腺相关病毒和共聚焦显微镜对器官脑切片胶质细胞器的可视化和实时成像
Summary
形成突促进了快速动作电位的传播和神经元的存活。这里描述的是一个协议的胶质特异表达荧光蛋白在器官脑切片与后续的延时成像。此外, 一个简单的程序, 可视化不髓鞘提出。
Abstract
神经元依赖于形成突的电绝缘和营养支持。尽管突的重要性, 目前用于研究神经元的先进工具, 只是部分被胶质研究人员所接受。细胞类型特异性染色的病毒转导是一个有用的方法来研究活体细胞动力学。本文介绍了一种在转录控制下利用腺相关病毒 (AAV) 携带线粒体靶向荧光蛋白的基因对器官脑切片中胶质线粒体进行可视化的协议髓鞘碱性蛋白启动子。包括制作器官冠状小鼠脑切片的协议。一个过程中的线粒体的延时成像, 然后跟随。这些方法可以转移到其他的细胞器, 可能是特别有用的研究细胞在髓鞘。最后, 我们描述了一个现成的技术, 可视化的活体切片不髓鞘的共聚焦反射显微镜 (核心)。核心不需要额外的设备, 并可以用于识别髓鞘鞘在现场成像。
Introduction
大脑的白质是由髓鞘包裹的神经细胞轴突组成的, 这是由突所形成的一种专门的扩展等离子体膜。髓鞘是需要快速和可靠的动作电位传播和长期生存的髓轴突, 并失去髓鞘可能导致神经功能障碍。尽管它们的重要性, 与神经元和星形胶质细胞相比, 突的性质是不为人知的。因此, 为研究突而开发的工具越来越少。
活体成像的细胞器, 如线粒体, endoplasmatic 网 (ER) 或不同的水泡结构, 可以用来研究动态变化, 随着时间的推移。传统上, 生活突的成像已经在单一1,2中执行。然而, 突在单养不显示致密髓鞘, 因此, 器官或急性脑切片可能是一个更好的选择, 在研究的本地化和运动的细胞器。由于髓轴突与周围髓鞘之间的短距离, 髓鞘内的小细胞器和蛋白质的定位可能具有挑战性。因此, 光是显微免疫程序就没有空间分辨率来区分髓鞘内的细胞器和髓轴突的细胞。这可以通过病毒转导与细胞型特异性促进剂驱动的器官靶向荧光蛋白的基因来解决。其优点是细胞特异性和稀疏表达式, 能够准确地评估器官的定位和动态。转基因动物也可以用来实现这样一个以细胞为目标的特定表达式3。然而, 转基因动物的生产和维护费用昂贵, 通常不提供可通过病毒方法实现的稀疏表达。
本文所描述的方法使用的病毒转导突与线粒体靶向荧光蛋白 (dsred 或绿色荧光蛋白, gfp) 驱动的髓鞘碱性蛋白促进剂 (细胞内 dsred 或绿细胞-gfp), 以可视化器官脑切片中的胶质线粒体。此外, 另一种荧光蛋白在细胞质中的表达 (无论是 gfp 与 dsred 或 tdtomato 与美图-gfp 一起使用) 是用来使细胞形态学的可视化, 包括髓鞘鞘的细胞质。该协议包括制作器官脑切片的程序 (由 De Simoni 和 Yu 描述的修改后的协议版本, 20064,5)。然后我们描述了用于研究线粒体运动的延时成像程序。这个过程使用一个直立的共焦显微镜与连续交换成像介质, 设置, 使易于应用的药物或其他媒体的变化, 在成像。延时成像程序可以在任何共聚焦显微镜下进行, 有些额外的设备用于维持活体切片, 如下所述。该协议还包含一些优化成像和减少光的提示。
最后, 本文介绍了一种快速、简便的共焦反射显微镜 (CoRe) 可视化不髓鞘的方法。这可能是有用的, 以确定髓鞘鞘的实时成像。近年来, 一些技术已经发展到图像髓鞘不需要任何染色, 但其中大多数需要特定的设备和专长6,7,8。这里描述的过程使用髓鞘的反射的物产并且是一个简单的 single-excitation 波长版本光谱共焦反射显微镜 (比分, 几 laser 波长被结合形象化髓鞘)9. 核心可以在具有 488 nm 激光和 470-500 nm 带通发射过滤器或可调谐发射过滤器的任何共焦显微镜上进行。
Protocol
此处所述的程序已得到挪威动物研究局的批准。文件末尾的材料清单中提供了消耗品和其他所需设备的供应商和目录号. 1. 器官切片的制备 注意: 此食谱在产后日 7-9 (p7-9) 使用两只小鼠幼崽, 在两个六良好的培养皿上分别产生24器官切片。除非另有说明, 所有的程序应在无菌罩和 nitril 或乳胶手套使用。只应使用细胞培养级成分. 高压釜瓶, 解剖…
Representative Results
器官脑切片的培养和转如上所述, 显示了稀疏分布的皮质突表达 mito_dsred 和 GFP。免疫与抗体抗 Olig2 和碱性髓鞘证实, 表达特定于突 (图 1)。 对于活体成像, 转突是公认的几个平行运行的髓鞘鞘的特征形态学 (图 1和图 2A)。通过在转突上执行延时成像, 可以监测主…
Discussion
此处描述的器官区域性的协议是由 De Simoni 和 Yu (2006)5所描述的协议的修改版本18 。下文概述了最重要的变化。培养基中添加了三个缓冲液, 在病毒转导和细胞培养基的变化过程中, 提高了孵化室外的切片成活率。纸屑的灭菌程序也发生了变化。虽然其他协议消毒五彩纸屑由灭菌, 我们不建议这样做, 因为它会导致几个五彩纸屑卷曲。避免使用卷曲的纸屑, 因为在?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
我们感谢琳达 Hildegard 博格森和 Magnar Bjørås 获得细胞实验室和设备, Janelia 分子生物学共享资源人员的质粒和病毒生产和公园 Vervaeke, 以协助激光功率测量。这项工作由挪威卫生协会资助, 挪威研究理事会和显微镜设备由 Norbrain 资助。
