线粒体和内质视网膜成像由相关光和体积电子显微镜
Summary
我们提出了一个方案,研究线粒体和内质神经在全细胞的分布后,使用相关的光和体积电子显微镜,包括抗坏血蜡过氧化物酶2和马萝卜过氧化物酶染色后,同一部分中具有或没有靶基因的细胞的序列切片,并通过电子显微镜进行序列成像。
Abstract
细胞细胞器,如线粒体和内质视网膜(ER),创建一个网络来执行各种功能。这些高度弯曲的结构被折叠成各种形状,形成一个动态网络,根据细胞条件。利用超高分辨率荧光成像和光显微镜,尝试对线粒体和ER之间的网络进行可视化;然而,有限的分辨率不足以详细观察线粒体和ER之间的膜。透射电子显微镜为细胞细胞器的观察提供了良好的膜对比度和纳米级分辨率;然而,在评估高度弯曲的细胞器的三维(3D)结构时,非常耗时。因此,我们利用增强的抗甲酰氨基甲酸酯过氧化物酶2和马萝卜过氧化物酶染色技术,通过相关的光电子显微镜(CLEM)和体积电子显微镜技术观察线粒体和ER的形态。应用了一种组染色方法、超薄序列切片(阵列断层扫描)和体积电子显微镜来观察三维结构。在此协议中,我们建议将CLEM和3D电子显微镜相结合,对线粒体和ER进行详细的结构研究。
Introduction
线粒体和内质视网膜(ER)是膜结合的细胞器。它们之间的联系对于它们的功能是必要的,并且与其网络相关的蛋白质已经描述了1。线粒体和ER之间的距离已报告约100纳米使用光显微镜2;然而,最近的超分辨率显微镜3和电子显微镜(EM)4的研究表明,它相当小,在大约10-25纳米。超分辨率显微镜的分辨率低于EM,需要进行特定的贴标。EM是一种合适的技术,用于线粒体和ER之间的连接结构研究,实现足够高分辨率的对比度。然而,缺点是有限的z轴信息,因为薄部分必须大约60纳米或更薄的传统传输电子显微镜(TEM)。对于足够的EM z轴成像,可以使用三维电子显微镜(3DEM)5。然而,这涉及到整个细胞的数百个薄序列部分的制备,这是非常棘手的工作,只有少数熟练的技术人员已经掌握。这些薄部分被收集在易碎的圆锥膜涂层单孔TEM网格上。如果薄膜在一个梁上断裂,则无法进行序列成像和体积重建。串行块面扫描电子显微镜 (SBEM) 是 3DEM 的一种常用技术,它使用扫描电子显微镜 (SEM) 真空室内的破坏性组合切片,使用金刚石刀 (Dik-SBEM) 或聚焦电束 (FIB-SEM)6.然而,由于这些技术并非在所有设施中都可用,我们建议阵列断层扫描7使用串行切片和 SEM。在阵列断层扫描中,使用超微缩的序列截取部分被转移到玻璃盖玻片上,而不是TEM网格,并通过光显微镜和SEM8进行可视化。为了增强背散射电子 (BSE) 成像的信号,我们使用了一个组合 EM 染色协议,该协议使用四氧化二氮(OsO 4) 固定细胞,其基质硫二氢化物 (TCH)9,使我们能够在不嵌入后双重染色。
此外,线粒体标记SCO1(细胞色素c氧化酶组装蛋白1)*抗坏药过氧化物酶2(APEX2)10分子标记用于在EM水平上可视化线粒体。APEX2约为28 kDa,来自大豆抗坏血球过氧化物酶11。它被开发为显示特定蛋白质在EM水平的详细位置,就像绿色荧光蛋白标记蛋白在光显微镜中使用一样。APEX2在存在过氧化氢(H2O2)的情况下,将3,3′ -二氨基苯甲酸(DAB)转换成标签部位的不溶性亲血性聚合物。APEX2 可用作 EM 中传统抗体标记的替代品,在整个细胞的深度进行蛋白质定位。换句话说,APEX2标记的蛋白质可以通过特定的渗透11可视化,无需免疫金标记和超低温切片后的渗透。马萝卜过氧化物酶(HRP)也是一个敏感标签,催化H2O2依赖性聚化DAB成局部沉淀,提供EM对比度后处理与OsO4。ER靶肽序列HRP-KDEL(裂化-白脂-葡-绿露)12应用于整个细胞内的ER可视化。为了评价我们使用遗传标记和组染色的协议,同时使用渗透效应,在rOTO群染色中比较了膜对比度,并且不使用每个遗传标记。 虽然具有阵列断层扫描和 DAB 染色的 3DEM 分别用于其他目的,但我们的协议是独一无二的,因为我们将 3DEM 阵列断层扫描和 DAB 染色相结合,用于线粒体和 ER 标记。具体来说,我们在同一部分展示了五个带有APEX标记基因和无APEX标记基因的细胞,这有助于研究基因改造对细胞的影响。
Protocol
Representative Results
Discussion
