Name: correlative light and electron microscopy to study microglial interactions with β-amyloid plaques
Uploaded: 2016-06-01T15:00:00.000+00:00
Duration: 10 min 52 s
Description: Watch this Scientific Journal Video about Correlative Light and Electron Microscopy to Study Microglial Interactions with Î²-Amyloid Plaques at JoVE.com

We are grateful to Dr. Sachiko Sato and Julie-Christine L&#233;vesque at the Bioimaging Platform of the Centre de recherche du CHU de Qu&#233;bec 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 Facult&#233; de m&#233;decine of Universit&#233; Laval.

注:所有实验均批准并根据机构的动物伦理委员会的指导方针与加拿大议会关于动物饲养的准则进行的，符合由拉瓦尔大学的动物护理委员会的管理。使用了4至21个月的年龄之间的APP-PS1雄性小鼠。 25℃，免费获得食物和水 - 这些动物在22一12小时明暗周期下安置。 1.甲氧基X04溶液制备<ol><li>由甲氧基X04溶解于含有10％二甲基亚砜（DMSO），45％丙二醇和45％磷酸钠缓冲盐水（在100mM磷酸缓冲液的0.9％NaCl溶液制备甲氧基X04 9的5毫克/毫升溶液，pH值7.4）。 <ol><li>使用微量天平，称量5毫克甲氧基X04化合物。根据通风橱，溶解甲氧基X04 DMSO中，并搅拌，直到获得澄清绿色溶液。依次添加丙二醇和ph值而与每个另外搅拌osphate缓冲盐水。 </li><li>搅拌在4℃旋转器上O / N的解决方案。获得黄绿色乳液。可以将溶液储存在4℃下长达两个月没有降解。 </li></ol></li></ol> 2.甲氧基X04注射液<ol><li>灌注前24小时，称重小鼠，并给各小鼠甲氧基X04的腹膜内注射，剂量为使用27个半ģ针10毫克/公斤体重。 </li></ol> 3.注射的小鼠的穿心灌注<ol><li>在灌注前一天，制备磷酸盐缓冲盐水（PBS），3.5％的丙烯醛，和4％低聚甲醛（PFA）的解决方案13的适当体积，并将其存储在4℃CO / N。 注意:这些解决方案将同时用于灌注和预嵌入免疫。与丙烯​​醛工作时要特别小心，因为它是有腐蚀性和有毒既研究人员和环境。此外，由于丙烯醛具有腐蚀性塑料，一定要使用合适的玻璃器皿准备丙烯醛的解决方案。 </li><li>上灌注的当天，通过用25微米的颗粒截留的粗滤纸过滤PFA和丙烯醛。 </li><li>在灌注，使用蠕动泵以提供15毫升PBS中，75毫升丙烯醛，和150ml的PFA依次到小鼠循环，以25毫升/分钟的流速。 谨慎使用。严格执行灌注在通风橱内，以避免PFA和丙烯醛的有害烟雾。 <ol><li>要设置泵，一端插入PBS液，补油管（持有约15毫升），用PBS和修复将25g翼采血针的另一端。在任何时候，要确保管道是任何截留气泡免费。 </li><li>直接放置在管中的丙烯醛瓶设置填充用PBS油管灌注泵后。 </li></ol></li><li>一个在腹膜内用27个半ģ针注射90毫克/公斤体重的剂量戊巴比妥钠的时间aesthetize一个鼠标。评估尾/脚趾捏响应。只有继续当鼠标不响应这样的厌恶，对疼痛刺激。 </li><li>用胶带前爪和后爪到工作表面固定在仰卧位（躺在用面向上的背面）的小鼠，并仔细暴露心脏，而不会造成损害其他器官。一定要刺破隔膜后迅速开展工作。 <ol><li>通过与沿尾开始向胸腔胸中线手术剪皮肤进行切口，并继续喙到锁骨。 </li><li>让另外两个侧面切口，沿腹胸腔的基础。轻轻移动和脚皮肤的两片，确保暴露了整个胸腔。 </li><li>保持钝钳剑突暴露胸腔和稳固尖剪刀秒。通过隔膜和胸腔切开，小心避免刺穿肺部，并继续切口喙锁骨。