Name: quantitative cell biology of neurodegeneration in drosophila through unbiased analysis of fluorescently tagged proteins using imagej
Uploaded: 2018-08-03T14:00:00
Description: Watch this Scientific Journal Video about Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ at JoVE.com

这项工作是支持希拉和大卫恩特神经病理性疼痛研究计划研究生奖学金 (J.M.B.), 露易丝教皇生活研究员计划 (铁匠, Y.Z., 和 J.M.B.), 斯奈德-鲁滨逊基金会 Predoctoral 奖学金 (铁匠), 约翰博士 T.麦克唐纳基金会 (铁匠), 合同, 国家卫生研究院 (NIH) HHSN268201300038C, R21GM119018 和 R56NS095893 (R.G.Z.) 的赠款, 并由泰山学者项目 (山东省, 中华人民共和国) (R.G.Z.)。

1. 图像分析实验设计的思考与准备
<ol><li>预先确定合适的解剖, 细胞, 或亚型标记, 以作为标志, 以标准化的兴趣区域 (ROI) 横跨不同的样本, 例如 4 ', 6-diamidino 2-苯基吲哚 (DAPI), 膜标记, 局部荧光蛋白等.
</li>
	<li>选择一个荧光标记, 它可以将离散 puncta 划分为兴趣结构以外的背景层。 注: 本协议是为蛋白质结构 (如错误折叠蛋白聚集) 和膜结合器 (如自噬记者) 进行优化的。为了?...

<ol>
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此处概述的协议可用于重现性定量细胞生物学过程的荧光成像。必须仔细考虑生物环境和技术限制, 以指导实验设计。荧光标记的兴趣, 无论是免疫组织化学, 染料基础, 或基因表达, 需要区分以上背景的形态学和强度。UAS/GAL4系统广泛应用于果蝇的靶向基因表达。这里的例子使用了携带 GAL4 驱动程序的转基因线激活荧光标记蛋白的转录在无人驾驶增强器的控制下, 以调查蛋白质聚集和?...

神经退行性疾病每年影响上百万人, 发病率随着人口老龄化1而增加。尽管每种神经退行性疾病都有一个独特的病因, 错误折叠蛋白的聚集和 proteostasis 网络的崩溃是许多这些疾病的常见病理特征。阐明这些基本和相互关联的过程的中断如何导致神经元功能障碍, 细胞死亡对于理解神经退行性疾病以及指导治疗干预是至关重要的。荧光成像技术允许对神经元的这些复杂和动态过程进行调查, 并极大地促进了我们对神经细胞生物学的理解。荧光标记蛋白的分析具有挑战性, 尤其是在体内进行实验时, 由于组织高度紧凑, 细胞类型多样, 形态学异质性。人工辅助量化是负担得起和直接的, 但往往是费时的, 并受人的偏见。因此, 需要有无偏见的、可重复的和易于获取的方法来从成像研究中提取量化数据。
我们概述了一个简单和适应性的工作流程使用斐济/ImageJ, 一个强大的和自由访问的图像处理软件2,3, 从荧光成像研究中提取定量数据的实验模型变性使用果蝇。通过遵循这一协议来量化蛋白质聚集和自噬性通量-两个细胞生物学特征, 这是高度相关的神经退行性疾病病理学-我们展示了敏感性和再现这种方法。荧光标记突变体杭丁顿蛋白 (Htt) 蛋白在果蝇视神经叶中的分析揭示了蛋白质聚集的数量、大小和强度。我们在果蝇视觉系统中可视化了自噬性通量的串联荧光记者, 它根据区划环境4显示不同的发射信号。比例分析的串联记者允许从 autophagosome 的形成, 成熟和运输到降解溶酶体的自噬溶酶通量的定量和全面的看法, 并进一步强调脆弱神经退行性疾病的车厢中断。重要的是, 在这两种分析中, 我们在协议中实现了半自动阈值和分割步骤, 以最大限度地减少无意识偏差, 增加采样功率, 并提供一个标准来促进类似研究之间的比较。简单的工作流旨在使强大的斐济/ImageJ 插件 (由计算机科学家根据数学算法开发) 更容易被 neurobiologists 和生命科学社区访问。

