Name: quantifying liver size in larval zebrafish using brightfield microscopy
Uploaded: 2020-02-02T13:00:00
Description: Watch this Scientific Journal Video about Quantifying Liver Size in Larval Zebrafish Using Brightfield Microscopy at JoVE.com

我们要感谢犹他大学的Maurine Hobbs和集中的斑马鱼动物资源（CZAR），他们提供了斑马鱼的饲养、实验室空间和设备，以开展部分研究。国家卫生研究院拨款 #1G20OD018369-01 部分支持 CZAR 的扩展。我们还要感谢罗德尼·斯图尔特、克洛伊·林、兰斯·格雷厄姆、科迪·詹姆斯、加勒特·尼克姆以及亨茨曼癌症研究所（HCI）斑马鱼护理设施。我们要感谢肯尼思·康帕斯在研发方面的帮助。这项工作的部分资金来自亨茨曼癌症基金会的赠款（连同授予亨茨曼癌症研究所P30 CA042014的赠款）和NIH/NCI R01CA222570（KJE）。

动物研究按照犹他大学机构动物护理和使用委员会（IACUC）批准的程序进行。
1. 修复幼虫
<ol><li>在受精后3~7天（dpf），用三联甲甲烷酸酯（0.03%）对幼虫实施安乐死使用玻璃移液器和移液器泵在 2 mL 管中收集多达 15 个幼虫。</li>
	<li>用1mL的冷（4°C）1x磷酸盐缓冲盐水（PBS）在冰上洗涤幼虫两次。每次清洗时，用玻璃移液器和移液器泵从管中取出尽可能多的液体，然后将 1 mL ?...

<ol>
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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Chemical+Inhibitors+of+&#946;-Catenin-Driven+Liver+Tumorigenesis+in+Zebrafish.">Identification of Chemical Inhibitors of &#946;-Catenin-Driven Liver Tumorigenesis in Zebrafish.</a> PLoS Genetics. 11 (7), 1005305 (2015).</li><li>Kalasekar, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneous+beta-catenin+activation+is+sufficient+to+cause+hepatocellular+carcinoma+in+zebrafish.">Heterogeneous beta-catenin activation is sufficient to cause hepatocellular carcinoma in zebrafish.</a> Biology Open. 8 (10), (2019).</li><li>Nguyen, A. 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肝脏大小的定量对于旨在了解肝脏发育、再生和肿瘤发生的研究至关重要。这里描述的方案是一种相对快速、简单和廉价的幼虫斑马鱼肝脏大小定量技术。在执行协议的某些方面时，适当谨慎行事有助于提高结果的准确性并减少挫折感。
正确固定幼虫对于获得保存完好的生物样本和防止其解体至关重要。在将 PFA 添加到冲洗的幼虫之前未完全去除 PBS 时，可能会出现 4% PFA 溶?...

肝细胞癌（HCC）是肝脏1最常见的原发性恶性肿瘤，是导致癌症相关死亡的第三大原因2。为了更好地了解肝癌的机理和识别潜在的HCC治疗，我们和其他人已经开发出转基因斑马鱼，其中肝细胞特异性表达的肿瘤基因，如β-卡泰宁3，4，克拉斯（V12）5，6，Myc7，或Yap18导致成年动物的HCC。在这些斑马鱼中，肝增增早在受精后6天（dpf），为测试药物和基因改变对卵基因驱动肝过度生长的影响提供了一个方便的平台。准确和精确测量幼虫肝大小对于确定这些操作的影响至关重要。
肝脏大小和形状可以半定量评估在固定斑马鱼幼虫由CY3-SA标签9或活斑马鱼幼虫使用肝细胞特异性荧光报告机和荧光解剖显微镜5，6。后一种方法比较快，使用图像处理软件7、10拍摄和测量每个肝脏的面积，可以解决其精度不足的问题。然而，在实验中统一定位所有活幼虫，使二维肝区准确表示肝脏大小，在技术上可能具有挑战性。一种类似的量化肝脏大小的技术包括使用光片荧光显微镜来量化幼虫肝体积8，当肝脏在不同维度上不均匀地扩张时，这可能更准确检测大小差异。荧光活化细胞分拣（FACS）可用于计算幼肝8、11中荧光标记肝细胞和其他肝细胞类型的数量。在这种方法中，幼虫肝被集中和分离，因此有关单个肝脏大小和形状的信息丢失。结合另一种肝大小测定方法，FACS 能够区分增加的细胞数（增生）和增加的细胞大小（肥大）。所有这些方法都采用昂贵的荧光技术（显微镜或细胞分拣机），除 CY3-SA 标签外，还需要用荧光报告器标记肝细胞。
在这里，我们详细介绍了使用明场显微镜和图像处理软件3，12，13，14量化斑马鱼幼肝区域的方法。该协议允许在原位对单个肝脏区域进行精确定量，而无需使用荧光显微镜。在分析肝脏大小时，我们盲注图像特征，以减少研究者的偏见，提高科学严谨性。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Camera for dissecting microscope</td>
			<td>Leica, for example</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Leica, for example</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fine (Dumont #5) forceps</td>
			<td>Fine Science Tools</td>
			<td>11254-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass pipets</td>
			<td>VWR</td>
			<td>14672-608</td>
			<td></td>
		</tr>
		<tr>
			<td>Image analysis software</td>
			<td>Image J/FIJI</td>
			<td></td>
			<td>ImageJ/FIJI can be dowloaded for free: <a href="https://imagej.net/Welcome" target="_blank">https://imagej.net/Welcome</a></td>
		</tr>
		<tr>
			<td>Methyl cellulose</td>
			<td>Sigma</td>
			<td>M0387</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma Aldrich</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline</td>
			<td>Various suppliers</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette pump</td>
			<td>VWR</td>
			<td>53502-233</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic Petri dishes</td>
			<td>USA Scientific Inc</td>
			<td>2906</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyrex 9-well round-bottom glass dish</td>
			<td>VWR</td>
			<td>89090-482</td>
			<td></td>
		</tr>
		<tr>
			<td>Software for blinding files</td>
			<td>R project</td>
			<td></td>
			<td>R can be downloaded for free: <a href="https://www.r-project.org/" target="_blank">https://www.r-project.org/</a></td>
		</tr>
		<tr>
			<td>Scientific graphing and statistics software</td>
			<td>GraphPad Prism</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spreadsheet program</td>
			<td>Microsoft Excel</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tricaine methanesulfonate (Tricaine-S)</td>
			<td>Western Chemical</td>
			<td>200-226</td>
			<td></td>
		</tr>
		<tr>
			<td>Very fine (Dumont #55) forceps</td>
			<td>Fine Science Tools</td>
			<td>11255-20</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantifying liver size in larval zebrafish using brightfield microscopy