一定要刺破隔膜后迅速开展工作。 </li></ol></li><li>轻轻撕钝钳心包囊。按住心脏稳定钝钳，剪开右心房，并开始PBS输液。立即将采血针进入左心室。 </li><li>灌注用PBS小鼠（在管道），随后丙烯醛3分钟（相当于75毫升），然后切换到的PFA为6分钟（相当于150ml）中。务必切换解决方案，以防止气泡进入管之前暂停泵的流量。 </li><li>斩首鼠标和提取固定脑并直接将其放入含有4％PFA中至少2小时，在4℃的玻璃小瓶中。 <ol><li>以提取大脑，用剪刀和镊子通过皮肤切割以暴露颅骨。小心打破头骨运烯在眼睛之间，并且削掉小块它以暴露下面的大脑。 </li><li>为继续切片vibratome之前，小心地取出大脑暴露和后修复它用4％PFA额外2小时。 </li></ol></li></ol> 4.脑切片使用振动<ol><li>用冷PBS洗涤固定大脑的3倍。用锋利的剃刀刀片，取出嗅球（除非该区域是调查中）和横向切脑入2 - 3个大致相等的高度，所有这些可以同时以加速的程序进行切片。 </li><li>胶脑组织块垂直固定上放入盘试样板。确保切面光洁坚定奉行样品板和切片过程中不会被抛下。补充足够的PBS液入盘，直到整个大脑表面被完全淹没。重要的是保持个很重要Ë脑片和其后的章节完全沉浸在PBS在整个这一步。 </li><li>放置在托盘中的vibratome，调节切片速度为0.5mm /秒，切片频率90赫兹，和进料厚度至50μm，以收率50微米厚的切片。然后将切片转移到使用细画笔含有冷冻保护剂溶液（40％的PBS，30％乙二醇和30％甘油）中加入20毫升玻璃小瓶中。在-20°C，直到进一步使用存放小瓶。可替代地，如在以下步骤中所述保持部分在PBS立即筛选。 </li></ol> 5.第筛选甲氧基X04染斑的存在<ol><li>用立体鼠脑的援助寰含有感兴趣的区域14中，选择脑切片，例如，如在本实施例中使用的海马CA1区。将每个部分插入一个孔含有足够的冷冻保护剂溶液中的24孔培养板内以便防止从晒出部分。 </li><li>检查每个部分依次，以避免干燥的部分中，使用以下步骤: <ol><li> PBS中的液滴添加到与一次性吸显微镜载玻片，并将其放置在液滴的部分。 </li><li>检查在荧光显微镜下的部分以鉴定含有甲氧基X04标记的Aβ斑块的区域。 注意:甲氧基X04可使用荧光紫外（UV）滤光器（激发340 - 380纳米）容易地可视化。 </li></ol></li><li>捕获荧光模式的明视场兴趣区域（ROI）的图像以及不移动显微镜载物台，由于每个图像必须以直接相关的结构区域中的斑块的存在表明在这两个领域的相同区域组织切片。 </li><li>保存并命名根据动物的数量拍摄的图像。也记录在该板的孔数和画面的TA领域肯。例如，例:9978A1B;动物编号:9978;井数:A1;现场:明亮。放置部分放回其指定以及一旦成像完成。 注意:在结束时，对检查的每个部分中，获得两个图象，一个在明场和另一个在紫外领域。 </li><li>根据公号码打开相同的ROI中的Image J两个图像（一个在明场，另一个在紫外域），以及使用该MosaicJ插件，对准两个图像的边缘，并保存对齐并组合图像它来自。 </li><li>与特定动物的数量将图片保存在一个单独的文件夹中。结合图像来识别和定位在组织部分中的斑块，并具有在两个不同场（亮和UV）全部的完整视图。 </li><li>筛选过程完成后，在-20℃存储检查切片在含有冷冻保护剂24孔培养板，直至免疫染色或进一步加工为Carried出来。 </li></ol> 6.预嵌入免疫染色IBA1 注:通过将平板上在室温下缓慢移动摇杆为IBA1执行免疫上选择自由浮动的部分。 <ol><li>彻底大约1ml的PBS，每次洗涤持续10分钟，洗第3次。 </li><li>淬火用10分钟的PBS溶液0.3％的过氧化氢的内源性过氧化物酶。此步骤防止非特异性过氧化物酶活性，以避免背景染色这是重要的。 </li><li>洗组织在PBS中3次，每次10分钟。 </li><li>孵育在PBS溶液中的0.1％的硼氢化钠的部分30分钟。这个步骤是重要的，以减少从固定步骤中的任何剩余的醛，并且如果丙烯醛在组织固定使用是特别重要的。 </li><li>洗PBS 3倍组织，每次10分钟，并确保消除所有的泡沫。 </li><li>阻断部分1小时。