Materials
| Agarose | Sigma | A9539 | |
| BD Microlance 19G | BD | 301500 | Needles used for in- and outlet of bath |
| Bioxide gas | AGA | 105701 | |
| Brand pipette bulbs | Sigma-Aldrich | Z615927 | Pipette bulbs |
| Bunsen burner (Liquid propane burner) | VWR | 89038-530 | |
| Cable assembly for heater controllers | Warner Instruments | 64-0106 | Temperature controller – thermometer part |
| CaCl2 | Fluka | 21100 | |
| CO2 | AGA | 100309 | CO2 for incubator |
| Cover glass, square Corning | Thermo Fischer Scientific | 13206778 | To attach under bath for live imaging. Seal with glue or petrolium jelly. |
| D-(+)-Glucose | Sigma | G7021 | |
| Delicate forceps | Finescience | 11063-07 | For dissection |
| Diamond scriber pen | Ted Pella Inc. | 54463 | |
| Disposable Glass Pasteur Pipettes 230 mm | VWR | 612-1702 | Glass pipettes |
| Double edge stainless steel razor blade | Electron Microscopy Sciences | #7200 | Razor blade for vibratome |
| Earle's Balanced Salt Solution (EBSS) | Gibco-Invitrogen | 24010-043 | |
| Filter paper circles | Schleicher & Schuell | 300,220 | Filter paper used for filtration of PFA |
| Fun tack | Loctite | 1270884 | Use to connect/adjust position of in- and outlets in bath |
| Hand towel C-Fold 2 | Katrin | 344388 | |
| Harp, Flat for RC-41 Chamber, | Warner Instruments | 64-1418 | Harp to hold down confetti in bath. Cut off strings before use with organotypic slices. 1.5 mm, 13mm, SHD-41/15 |
| HEPES, FW: 260.3 | Sigma | H-7006 | |
| Holten LaminAir, Model 1.2 | Heto-Holten | 96004000M | Laminar flow hood |
| Horse serum, heat inactivated | Gibco-Invitrogen | 26050-088 | |
| KCl | Sigma | P9541 | |
| LCR Membrane, PTFE, | Millipore | FHLC0130 | Confetti |
| Leica VT1200 | Leica | 14048142065 | Vibratome |
| MEM-Glutamax with HEPES | Thermo Fischer Scientific | 42360024 | |
| MgCl2 | R.P. Normapur | 25 108.295 | |
| Micro Spoon Heyman Type B | Electron Microscopy Sciences | 62411-B | Small, rounded spatula with sharpened end for dissection |
| Millex-GP filter unit | Millipore | SLGPM33RA | Syringe filter unit |
| Millicell cell culture insert, 30 mm | Millipore | PICM03050 | Cell culture inserts |
| Minipuls 3 Speed Control Module | GILSON | F155001 | Peristaltic pump for live imaging – Control module part (connect to two-cannel head) |
| Na2HPO4 | Sigma-Aldrich | S7907 | |