使用EM以纳米分辨率确定特定蛋白质的细胞定位对于了解蛋白质的细胞功能至关重要。通常,有两种技术可以研究通过EM定位靶蛋白。一种是免疫黄金技术,自1960年以来一直用于EM,另一种是使用最近开发的遗传编码标签16的技术。传统的免疫金技术使用抗体结合的金颗粒或量子点来显示标记蛋白的位置。然而,由于对高质量抗体的要求和受树脂和固定性抗体影响的抗体的渗透效率,该?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项研究得到了KBRI基础研究计划的支持,该计划由韩国脑研究所资助,由科学和ICT部(19-BR-01-08)和韩国国家研究基金会(NRF)资助,由韩国政府(MSIT)资助(NO.2019R1A2C1010634. SCO1-APEX2和HRP-KDEL质粒由玄武瑞(首尔国立大学)提供。TEM数据是在KBRI的大脑研究核心设施获得的。
Materials
| Glutaraldehyde | EMS | 16200 | Use only in fume hood |
| Paraformaldehyde | EMS | 19210 | Use only in fume hood |
| Sodium cacodylate | EMS | 12300 | |
| Osmium tetroxide 4 % aqueous solution | EMS | 16320 | Use only in fume hood |
| Epon 812 | EMS | 14120 | EMbed 812- 20 ml/ DDSA- 16 ml/ NMA- 8 ml/ DMP-30 – 0.8 ml |
| Ultra-microtome Leica ARTOS 3D | Leica | ARTOS 3D | |
| Uranyl acetate | EMS | 22400 | Hazardous chemical |
| Lead citrate | EMS | 17900 | |
| 35mm Gridded coverslip dish | Mattek | P35G-1.5-14-CGRD | |
| Glow discharger | Pelco | easiGlow | |
| Formvar carbon coated Copper Grid | Ted Pella | 01805-F | |
| Hydrochloric acid | SIGMA | 258148 | |
| Fugene HD | Promega | E2311 | |
| Glycine | SIGMA | G8898 | |
| 3,3′ -diaminobenzamidine (DAB) | SIGMA | D8001 | Hazardous chemical |
| 30% Hydrogen peroxide solution | Merck | 107210 | |
| Potassium hexacyanoferrate(II) trihydrate | SIGMA | P3289 | |
| 0.22 um syringe filter | Sartorius | 16534 | |
| Thiocarbonyldihydrazide | SIGMA | 223220 | Use only in fume hood |
| Potassium hydroxide | Fluka | 10193426 | |
| L-aspartic acid | SIGMA | A9256 | |
| Ethanol | Merck | 100983 | |
| Transmission electron microscopy | FEI | Tecnai G2 | |
| Indium tin oxide (ITO) coated glass coverslips | SPI | 06489-AB | fragil glass |
| Isopropanol | Fisher Bioreagents | BP2618-1 | |
| Diamond knife | Leica | AT-4 | |
| Inveted light microscopy | Nikon | ECLipse TS100 | |
| Scanning electron microscopy | Zeiss | Auriga |
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