不同的抗体，可能需要不同的阻挡时间和解决方案。为IBA1，制备阻断含有10％胎牛血清，3％牛血清白蛋白和0.01％的Triton X-100的50mM Tris缓冲盐水缓冲液（TBS; pH7.4）中。 注意:阻断步骤是为了防止第一抗体的非特异性结合。的Triton X-100的低浓度允许更好染色膜的轻微透，并足够低以保持下的EM组织的最超微结构特征。 </li><li>从组织去除封闭缓冲液，并在一次抗体溶液孵育（兔抗IBA1，稀释[1:1000]在封闭缓冲液）于4℃CO / N。 </li><li>在TBS中洗3次，每次10分钟。 </li><li>孵育在二次抗体（山羊抗兔缀合于生物素）[1:200]在0.05M TBS 90分钟。 </li><li>在TBS中洗5次，每次5分钟。 </li><li>孵育在抗生物素蛋白 - 生物素复合物溶液（抗生物素蛋白[1:100]，生物素[1:100]）的TBS解1小时。 </li><li>在TBS中洗5次，每次5分钟。 </li><li>揭示了用0.05％二氨基联苯胺（DAB）和在TBS中的0.015％的过氧化氢为8分钟的IBA1染色。 注:DAB发展的时机可能会有所不同的协议，以协议，应该由快速检查光显微镜下染色的切片来确定。 注意:对于IBA1，应该能够在20X清楚地显现IBA1染色小胶质细胞的细胞体和过程（参见图2为代表例）。请谨慎采用DAB，因为它是一种已知的致癌物质。 </li><li>避免由在此步骤中仔细监测部分（如上所述）过显影。通过洗涤段中冷冻PB 5次，每次5分钟，使反应停止。 </li></ol> 7.处理电子显微镜<ol><li>准备在玻璃小瓶中在PBS的1％四氧化锇溶液中。四氧化锇是感光;覆盖的G用铝箔小姑娘小瓶，以保护从光的解决方案。 要谨慎使用四氧化锇，因为它是一个非常危险的化学品。执行此并在通风橱内的以下步骤。 </li><li>从部分删除PBS和平板用细的画笔传播他们。在一个时间执行此步骤一个阱，以保持部分干燥。一定立即加入四氧化锇，如在章节任何褶皱将成为永久性的，试图弄平组织交锇固定只会破坏部之前变平的部分。 </li><li>沉浸在四氧化锇的部分在RT下30分钟后，在用移液管每次添加四氧化锇一滴，防止部分从折叠。用铝箔覆盖井，以保护部分从光。 注意:此步骤修复段内的脂类。该组织会出现锇后固定后非常黑暗。 </li><li>虽然部分是在锇tetro西德，通过将20 G成分中的A的一次性烧杯制备塑料树脂，将20g的组分B，0.6 G分量℃，和0.4g组分D.的分量组合在一起以上述顺序和用10ml的血清吸管以及它们混​​合直至得到均匀的颜色。 </li><li>准备好的混合物转移到铝称重菜肴。它们将接收到的组织切片一旦被脱水。 </li><li>脱水在增加的乙醇浓度为2分钟以下顺序的部分:35％，35％，50％，70％，80％，90％，100％，100％，100％。 </li><li>对于每次2分钟除去残留的乙醇，在环氧丙烷浸入第3次。除去从24孔培养板中的脱水区段20毫升玻璃小瓶中。与环氧丙烷时，它溶解塑料总是使用的玻璃小瓶，并保持谨慎，因为这是危险的。 </li><li>使用热弯玻璃枪头，或罚款的画笔传递由环氧丙烷soluti的章节在进入树脂和离开O / N渗透在室温。要小心，不要稀释环氧丙烷的树脂。 </li><li>嵌入与聚氯三氟乙烯（PCTFE）filmsheets树脂中的部分: <ol><li>放置装有样品的铝称重菜到50 - 60℃的烘箱中进行10 - 之前嵌入上PCTFE filmsheets切片15分钟。 </li><li>当嵌入部分，用一个铝在一次称量皿工作。用细画笔，涂料树脂的薄层到一个PCTFE膜片。 </li><li>动组织的一个部分在从铝称重盘的PCTFE膜片的时间。来自各地的组织去除多余的树脂，小心不要去打扰它。 </li><li>所有章节从一个移动称重盘到PCTFE薄膜片后，将第二PCTFE板在第一，创造组织的三明治和2片之间树脂英寸</li></ol></li><li>聚合在培养箱的树脂3天55 - 60℃。 </li><li>该材料现在准备超薄切片和超微结构检查，这往往是由EM核设施进行专门的技术。存储在RT安全PCTFE膜片之间的样品没有超微结构降解。 注意:超薄切片透射电子显微镜检查后续的步骤在单独的协议13进行说明。 </li></ol>