| NaCl | Sigma-Aldrich | S9888 | |
| NaH2PO4 | Sigma-Aldrich | S8282 | |
| NaHCO3 | Fluka | 71628 | |
| Nunclon Delta Surface | Thermo Fischer Scientific | 140675 | Culture plate |
| Nystatin Suspension | Sigma-Aldrich | N1638 | |
| Objective W "Plan-Apochromat" 40x/1.0 DIC | Zeiss | 441452-9900-000 | Water immersion objective used for live imaging. (WD=2.5mm), VIS-IR |
| Parafilm | VWR | 291-1211 | |
| Paraformaldehyde, granular | Electron Microscopy Sciences | #19208 | |
| PC-R perfusion chamber | SiSkiYou | 15280000E | Bath for live imaging |
| Penicillin-Streptomycin, liquid | Invitrogen | 15070-063 | |
| Petri dish 140 mm | Heger | 1075 | Large Petri dish |
| Petri dish 92×16 mm | Sarstedt | 82.1473 | Medium Petri dish |
| Petridish 55×14,2 mm | VWR | 391-0868 | Small Petri dish |
| Phosphate buffered saline (PBS) | Sigma | P4417 | PBS tablets |
| R2 Two Channel Head | GILSON | F117800 | Peristaltic pump for live imaging – Two channel head part (requires control module) |
| Round/Flat Spatulas, Stainless Steel | VWR | 82027-528 | Large spatula for dissection |
| Sand paper | VWR | MMMA63119 | Optional, for smoothing broken glass pipettes |
| Scissors, 17,5 cm | Finescience | 14130-17 | Large scissors for dissection |
| Scissors, 8,5 | Finescience | 14084-08 | Small, sharp scissors for dissection |
| Single edge, gem blade | Electron Microscopy Sciences | #71972 | Single edge razor blade |
| Single inline solution heater SH-27B | Warner Instruments | 64-0102 | Temperature controller – heater part |
| Steritop-GP Filter unit, 500 ml , 45mm | Millipore | SCGPT05RE | Filter to sterilize solutions |
| Super glue precision | Loctite | 1577386 | |
| Surgical scalpel blade no. 22 | Swann Morton Ltd. | 209 | Rounded scalpel blade |
| Temperature controller TC324B | Warner Instruments | 64-0100 | Temperature controller for live imaging (requires solution heater and cable assembly) |
| Trizma base | Sigma | T1503 | |
| Trizma HCl | Sigma | T3253 | |
| Water jacketed incubator series II | Forma Scientific | 78653-2882 | Incubator |
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Citer Cet Article
Kennedy, L. H., Rinholm, J. E. Visualization and Live Imaging of Oligodendrocyte Organelles in Organotypic Brain Slices Using Adeno-associated Virus and Confocal Microscopy. J. Vis. Exp. (128), e56237, doi:10.3791/56237 (2017).