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Abstract
Introduction
Protocol
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신경과학

人 Intraepidermal 神经纤维内线粒体三维成像及分析

Published: September 29, 2017
doi:
10.3791/53369
, , ,
1University of Michigan Medical School, 2Department of Neurology,University of Michigan, 3Department of Internal Medicine,University of Michigan

Summary

该协议使用三维 (3D) 成像和分析技术来可视化和量化神经特异性线粒体。这些技术适用于在其他情况下, 一个荧光信号被用来隔离一个子集的数据, 从另一个荧光信号。

Abstract

本议定书的目的是研究 intraepidermal 神经纤维内的线粒体。因此, 3D 成像和分析技术的发展, 以隔离神经特异性线粒体和评估疾病诱导的线粒体远端的感觉神经的变化。该协议结合荧光免疫组织化学、共聚焦显微镜和3D 图像分析技术来可视化和量化神经特异性线粒体。详细的参数在整个过程中定义, 以提供一个具体的例子, 如何使用这些技术来隔离神经特异性线粒体。抗体被用来标记的神经和线粒体信号的组织切片的皮肤穿孔活检, 其次是间接免疫荧光可视化的神经和线粒体与绿色和红色的荧光信号分别。利用共焦显微镜对 Z 系列图像进行采集, 并采用3D 分析软件对信号进行处理和分析。没有必要按照内部描述的确切参数, 但重要的是要与整个染色, 获取和分析步骤中选择的一致。该协议的优点是, 它适用于各种情况, 其中一个荧光信号被用来隔离其他的信号, 否则是不可能单独研究。

Introduction

线粒体提供重要的细胞功能, 包括产生细胞能量, 缓冲钙, 调节坏死和凋亡细胞死亡1,2,3。神经系统具有很高的新陈代谢率, 与身体的4相比, 这表明神经元通过线粒体呼吸以三磷酸腺苷 (ATP) 的形式产生高度的细胞能量。许多证据证明, 神经元函数依赖于 ATP5, 特别是在突触的6。因此, 神经元内线粒体的分布是很重要的。

在过去的10年里, 大量的信息表明, 神经元线粒体的贩运和对接受到高度的管制。运动蛋白参与在神经元中分布线粒体到特定的细胞隔间。线粒体的贩运是特别重要的, 因为神经元项目轴突和树突远离躯体。驱动马达蛋白质主要直接顺 (远离体细胞) 贩运的线粒体沿微管, 而蛋白马达蛋白直接逆行 (向 soma) 运动7,8,9,10. 有细胞信号, 这样的线粒体膜电位和冲动传导, 影响线粒体贩运的存在和方向11,12,13。

除了传送线粒体外, 还有专门的蛋白质将线粒体定位到具有高能量需求的特定蜂窝室, 如郎和突触的节点8,14,17. 实际上, 轴突内的大部分线粒体是 non-motile9、13、18。专门的蛋白质像 syntaphilin 锚定线粒体的微管沿轴突, 而其他蛋白质锚定线粒体的肌动蛋白细胞骨架19–21。生长因子和钙等离子被报告支持停止线粒体运动, 将它们本地化为需要的区域21,22,23。

两者结合在一起, 线粒体的贩运和对接对神经元的正常功能至关重要。为支持这一点, 线粒体贩运的中断与包括阿尔茨海默病、肌萎缩侧索硬化症、腓骨-玛丽牙病、亨廷顿氏病、遗传性痉挛截瘫, 和视神经萎缩15,24,25,26,27。最近的研究侧重于线粒体功能障碍和病理作为糖尿病神经病变的潜在机制, 与糖尿病相关的感觉丧失28,29,30,31 ,32,33。假设是糖尿病改变了皮肤神经末端感官投射中线粒体的分布。因此, 在 intraepidermal 神经纤维 (IENFs), 背根神经节感觉传入的远端提示中, 开发了一种技术来可视化和量化线粒体。该技术将特定线粒体和神经纤维标签的荧光免疫组化与共焦显微镜相结合, 用强大的3D 图像分析软件对信号进行 z 系列采集, 测量神经特异性的分布线粒体从人体皮肤穿孔活检达到这个目的。

Protocol

在犹他州糖尿病中心 (盐湖城), 从一个大型社区初级保健网络招募的受试者中获得了皮肤穿孔活检。这项研究得到了密歇根大学机构审查委员会的批准, 并遵守了《赫尔辛基宣言》的宗旨。在测试之前, 从每个主题获得书面知情同意. 1. 荧光免疫组化 为 intraepidermal 神经纤维免疫组织化学制备穿孔活检: 由医务人员进行3毫米皮肤活检, 并将整个活…

Representative Results

人 IENFs 中线粒体的可视化与量化 荧光免疫组织化学允许在人体皮肤活检中同时标记多个信号, 以可视化神经、线粒体和细胞核。96井板是组织免疫组化过程中的步骤的一种简便方法。图 1显示此配置占了多达8节, 可通过12阶段的解决方案进行处理。自由浮动的方法, 加上温和的搅拌与平摇杆, ?…