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		<tr height="38">
			<td height="38" style="height:38px;width:347px;">SYLGARD(R) 184 Silicone Elastomer Kit</td>
			<td style="width:164px;">Dow Corning Corporation</td>
			<td style="width:243px;">PF184250</td>
			<td style="width:349px;">Dissection dish</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:347px;">Falcon 35 mm Not TC-Treated Easy-Grip Style Bacteriological Petri Dish</td>
			<td style="width:164px;">Corning</td>
			<td style="width:243px;">351008</td>
			<td style="width:349px;">Dissection dish</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Dumont #5 Forceps</td>
			<td style="width:164px;">Fine Science Tools</td>
			<td style="width:243px;">11251-20</td>
			<td style="width:349px;">Dissection tool</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Sodium Chloride</td>
			<td style="width:164px;">Sigma</td>
			<td style="width:243px;">S3014</td>
			<td style="width:349px;">PBS solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Sodium Phosphate Dibasic&#160;</td>
			<td style="width:164px;">Sigma</td>
			<td style="width:243px;">S5136</td>
			<td style="width:349px;">PBS solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Potassium Phosphate Monobasic</td>
			<td style="width:164px;">Sigma</td>
			<td style="width:243px;">P5655</td>
			<td style="width:349px;">PBS solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Triton&#160;X-100</td>
			<td style="width:164px;">Sigma</td>
			<td style="width:243px;">T9284</td>
			<td style="width:349px;">Washing and antibody incubaton solution.&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">37% Formaldehyde</td>
			<td style="width:164px;">VWR</td>
			<td style="width:243px;">10790-710</td>
			<td style="width:349px;">Fixation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Disposable Microcentrifuge Tubes (0.5mL, blue)</td>
			<td style="width:164px;">VWR</td>
			<td style="width:243px;">89000-022</td>
			<td style="width:349px;">Fixation, washing, and antibody incubaton.&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Plain and Frosted Micro Slides (25&#215;75mm)</td>
			<td style="width:164px;">VWR</td>
			<td style="width:243px;">48312-004</td>
			<td style="width:349px;">Slides for confocal imaging</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Micro Cover Glasses, rectangular (22&#215;40mm)</td>
			<td style="width:164px;">VWR</td>
			<td style="width:243px;">48393-172</td>
			<td style="width:349px;">Slides for confocal imaging</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Rubber Cement</td>
			<td style="width:164px;">Slime</td>
			<td style="width:243px;">1051-A</td>
			<td style="width:349px;">Mounting</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:347px;">VECTASHIELD Antifade Mounting Medium</td>
			<td style="width:164px;">Vector Laboratories, Inc.</td>
			<td style="width:243px;">H-1000</td>
			<td style="width:349px;">Mounting</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Scotch Magic 810 Invisible Tape (19mm&#215;25.4m)</td>
			<td style="width:164px;">3M Company</td>
			<td style="width:243px;">810</td>
			<td style="width:349px;">Mounting</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:347px;">Normal Goat Serum</td>
			<td style="width:164px;">Thermo Fisher Scientific</td>
			<td style="width:243px;">PCN5000</td>
			<td style="width:349px;">Antibody incubaton</td>
		</tr>
		<tr height="76">
			<td height="76" style="height:76px;width:347px;">DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)&#160;</td>
			<td style="width:164px;">Thermo Fisher Scientific</td>
			<td style="width:243px;">D1306</td>
			<td style="width:349px;">Nucleic acid staining. Dissolve in deionized water to make a 5 mg/mL stock solution and store at -80&#176;C. Dilute to a working concentration of 10-20 &#956;g/mL in PBTx.</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:347px;">3.5X-90X Stereo Zoom Inspection Industrial Microscope</td>
			<td style="width:164px;">AmScope</td>
			<td style="width:243px;">SM-1BNZ</td>
			<td style="width:349px;">Dissection scope. Equipped with 6W LED Dual Gooseneck Illuminator</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">ImageJ/Fiji</td>
			<td style="width:164px;">NIH</td>
			<td style="width:243px;">v1.51u</td>
			<td style="width:349px;">With SCF_MPI_CBG plugins (version 1.1.2)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">FV1000-IX81 Confocal-laser Scanning Microscope</td>
			<td style="width:164px;">Olympus</td>
			<td style="width:243px;"></td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Recombinant Construct</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td style="width:243px;">BL37749</td>
			<td style="width:349px;"></td>
		</tr>
	</tbody>
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quantitative cell biology of neurodegeneration in drosophila through unbiased analysis of fluorescently tagged proteins using imagej

