相关光学和电子显微镜，研究与β淀粉样蛋白斑块的小神经胶质互动
Summary
本文介绍了使用甲氧基X04，它穿过血 – 脑屏障，并选择性地结合β褶的致密核心斑块Aβ发现张在阿尔茨海默氏症小鼠模型可视化淀粉样蛋白斑块Aβ的协议。它允许的前免疫染色和处理电子显微镜含斑块组织切片预检。
Abstract
详细协议这里提供给前从阿尔茨海默氏病小鼠模型中确定在脑切片的淀粉样蛋白的Aβ斑块的预嵌入免疫染色（特别为离子化的钙结合衔接分子1（IBA1），由小胶质细胞中表达的钙结合蛋白）和组织处理电子显微镜（EM）。甲氧基X04是荧光染料跨越血 – 脑屏障和选择性结合在致密核心的Aβ斑块中发现β-打褶纸。用甲氧基X04动物注射前牺牲和脑定影允许预筛分和含斑块的脑切片进行进一步的处理与费时的操作的选择。研究可能含有非常少的斑块，本仅在部分的一小部分的特定的脑区域或层内早期AD病理时，这是特别有帮助。刚果红，硫磺素S和Thiofl组织切片的验尸处理AVIN T（或者甚至与甲氧基X04）可以标记β-折叠片，但需要用乙醇广泛清算去除多余的染料和这些程序与超微结构保存不兼容。它也将是低效率的，以对Aβ（和其它细胞标记诸如IBA1）对感兴趣的区域的所有的脑切片进行标记，只得到含有的Aβ斑块在合适位置的一小部分。重要的是，Aβ斑块仍然可见的EM组织处理后，允许对区域的精确识别（一般下降到几平方毫米），用电子显微镜检查。
Introduction
淀粉样蛋白的Aβ斑块的形成是阿尔茨海默氏病（AD）的主要神经病理学特征。然而越来越多的证据表明，在疾病进展1,2-免疫系统的重要的作用。特别是，从临床前和临床研究的新的数据建立的免疫功能障碍为主要驱动和贡献者AD病理学。有了这些发现，中枢及外周免疫细胞已成为有前途的治疗靶点AD 3。以下方案结合了光学和电子显微镜（EM），以产生新的见解的Aβ斑块沉积和在公元小胶质细胞表型改变之间的关系。该协议允许的Aβ斑块在AD小鼠模型中使用的荧光染料甲氧基X04的体内注射的标记。甲氧基X04是，可以很容易穿越血 – 脑屏障进入脑实质并结合以高的β型褶片材的刚果红衍生物ffinity。因为染料是荧光的，它可用于Aβ斑块沉积的体内检测用双光子显微镜4。一旦绑定到Aβ，甲氧基X04不会离解或斑块重新分配了，而且随着时间的推移保留其荧光。它一般给药外周，以允许大脑动力学5的非侵入性成像。荧光也保持以下醛固定，允许相关验尸分析，包括在Aβ斑块6附近的神经元死亡的调查。
这个协议利用甲氧基X04的性质来选择由APP SWE / PS1A 246E小鼠脑切片（APP-PS1;共表达在APP基因Lys670Asn / Met671Leu一个双突变，和人类早老PS1-A264E变体）表现出Aβ7感兴趣的特定区域（海马CA1，地层radiatum和腔隙，moleculare斑）预嵌入对小胶质细胞标记钙离子结合适配器分子1（IBA1免疫）可视化小胶质细胞机构和EM进程之前。将小鼠通过穿心灌注给定的甲氧基X04溶液，脑定影前24小时腹膜内注射。使用振动得到冠状脑切片。含有海马CA1节为Aβ斑块在地层radiatum和腔隙-moleculare存在下在荧光显微镜下进行筛选。免疫染色IBA1，四氧化锇后固定，塑料树脂包埋在所选脑切片随后进行。在该协议的结束时，部分可以在没有进一步的超微结构降解被存档，准备超薄切片和超微结构检查。重要的是，斑块仍与不同的抗体的免疫染色，例如IBA1如在本协议之后荧光。他们变得更暗Ť汉其周围神经毡以下锇酸后固定，独立地甲氧基X04染色，这有助于准确地识别感兴趣的区域，一般下降到几平方毫米，以用透射电子显微镜进行检查。
这种关联方法提供了一个有效的方式来确定具体的脑切片在超微结构层面来考察。研究早期AD病理学，特定脑区域或层内，可能只包含几个Aβ斑块中，存在于只有组织切片的一小部分时，这是特别有帮助。在这些时候尤其，这将是低效率的用于Aβ免疫染色（和双标记用于其它细胞标记诸如IBA1）在几个脑切片简单地，得到含有的Aβ斑块在合适位置的一小部分。此外，注射活体小鼠与甲氧基X04牺牲和组织处理没有做之前，仲裁协议Ë超微结构保存。替代方法，如刚果红，硫磺素S，硫黄素T或甲氧基X04验染色固定组织切片需要乙醇染色分化，8-11导致渗透压和破坏的超微结构。刚果红也是已知的人类致癌物质12。
Protocol
Representative Results
Discussion
该协议解释了使用EM针对致密核心Aβ斑块相关办法。甲氧基X04 体内注入允许包含特定区域内的Aβ斑块和感兴趣的层，例如海马CA1区，地层radiatum和腔隙-moleculare脑切片的快速选择。在本例中，甲氧基X04预检用嵌入预免疫染色IBA1研究小神经胶质表型如何不同与在致密核心的Aβ斑块的存在超微结构水平突触相互作用结合。
小胶质细胞是令人难以置信的响应他们的环境和他们…
Declarações
The authors have nothing to disclose.