Discussion

本议定书的目的是分离, 量化和分析的大小和分布的神经特异性线粒体在 IENFs 3D 从人体皮肤活检。协议中有几个关键步骤。自由浮动荧光免疫组织化学设计用于对每个样本中的多个信号进行着色和分析, 为探索性研究提供了一种更加通用的方法44,45。这一过程允许抗体渗入组织, 以最大限度地获得图像整个50µm 部分, 这是必要的获得共聚焦显微镜图像和3D …

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项工作得到了国家卫生研究院的资助, K08 NS061039-01A2, 神经病学研究和 #38; 发现, 和密歇根大学 Taubman 医学研究所的 a。这项工作使用的形态学和图像分析核心的密歇根糖尿病研究中心, 由国家卫生研究院资助 5P90 DK-20572 来自国家糖尿病和消化系统和肾脏疾病研究所。作者要感谢 j. 鲁滨逊单身人士和 a. 戈登？史密斯 (犹他大学) 慷慨捐献人体皮肤样本。

Materials

2% Zamboni's Fixative Newcomer Supply, Middleton, WI  1459A 2% paraformaldehyde, 0.2% saturated picric acid in phosphate buffered saline (PBS), pH 7.4
10X Phosphate Buffered Saline (PBS)  Fisher Scientific, Pittsburgh, PA BP399-4 To make up 1X PBS
Image-iT FX Signal Enhancer ThermoFisher Scientific, Waltham, Massachusetts I36933 enhances Alexa Fluor dye signals by reducing nonspecific binding
Anti-Protein Gene Product 9.5 Antibody (Rabbit Polyclonal) Proteintech Group Inc. Rosemont, IL 14730-1-AP abbreviated as PGP9.5, replaces discontinued AbD Serotec (Cat. No. 7863-0504) antibody
Anti-Pyruvate Dehydrogenase E2/E3bp Antibody (Mouse Monoclonal) abcam, Cambridge, MA ab110333 abbreviated as PDH
Goat anti-mouse Secondary antibody Alexa Fluor 594 conjugate ThermoFisher Scientific, Waltham, Massachusetts A-11034 red-fluorescent conjugated secondaryantibody
Goat anti-rabbit Secondary antibody Alexa Fluor 488 conjugate ThermoFisher Scientific, Waltham, Massachusetts A-11032 green-fluorescent conjugated secondaryantibody
Albumin, from Bovine Serum Sigma-Aldrich, St. Louis, MO A7906-100 abbreviated as BSA
Triton X- 100 Sigma-Aldrich, St. Louis, MO T9284 abbreviated as TX-100
0.22 µm Filter EMD Millipore, Billerica
MA		 MILLEX GP SLGP 033NS 0.22 µm Millipore filter
Parafilm M Fisher Scientific, Pittsburgh, PA 13-374-10 Curwood Wisconsin LLC Parafilm M (PM-996)
Non-calibrated Loop Fisher Scientific, Pittsburgh, PA 22-032092 inoculating Loop by Decon LeLoop (MP 199-25)
96-well Assay Plate Corning Incorporated, Corning, NY 3603 96-well flat bottom plate
Prolong Gold antifade reagent with DAPI ThermoFisher Scientific, Waltham, Massachusetts P-36931 DAPI staining of nuclei
Microscope Cover Glass 50 x 24 mm Fisher Scientific, Pittsburgh, PA 12-544E Coverslips
Superfrost Plus Microscope Slides Fisher Scientific, Pittsburgh, PA 12-550-15 Microscope Slides
Leica SP5 Laser Scanning Confocal Microscope Leica Microsystems, Buffalo Grove, IL SP5 Confocal Microscope
Volocity x64 Software  Perkin Elmer, Waltham , MA version 4.4.0 Volocity software is used for Steps 3.1 and 3.2 in the protocol for image processing
Imaris x64 3 Dimensional Analysis Software Bitplane, Concord, MA version 7.7.1 Imaris software is used for Steps 3.3 through 3.5 in the protocol for image analysis
Excel Microsoft, Redmond, WA version Office 2013 Excel spreadsheet software is used for Step 3.6 in the protocol to summarize morphometric features
Optimum Cutting Temperature Compound Sakura Finetek USA, Inc., Torrance, CA 4583 abbreviated as OCT
Leica Cryostat Leica Biosystems, Buffalo Grove, IL CM1850 Cryostat for cutting 50 µm sections
CellLight Mitochondria-GFP, BacMam 2.0 ThermoFisher Scientific, Waltham, Massachusetts C10600 Used as a postive control to label mitochondria with a green fluorescent signal
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Tags

Keywords: 3D ImagingMitochondriaIntraepidermal Nerve FibersFluorescence ImmunohistochemistryPGP9.5PDHSecondary AntibodiesSignal EnhancementEpidermal Nerve FibersNeurological Complications
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Hamid, H. S., Hayes, J. M., Feldman, E. L., Lentz, S. I. Three-dimensional Imaging and Analysis of Mitochondria within Human Intraepidermal Nerve Fibers. J. Vis. Exp. (127), e53369, doi:10.3791/53369 (2017).
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