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E., Riad, M., Majewska, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+mouse+brain+tissue+for+immunoelectron+microscopy.">Preparation of mouse brain tissue for immunoelectron microscopy.</a> J Vis Exp. (41), (2010).</li><li>Konsman, J. -. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mouse+brain+in+stereotaxic+coordinates.">The mouse brain in stereotaxic coordinates.</a> Psychoneuroendocrinology. 28 (6), (2003).</li><li>Norden, D. M., Muccigrosso, M. M., Godbout, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglial+priming+and+enhanced+reactivity+to+secondary+insult+in+aging,+and+traumatic+CNS+injury,+and+neurodegenerative+disease.">Microglial priming and enhanced reactivity to secondary insult in aging, and traumatic CNS injury, and neurodegenerative disease.</a> Neuropharmacology. 96 ((Pt A)), 29-41 (2014).</li><li>Tremblay, M. -. E., Stevens, B., Sierra, A., Wake, H., Bessis, A., Nimmerjahn, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+microglia+in+the+healthy+brain.">The role of microglia in the healthy brain.</a> J. Neurosci. 31 (45), 16064-16069 (2011).</li><li>&#352;i&#353;kov&#225;, Z., Tremblay, M. &. #. 2. 3. 2. ;. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+and+synapse:+Interactions+in+health+and+neurodegeneration.">Microglia and synapse: Interactions in health and neurodegeneration.</a> Neural Plast. 2013, 425845 (2013).</li><li>DeKosky, S. T., Scheff, S. W., Styren, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+correlates+of+cognition+in+dementia:+quantification+and+assessment+of+synapse+change.">Structural correlates of cognition in dementia: quantification and assessment of synapse change.</a> Neurodegeneration. 5 (4), 417-421 (1996).</li><li>Terry, R. D., Masliah, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+basis+of+cognitive+alterations+in+Alzheimer's+disease:+Synapse+loss+is+the+major+correlate+of+cognitive+impairment.">Physical basis of cognitive alterations in Alzheimer's disease: Synapse loss is the major correlate of cognitive impairment.</a> Ann. Neurol. 30 (4), 572-580 (1991).</li></ol>