随着神经退行性疾病患病率的上升, 了解导致神经元功能障碍和丧失的潜在病理生理学越来越重要。基于荧光的成像工具和技术能够对亚细胞神经生物学过程进行空前的分析, 但仍然需要有无偏见、可重现和易于获取的方法来从成像研究中提取量化数据。.我们开发了一个简单而适应的工作流程, 从荧光成像研究中提取定量数据, 利用果蝇模型变性。具体来说, 我们描述了一个易于跟踪, 半自动化的方法, 使用斐济/ImageJ 分析两个细胞过程: 首先, 我们用荧光标记突变体量化果蝇光学叶中蛋白质的总含量和剖面。杭丁顿蛋白蛋白;第二, 我们评估果蝇视觉系统的自噬溶酶通量, 并以比例为基础, 对一个串联荧光报告器进行定量化。重要的是, 这里概述的协议包括一个半自动的分割步骤, 以确保所有的荧光结构进行分析, 以尽量减少选择偏差, 并增加微妙比较的分辨率。这种方法可以推广到分析其他细胞生物学结构和过程中涉及变性, 如蛋白质 puncta (应力颗粒和突触复合物), 以及膜约束的车厢 (线粒体和膜贩运囊泡)。该方法为图像分析和量化提供了一个标准化的、可适应的参考点, 并能促进整个领域的可靠性和重现性, 最终增强了对变性的机械理解。

果蝇荧光标记突变 Htt 集的数量、面积和强度的量化
为了研究亨廷顿氏病果蝇模型的中枢神经系统中的错误折叠蛋白聚集, 用 RFP 标记的突变体 Htt 非病理 (UAS-RFP-hHttQ15) 或病理扩张 (UAS-RFP-hHttQ138) 和膜 GFP (UAS-mCD8::GFP)11是共同表达下一个泛神经元驱动elav-GAL4。根据《议定书?...

Watch this Scientific Journal Video about Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ at JoVE.com

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

荧光标记蛋白 ImageJ 的无偏分析在果蝇变性定量细胞生物学中的应用

我们开发了一个简单且适应性强的工作流, 从荧光成像的细胞生物学研究中提取定量数据自噬性果蝇模型中中枢神经系统蛋白质聚集和通量变性.

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal ...

Research

JoVE Journal

Neuroscience

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

Automated Sholl Analysis of Digitized Neuronal Morphology at Multiple Scales

肖尔自动数字化的神经元形态分析，多尺度

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3.  Specifically, it affects the ability of neurons to ...

科研

神经科学

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

含量的运动能力，学习和记忆的赤字果蝇</em神经退行性疾病>模型

Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system1. Most of ...

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

使用调频染料培养的小脑颗粒神经突触小泡池补充的定量分析

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

Spectral Confocal Imaging of Fluorescently tagged Nicotinic Receptors in Knock-in Mice with Chronic Nicotine Administration

光谱共焦成像的荧光标记的尼古丁受体敲在慢性尼古丁政府小鼠

Ligand-gated ion channels in the central nervous system (CNS) are implicated in numerous conditions with serious medical and social consequences.  For ...

Quantitative Analysis of Climbing Defects in a Drosophila Model of Neurodegenerative Disorders

攀登缺陷的神经退行性疾病的果蝇模型定量分析

Locomotive defects resulting from neurodegenerative disorders can be a late onset symptom of disease, following years of subclinical degeneration, and ...

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

体内基因转移到雪旺细胞在啮齿动物坐骨神经电穿孔

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

Mass Histology to Quantify Neurodegeneration in Drosophila

Mass Histology to Quantify Neurodegeneration in Drosophila

大众组织学量化在神经退行性变<em&gt;果蝇</em

Progressive neurodegenerative diseases like Alzheimer&#39;s disease (AD) or Parkinson&#39;s disease (PD) are an increasing threat to human health ...

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

用线性琼脂糖渠道研究<em&gt;果蝇</em&gt;幼虫爬行行为

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat ...

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

嗜德维人神经元树突状树突状复杂度的定量分析

Dendrites are the branched projections of a neuron, and dendritic morphology reflects synaptic organization during the development of the nervous system. ...

An Unbiased Approach of Sampling TEM Sections in Neuroscience

神经科学中透射电镜切片采样的一种无偏方法

Investigations of the ultrastructural features of neurons and their synapses are only possible with electron microscopy. Especially for comparative ...