在肝细胞癌（HCC）的几个转基因斑马鱼模型中，在早期幼虫阶段可以观察到肝球菌。在斑马鱼HCC模型中量化幼虫肝大小提供了一种快速评估药物和其他操作对与基因相关的表型的影响的方法。在这里，我们展示如何修复斑马鱼幼虫，解剖肝脏周围的组织，使用明亮的显微显微镜拍摄肝脏，测量肝脏面积，并分析结果。该协议能够快速、精确地定量肝脏大小。由于这种方法涉及测量肝脏面积，它可能会低估肝脏体积的差异，并且需要补充方法来区分细胞大小的变化和细胞数的变化。本文描述的解剖技术是一个很好的工具，用于可视化肝脏、肠道和胰腺的自然位置，用于各种下游应用，包括免疫荧光染色和原位杂交。所述的量化幼肝大小的策略适用于肝脏发育和再生的许多方面。

转基因斑马鱼表达肝细胞特异性活性β-卡特宁（Tg（fabp10a：pt-α-cat）斑马鱼3，非转基因对照兄弟姐妹在6dpf和肝脏区域使用明场显微镜和图像处理软件进行定量。转基因斑马鱼与非转基因兄弟姐妹相比，肝脏尺寸显著增加（0.0006厘米2），（0.0004厘米2，p<0.0001; 图 1.
<img alt="Figu...

Watch this Scientific Journal Video about Quantifying Liver Size in Larval Zebrafish Using Brightfield Microscopy at JoVE.com

Quantifying Liver Size in Larval Zebrafish Using Brightfield Microscopy

在这里，我们演示了一种量化幼虫斑马鱼肝脏大小的方法，提供了一种评估遗传和药理操作对肝脏生长和发育的影响的方法。

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 ...