The authors have nothing to disclose.

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...

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。

<table><tbody><tr><td>Methoxy-X04</td><td>Tocris Bioscience</td><td>4920</td><td>10 mg substrate per tablet</td></tr><tr><td>Propylene glycol</td><td>Sigma Aldrich</td><td>W294004&amp;nbsp;</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Fisher BioReagents</td><td>BP231-1</td><td>Caution: toxic</td></tr><tr><td>Sodium Chloride NaCl</td><td>Sigma</td><td>S9625</td><td></td></tr><tr><td>Sodium phosphate monobasic monohydrate</td><td>Sigma</td><td>S9638</td><td></td></tr><tr><td>Sodium phosphate dibasic</td><td>Sigma</td><td>S0876</td><td></td></tr><tr><td>Tris Hydrochloride</td><td>Fisher BioReagents</td><td>BP153500</td><td></td></tr><tr><td>Acrolein</td><td>Sigma</td><td>110221</td><td>Caution: Toxic</td></tr><tr><td>Paraformaldehyde Granular</td><td>Electron Microscopy Sciences</td><td>19210</td><td>Caution: Toxic</td></tr><tr><td>Filter paper</td><td>Fisher</td><td>09-790-14F</td><td></td></tr><tr><td>Peristaltic Pump with Tubing</td><td>ColeParmer</td><td>cp.78023-00</td><td></td></tr><tr><td>Excel Winged blood Collection Set Needles 25&amp;nbsp;G</td><td>Becton Dickinson</td><td>367341</td><td></td></tr><tr><td>Extrafine Forceps</td><td>F.S.T</td><td>11152-10</td><td>Tip shape: curved</td></tr><tr><td>Scissors</td><td>F.S.T</td><td>14090-09</td><td>Tip shape: straight</td></tr><tr><td>Hartman Hemostats</td><td>F.S.T</td><td>13003-10</td><td>Tip shape: curved</td></tr><tr><td>Surgical Scissors</td><td>F.S.T</td><td>14004-16</td><td>Tip shape: straight</td></tr><tr><td>Micro Dissecting Scissors</td><td>ROBOZ surgical store</td><td>5818</td><td></td></tr><tr><td>Glass scintillation vials</td><td>Fisher Scientific</td><td>74515-20</td><td></td></tr><tr><td>Vibrating Blade Microtome Leica VT1000 S</td><td>Leica Biosystems&amp;nbsp;</td><td>14047235612</td><td></td></tr><tr><td>Vibratome blades</td><td>Electron microscopy Sciences</td><td>71990</td><td></td></tr><tr><td>Microscope Slides</td><td>Fisher Scientific</td><td>12-550-15</td><td></td></tr><tr><td>24-well Tissue Culture Plates</td><td>Fisher Scientific</td><td>353047</td><td></td></tr><tr><td>Ethylene Glycol</td><td>Fisher BioReagents</td><td>BP230-4</td><td></td></tr><tr><td>Glycerol</td><td>Fisher BioReagents</td><td>BP229-4</td><td></td></tr><tr><td>Hydrogen Peroxide, 30%</td><td>J.T.BAKER</td><td>cat: 2186-01</td><td></td></tr><tr><td>Sodium borohydride</td><td>Sigma</td><td>480886</td><td></td></tr><tr><td>Tris HCl</td><td>Fisher</td><td>BP153-500ML</td><td></td></tr><tr><td>Fetal bovine Serum (FBS)</td><td>Sigma Aldrich</td><td>F1051</td><td></td></tr><tr><td>Bovine serum albumin (BSA), fraction V</td><td>Thomas Scientific</td><td>C001H24</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T8787</td><td></td></tr><tr><td>Anti IBA1, Rabbit&amp;nbsp;</td><td>WAKO</td><td>019-19741</td><td></td></tr><tr><td>Goat Anti-Rabbit IgG</td><td>Jackson Immunoresearch</td><td>111066046</td><td></td></tr><tr><td>VECTASTAIN Elite ABC Kit (Standard)</td><td>Vector Labs</td><td>PK-6100</td><td></td></tr><tr><td>3.3&#039;-Diaminobenzidine tetra-hydrochloride (DAB)</td><td>Sigma</td><td>D5905-50TAB</td><td>Caution: toxic</td></tr><tr><td>Osmium tetroxide, 4% solution</td><td>Electron Microscopy Sciences</td><td>19150</td><td>Caution: toxic</td></tr><tr><td>Durcupan&amp;trade; ACM single component A</td><td>Sigma</td><td>44611</td><td>Resin Caution: Toxic</td></tr><tr><td>Durcupan&amp;trade; ACM single component B</td><td>Sigma</td><td>44612</td><td>Hardener Caution: Toxic</td></tr><tr><td>Durcupan&amp;trade; ACM single component C</td><td>Sigma</td><td>44613</td><td>Plasticizer Caution: Toxic</td></tr><tr><td>Durcupan&amp;trade; ACM single component D</td><td>Sigma</td><td>44614</td><td>Accelerator Caution: Toxic</td></tr><tr><td>Ethanol</td><td>LesAlcoolsdeComerce</td><td>151-01-15N</td><td></td></tr><tr><td>Propylene oxide</td><td>Sigma</td><td>110205</td><td>Caution: corrosive</td></tr><tr><td>Aluminum weigh dishes</td><td>Electron Microscopy Sciences</td><td>70048-01</td><td></td></tr><tr><td>ACLAR&amp;reg;&amp;ndash;Fluoropolymer Films</td><td>Electron Microscopy Sciences</td><td>50425</td><td></td></tr><tr><td>Oven/Incubator</td><td>VWR</td><td></td><td></td></tr></tbody></table>

correlative light and electron microscopy to study microglial interactions with &#946;-amyloid plaques

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。

此部分示出了可以在协议的不同的关键步骤得到的结果。特别是，结果显示含有在特定区域甲氧基X04染色斑块和感兴趣的层的脑切片的例子:海马CA1区，地层radiatum和腔隙-moleculare。的斑块和海马区域/层状组织使用UV和亮场滤波器（ 图1）的组合依次显示。所选的脑切片随后免疫染色并处理的EM同时跟踪其的Aβ斑块的，考虑到它们仍然荧光以下免疫染色，并在与?...