Acknowledgements
We are grateful to Dr. Sachiko Sato and Julie-Christine Lévesque at the Bioimaging Platform of the Centre de recherche du CHU de Québec for their technical assistance. Grants from the Natural Sciences and Engineering Research Council of Canada (NSERC) RGPIN-2014-05308, The Banting Research Foundation, and The Scottish Rite Charitable Foundation of Canada to M.E.T supported this work.
H.E.H. is recipient of a scholarship from the Lebanese Ministry of Education and Higher Education, and K.B. from the Faculté de médecine of Université Laval.
Materials
| Methoxy-X04 | Tocris Bioscience | 4920 | 10 mg substrate per tablet |
| Propylene glycol | Sigma Aldrich | W294004 | |
| Dimethyl sulfoxide (DMSO) | Fisher BioReagents | BP231-1 | Caution: toxic |
| Sodium Chloride NaCl | Sigma | S9625 | |
| Sodium phosphate monobasic monohydrate | Sigma | S9638 | |
| Sodium phosphate dibasic | Sigma | S0876 | |
| Tris Hydrochloride | Fisher BioReagents | BP153500 | |
| Acrolein | Sigma | 110221 | Caution: Toxic |
| Paraformaldehyde Granular | Electron Microscopy Sciences | 19210 | Caution: Toxic |
| Filter paper | Fisher | 09-790-14F | |
| Peristaltic Pump with Tubing | ColeParmer | cp.78023-00 | |
| Excel Winged blood Collection Set Needles 25G | Becton Dickinson | 367341 | |
| Extrafine Forceps | F.S.T | 11152-10 | Tip shape: curved |
| Scissors | F.S.T | 14090-09 | Tip shape: straight |
| Hartman Hemostats | F.S.T | 13003-10 | Tip shape: curved |
| Surgical Scissors | F.S.T | 14004-16 | Tip shape: straight |
| Micro Dissecting Scissors | ROBOZ surgical store | 5818 | |
| Glass scintillation vials | Fisher Scientific | 74515-20 | |
| Vibrating Blade Michrotome Leica VT1000 S | Leica Biosystems | 14047235612 | |
| Vibratome blades | Electron microscopy Sciences | 71990 | |
| Microscope Slides | Fisher Scientific | 12-550-15 | |
| 24-well Tissue Culture Plates | Fisher Scientific | 353047 | |
| Ethylene Glycol | Fisher BioReagents | BP230-4 | |
| Glycerol | Fisher BioReagents | BP229-4 | |
| Hydrogen Peroxide, 30% | J.T.BAKER | cat: 2186-01 | |
| Sodium borohydride | Sigma | 480886 | |
| Tris HCl | Fisher | BP153-500ML | |
| Fetal bovine Serum (FBS) | Sigma Aldrich | F1051 | |
| Bovine serum albumin (BSA), fraction V | Thomas Scientific | C001H24 | |
| Triton X-100 | Sigma | T8787 | |
| Anti IBA1, Rabbit | WAKO | 019-19741 | |
| Goat Anti-Rabbit IgG | Jackson Immunoresearch | 111066046 | |
| VECTASTAIN Elite ABC Kit (Standard) | Vector Labs | PK-6100 | |
| 3.3'-Diaminobenzidine tetra-hydrochloride (DAB) | Sigma | D5905-50TAB | Caution: toxic |
| Osmium tetroxide, 4% solution | Electron Microscopy Sciences | 19150 | Caution: toxic |
| Durcupan™ ACM single component A | Sigma | 44611 | Resin Caution: Toxic |
| Durcupan™ ACM single component B | Sigma | 44612 | Hardener Caution: Toxic |
| Durcupan™ ACM single component C | Sigma | 44613 | Plasticizer Caution: Toxic |
| Durcupan™ ACM single component D | Sigma | 44614 | Accelerator Caution: Toxic |
| Ethanol | LesAlcoolsdeComerce | 151-01-15N | |
| Propylene oxide | Sigma | 110205 | Caution: corrosive |
| Aluminum weigh dishes | Electron Microscopy Sciences | 70048-01 | |
| ACLAR®–Fluoropolymer Films | Electron Microscopy Sciences | 50425 | |
| Oven/Incubator | VWR |
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