这种方法提供了一种量化幼虫斑马鱼肝脏大小的方法。它还允许评估遗传和药理操作对肝脏生长发育的影响。这项技术提供了一种快速和精确的方式来可视化和测量肝脏以及肠道和胰腺。除了量化肝脏大小外，这种解剖方法还有助于准备肝脏、肠道和胰腺进行原位杂交或共体成像。在尝试对实验幼虫进行桌面分析之前，请确保在非实验样本上花费大量时间练习，并且具有显著的成功率。要可视化斑马鱼幼虫和钳子的正确方向和运动，以便在不干扰周围器官的情况下去除皮肤，这一点至关重要。在受精后三至七天，在安乐死幼虫后，使用玻璃移液器和移液器泵在两毫升管中收集多达15个幼虫。用一毫升的冷1XPBS在冰上清洗幼虫两次。使用玻璃移液器和移液器泵清除尽可能多的 PBS，并在 PBS 中加入 1 毫升 4% PFA 的冷液。在四摄氏度下孵育，至少一夜轻轻的摇动。使用玻璃移液器和移液器泵，从管子上取出 PFA，然后用一毫升冷 PBS 冲洗三次。在冲洗之间摇动五分钟。在PBS中将几个幼虫放入一个九井圆底玻璃盘的井中。在显微镜下，用细钳子去除肝脏周围的皮肤，将一个幼虫抱在背部，尽可能轻轻地抓住头部两侧。然后用非常精细的钳子在另一只手抓住皮肤只是过度心脏。对角线向下拉皮，对角向鱼左侧或右侧菜底的鱼尾。对另一侧重复。继续抓皮瓣和拉下，直到所有的皮肤和黑色素覆盖或肝脏附近已被删除。如果蛋黄在受精后有五到六天的幼虫，则将蛋黄放在一块中，将鱼背上的细钳子抱住，并使用非常细的钳子轻轻搅拌蛋黄。对于三到四个DPF幼虫，用细钳子握住鱼的背部，用非常细的钳子从腹侧开始抚摸蛋黄。把蛋黄刮成碎片。使用玻璃移液器和移液器泵将解剖的幼虫放入两毫升管中，放入新鲜、冰冷的 PBS 中。要安装幼虫，将几毫升百分之三的甲基纤维素倒在干净的塑料培养皿的盖子上。使用玻璃移液器和移液器泵将幼虫添加到甲基纤维素中，与幼虫一起添加尽可能少的 PBS。在低放大倍率下的解剖显微镜下，使用细钳来定位鱼，使鱼朝右。确认要拍摄的鱼与一只眼睛直接放在另一只眼睛上完美对齐。如果鱼的尾巴弯曲，用钳子用力捏它来取下尾巴，使鱼平放。放大到高放大倍率并聚焦在肝脏上，确保肝脏的轮廓清晰可见。按"捕获图像"按钮捕捉图片并保存文件。对所有鱼重复，确保每张图片的放大倍率相同。使用相同的放大倍率拍摄千分尺的照片。使用程序R来盲化所有肝脏图片，以避免潜在的研究者偏见，并促进科学严谨性，随机重命名文件，并创建一个随机化文件，其中包含原始文件名和相应的盲化文件名的列表。按顺序打开随机文件，从文件 1 开始。选择手绘选择工具并勾勒出每个肝脏的轮廓。按 Control-M 测量每个肝脏的面积。对于任何无法准确测量的肝脏，因为它们有残留的皮肤，蛋黄，没有焦点，或不完好无损，插入占位符测量。将测量值保存在文本文件中。要解盲和分析数据，请打开电子表格程序中的测量文件和随机化文件。在测量文件中插入新列，并使用随机化文件为盲文件添加原始文件名。将此文件另存为"未盲测量"文件。按原始文件名对数据进行排序。排除任何肝脏测量图片不足。如有必要，将测量值转换为所需的刻度（以平方毫米为单位）。打开图像处理软件中的"缩放"栏。使用直线工具在刻度栏上测量一毫米。然后，图像处理软件与肝脏以相同的单位进行测量，从而给出转换因子。使用转换系数在"无盲测量"文件中转换测量值。确定平均值和标准差并计算 P 值。在受精后六天，非转基因斑马鱼幼虫显示出肝脏覆盖肠道的自然位置和形状。与非转基因的兄弟姐妹相比，转基因斑马鱼的肝脏大小显著增加。此处显示了图像和千分尺不足的一些示例。幼虫用皮肤覆盖肝脏，幼虫用蛋黄遮盖肝脏，幼虫与部分肝脏挤压脱落，幼虫与肝脏脱位脱落，幼虫与缺失的肝脏，幼虫与肝脏轮廓难以识别，幼虫与不当定位。当尝试这个程序时，重要的是使用小和受控的运动，以防止损害肝脏。除了明亮的场显微镜，我们还可以使用这种解剖的幼虫，用于共和显微镜，可视化荧光记者蛋白或免疫荧光染色，以及原位杂交。甲醛或PFA是一种刺激性物质和疑似致癌物质。处理 PFA 时应佩戴手套，浓缩溶液应在化学烟气罩中处理。

Research

JoVE Journal

Developmental Biology

Live Imaging of Innate Immune and Preneoplastic Cell Interactions Using an Inducible Gal4/UAS Expression System in Larval Zebrafish Skin

先天免疫和癌前细胞间的相互作用使用可诱导的Gal4 / UAS表达系统在斑马鱼幼体皮肤实时成像

Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ERT2/UAS ...

科研

发育生物学

Capturing Tissue Repair in Zebrafish Larvae with Time-lapse Brightfield Stereomicroscopy

捕获组织修复的斑马鱼幼虫与时间推移明视立体显微镜

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two ...

Hepatocyte-specific Ablation in Zebrafish to Study 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 ...

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

配备了光学切片与成像定时使用解剖显微镜斑马鱼肾脏发展分析

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method ...

Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development

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 ...

Dissection of Larval Zebrafish Gonadal Tissue

幼虫斑马鱼性腺组织的解剖

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex ...

Histological Analyses of Acute Alcoholic Liver Injury in Zebrafish

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 ...

Correlative Super-resolution and Electron Microscopy to Resolve Protein Localization in Zebrafish Retina

斑马鱼视网膜蛋白定位的相关超分辨和电镜观察

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and ...

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

斑马鱼幼虫动物模型的建立研究锥虫运动性体内

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding ...

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

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 ...