超分辨率成像研究原发神经元中蛋白质和突触标记的共同定位
Summary
该协议演示了如何使用超分辨率显微镜来研究蛋白质在原发神经元培养物中的共定位。
Abstract
突触是神经元的功能元素，其缺陷或损失是几种神经退行性和神经性疾病的基础。成像研究被广泛用于研究其在生理和病理条件下的功能和可塑性。由于其尺寸和结构，蛋白质的定位研究需要高分辨率成像技术。在该协议中，我们描述了一个程序，用于研究在初级神经元中使用结构化照明显微镜 （SIM） 在超分辨率级别使用突触标记的目标蛋白质的共同定位。SIM 是一种图案照明技术，可使广场显微镜的空间分辨率翻倍，细节达到 100 nm 左右。该协议指示强大的联合本地化研究所需的控制和设置，并概述正确分析成像数据的统计方法。
Introduction
自从1897年福斯特和谢林顿在1897年第一次描述突触以来，对突触的理解和看法发生了巨大的变化。从那时起，我们对神经元交流的知识及其背后的分子过程呈指数级增长。很明显，突触可以被认为是一个双隔间系统：一个包含用于释放神经递质的囊泡的突触前隔间，以及一个具有受体3的突触后隔间。在过去的二十年里，这种简单化的观点已经演变成一个复杂的蛋白质网络，这些蛋白质是将发射机结合成信号4所需的蛋白质。
理解的收获部分是由于超分辨率技术，克服了传统光显微镜的衍射极限，以适应突触的尺寸更好5，6，7，8，9，10。6,7,8,9,105由于衍射限制，光学显微镜不能达到分辨率超过200纳米横向11，12。11,为了绕过这个限制，建立了超分辨率技术，使用不同的方法，并达到不同的子衍射极限分辨率：SIM，STED（刺激发射耗竭显微镜），PALM（照片激活定位显微镜）和风暴（随机光学重建显微镜）13，,14。SIM通过将衍射光栅插入激励光束路径15，使基于激光的广场显微镜系统的空间分辨率翻倍。可移动光栅可衍射激光束，形成已知的照明模式，通常是条纹。这种有目的的结构光模式叠加到荧光染料（样品）的未知空间分布上。由两种模式形成的干扰边缘编码，否则无法区分的精细细节与正常的宽场显微镜。最终的超解析图像是通过结合和解码与数学方法相同的样本的多个原始图像通过衍射光栅的平移和旋转获得的。超分辨率图像的分辨率达到100纳米的横向和500纳米的轴向2D-SIM15或100nm在横向和250纳米的轴向为3D-SIM16。16
由于许多神经系统疾病，突触功能障碍在发病和进展17，18中起着主要作用，对突触的新理解更为重要。阿尔茨海默病、唐氏综合症、帕金森病、普里昂病、癫痫、自闭症谱系障碍和易碎X综合征等都与突触组成、形态和功能19、20、21、22,20,21,的异常有关。
最近，我们使用一组SUMO特异性抗体，使用SIM显示SUMO蛋白质的原生海马神经元与突触前和后突触标记突触物理和PSD95在超分辨率水平23的共同定位。这使我们能够确认SSUMO在神经元中的定位的生化和共体显微镜证据。
在这里，我们描述了一个协议，研究蛋白质在小鼠海马原神经元的定位。同时，此协议可适应不同类型的原发神经元培养。
Protocol
1. 主要文化 培养小鼠海马原神经元在室盖玻片（如伊比迪μ-滑动8井或N得不好实验室-Tek室盖玻璃），符合#1.5（0.17毫米）盖玻片厚度的客观要求。 涂覆室盖玻片，带 100 μL 聚 L-lysine （100 μg/mL）。 第二天，用无菌磷酸盐缓冲盐水（PBS）清洗两次室盖玻片。 要获得小鼠原发神经元，请将海马从P1-P4幼崽23中分离。 将解剖的海马放在10 mL的解剖?…
Representative Results
我们在这里介绍研究神经元蛋白共同定位的标准工作流程。我们首先校准了显微镜，然后对样品进行了 SIM 分析。为了校准系统，我们使用直径为0.1 μm的荧光微球。在获取珠子的超解析 3D-SIM 图像后，对底层图像数据进行 Fourier 转换，以将其重新转换为空间频率表示。在图2A中，不同的花型显示为超分辨率细节水平的指示。接下来，我们测量了通过计算单个珠的强度轮廓峰值…
Discussion
阐明突触的结构和组成对于理解调节记忆和认知的生理和病理过程至关重要。虽然在正常状态下，突触是记忆的积木，它们也是复杂的神经系统疾病（如阿尔茨海默氏症32）的根据。此处描述的协议用于研究神经元蛋白与称为 SIM 的超分辨率显微镜技术的共同定位。使用特定的照明模式，SIM 可以达到约 0.1 μm 的分辨率，这适用于研究突触，通常测量在 0.03 和 0.15 μm 之间。对于更?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
作者感谢爱德华多·米科蒂对手稿的建设性批评。这项研究得到了B brightFocus A2019296F、Fondo di Beneficenza – Gruppo Intesa Sanpaolo （LC）、Fdazione 区域每拉赖斯卡生物医学组织 （Care4NeuroRare CP_20/2018） （CN） 和玛丽·斯科多夫斯卡-居里创新培训网络 （JK） 的支持。
Materials
