the uterine form in mesh and solid 3-D images.

Mice are widely used to study gestational biology. However, pregnancy termination is required for such studies which precludes longitudinal investigations and necessitates the use of large numbers of animals. Therefore, we describe a non-invasive technique of high-frequency ultrasonography for early detection and monitoring of post-implantation events in the pregnant mouse.

三维高频超声对小鼠早期妊娠特征的新应用

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is ...

A Familial Hypercholesterolemia Human Liver Chimeric Mouse Model Using Induced Pluripotent Stem Cell-derived Hepatocytes

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset cardiovascular disease due to marked elevation of LDL cholesterol (LDL-C) in blood. Statins are the first line of lipid-lowering drugs for treating FH and other types of hypercholesterolemia, but new approaches are emerging, in particular PCSK9 antibodies, which are now being tested in clinical trials. To explore novel therapeutic approaches for FH, either new drugs or new formulations, we need appropriate in vivo models. However, differences in the lipid metabolic profiles compared to humans are a key problem of the available animal models of FH. To address this issue, we have generated a human liver chimeric mouse model using FH induced pluripotent stem cell (iPSC)-derived hepatocytes (iHeps). We used Ldlr-/-/Rag2-/-/Il2rg-/- (LRG) mice to avoid immune rejection of transplanted human cells and to assess the effect of LDLR-deficient iHeps in an LDLR null background. Transplanted FH iHeps could repopulate 5-10% of the LRG mouse liver based on human albumin staining. Moreover, the engrafted iHeps responded to lipid-lowering drugs and recapitulated clinical observations of increased efficacy of PCSK9 antibodies compared to statins. Our human liver chimeric model could thus be useful for preclinical testing of new therapies to FH. Using the same protocol, similar human liver chimeric mice for other FH genetic variants, or mutations corresponding to other inherited liver diseases, may also be generated.

Here, we present a protocol to generate a human liver chimeric mouse model of familial hypercholesterolemia using human induced pluripotent stem cell-derived hepatocytes. This is a valuable model for testing new therapies for hypercholesterolemia.

诱导多潜能干细胞源性肝细胞的家族性高胆固醇血症人肝嵌合小鼠模型

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset ...

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 ...

Generation of Organoids from Mouse Extrahepatic Bile Ducts

Cholangiopathies, which affect extrahepatic bile ducts (EHBDs), include biliary atresia, primary sclerosing cholangitis, and cholangiocarcinoma. They have no effective therapeutic options. Tools to study EHBD are very limited. Our purpose was to develop an organ-specific, versatile, adult stem cell-derived, preclinical cholangiocyte model that can be easily generated from wild type and genetically engineered mice. Thus, we report on the novel technique of developing an EHBD organoid (EHBDO) culture system from adult mouse EHBDs. The model is cost-efficient, able to be readily analyzed, and has multiple downstream applications. Specifically, we describe the methodology of mouse EHBD isolation and single cell dissociation, organoid culture initiation, propagation, and long-term maintenance and storage. This manuscript also describes EHBDO processing for immunohistochemistry, fluorescent microscopy, and mRNA abundance quantitation by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). This protocol has significant advantages in addition to producing EHBD-specific organoids. The use of a conditioned medium from L-WRN cells significantly reduces the cost of this model. The use of mouse EHBDs provides almost unlimited tissue for culture generation, unlike human tissue. Generated mouse EHBDOs contain a pure population of epithelial cells with markers of endodermal progenitor and differentiated biliary cells. Cultured organoids maintain homogenous morphology through multiple passages and can be recovered after a long-term storage period in liquid nitrogen. The model allows for the study of biliary progenitor cell proliferation, can be manipulated pharmacologically, and may be generated from genetically engineered mice. Future studies are needed to optimize culture conditions in order to increase plating efficiency, evaluate functional cell maturity, and direct cell differentiation. Development of co-culture models and a more biologically neutral extracellular matrix are also desirable.

This protocol describes the production of a mouse extrahepatic bile duct 3-dimensional organoid system. These biliary organoids can be maintained in culture to study cholangiocyte biology. Biliary organoids express markers of both progenitor and biliary cells and are composed of polarized epithelial cells.

小鼠肝外胆管有机体的产生

Cholangiopathies, which affect extrahepatic bile ducts (EHBDs), include biliary atresia, primary sclerosing cholangitis, and cholangiocarcinoma. They have ...