| 0.4% Trypan blue solution | Thermo Fisher Scientific | 15250061 | Chemical |
| 70 µm filter | Corning | 352350 | Equiment |
| Alexa | Thermo Fisher Scientific | – | Antibody |
| Antibody SENP1 | Santa Cruz | sc-271360 | Antibody |
| B27 Supplement | Life Technologies | 17504044 | Chemical |
| Bovine serum albumin | Merck | 5470 | Chemical |
| CaCl2 | Merck Life Science | 21115 | Chemical |
| Chambered coverslips | Ibidi | 80826 | Equiment |
| DyLight | Thermo Fisher Scientific | – | Antibody |
| FBS (Hyclone) | GIBCO | SH3007002 (CHA1111L) | Serum |
| FluoSpheres carboxylate-modified microspheres, 0.1 μm, yellow–green fluorescent | Thermo Fisher Scientific | F8803 | Equiment |
| Glucose | Merck Life Science | G8769 | Chemical |
| Glutamax | GIBCO | 35050061 | Chemical |
| HEPES | Merck Life Science | H3537 | Chemical |
| L-Cystein | Merck Life Science | C6852-25g | Chemical |
| MAP2 | Merck | AB15452 | Antibody |
| MEM | Life Technologies | 21575022 | Medium |
| MgCl | Merck Life Science | M8266 | Chemical |
| NaOH | VWR International | 1,091,371,000 | Chemical |
| Neurobasal A | Life Technologies | 10888022 | Medium |
| N-SIM Super Resolution Microscope | Nikon | – | Instrument |
| Papain | Merck Life Science | P-3125 | Chemical |
| paraformaldehyde | Thermo Fisher Scientific | 28908 | Chemical |
| Pen/Strep 10x | Life Technologies | 15140122 | Chemical |
| phosphate-buffered saline | Gibco | 10010023 | Chemical |
| Poly-L lysine | Sigma | P2636 | Chemical |
| ProLong Diamond Glass Antifade Mountant | Thermo Fisher Scientific | P36970 | Chemical |
| PSD95 | NeuroMab | K28/43 | Antibody |
| Round coverglass | Thermo | 12052712 | Equiment |
| SUMO1 | Abcam | ab32058 | Antibody |
| Synaptophysin | Merck | S5768 | Antibody |
| Triton X-100 | Merck | T8787 | Chemical |
| Trypsin inhibitor | Merck Life Science | T9003-500MG | Chemical |
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Citazione di questo articolo
Russo, L., Natale, C., Conz, A., Kelk, J., Restelli, E., Chiesa, R., Salmona, M., Fioriti, L., Colnaghi, L. Super-Resolution Imaging to Study Co-Localization of Proteins and Synaptic Markers in Primary Neurons. J. Vis. Exp. (164), e61434, doi:10.3791/61434 (2020).