Watch this Scientific Journal Video about Correlative Light and Electron Microscopy to Study Microglial Interactions with Î²-Amyloid Plaques at JoVE.com

Correlative Light and Electron Microscopy to Study Microglial Interactions with &#946;-Amyloid Plaques

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。

A detailed protocol is provided here to identify amyloid A&#946; plaques in brain sections from Alzheimer&#39;s disease mouse models before pre-embedding ...

correlative-light-electron-microscopy-to-study-microglial

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

11.3K 次观看.  CHU de Québec Research Center. 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。 

视频: Correlative Light and Electron Microscopy to Study Microglial Interactions with β-Amyloid Plaques

Imaging Amyloid Tissues Stained with Luminescent Conjugated Oligothiophenes by Hyperspectral Confocal Microscopy and Fluorescence Lifetime Imaging

Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find neurodegenerative diseases such as Alzheimer&#39;s and Parkinson&#39;s disease afflicting primarily the central nervous system, and systemic amyloidosis where serum amyloid A, transthyretin and IgG light chains deposit as amyloid in liver, carpal tunnel, spleen, kidney, heart, and other peripheral tissues. Amyloid has been known and studied for more than a century, often using amyloid specific dyes such as Congo red and Thioflavin T (ThT) or Thioflavin (ThS). In this paper, we present heptamer-formyl thiophene acetic acid (hFTAA) as an example of recently developed complements to these dyes called luminescent conjugated oligothiophenes (LCOs). hFTAA is easy to use and is compatible with co-staining in immunofluorescence or with other cellular markers. Extensive research has proven that hFTAA detects a wider range of disease associated protein aggregates than conventional amyloid dyes. In addition, hFTAA can also be applied for optical assignment of distinct aggregated morphotypes to allow studies of amyloid fibril polymorphism. While the imaging methodology applied is optional, we here demonstrate hyperspectral imaging (HIS), laser scanning confocal microscopy and fluorescence lifetime imaging (FLIM). These examples show some of the imaging techniques where LCOs can be used as tools to gain more detailed knowledge of the formation and structural properties of amyloids. An important limitation to the technique is, as for all conventional optical microscopy techniques, the requirement for microscopic size of aggregates to allow detection. Furthermore, the aggregate should comprise a repetitive &#946;-sheet structure to allow for hFTAA binding. Excessive fixation and/or epitope exposure that modify the aggregate structure or conformation can render poor hFTAA binding and hence pose limitations to accurate imaging.

Amyloid deposition is a hallmark of different diseases and afflicts many different organs. This paper describes the application of luminescent conjugated oligothiophene fluorescence staining in combination with fluorescence microscopy techniques. This staining method represents a powerful tool for detection and exploration of protein aggregates in both clinical and scientific setups.

用高光谱共聚焦显微镜和荧光寿命成像对发光共轭吩染色的淀粉样组织的成像

Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find ...

科研

生物化学

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, direct visualization of individual viral complexes in their host cellular environment with cryoET is challenging2, due to the infrequent and dynamic nature of viral entry, particularly in the case of HIV-1. While time-lapse live-cell imaging has yielded a great deal of information about many aspects of the life cycle of HIV-13-7, the resolution afforded by live-cell microscopy is limited (~ 200 nm). Our work was aimed at developing a correlation method that permits direct visualization of early events of HIV-1 infection by combining live-cell fluorescent light microscopy, cryo-fluorescent microscopy, and cryoET. In this manner, live-cell and cryo-fluorescent signals can be used to accurately guide the sampling in cryoET. Furthermore, structural information obtained from cryoET can be complemented with the dynamic functional data gained through live-cell imaging of fluorescent labeled target.
In this video article, we provide detailed methods and protocols for structural investigation of HIV-1 and host-cell interactions using 3D correlative high-speed live-cell imaging and high-resolution cryoET structural analysis. HeLa cells infected with HIV-1 particles were characterized first by confocal live-cell microscopy, and the region containing the same viral particle was then analyzed by cryo-electron tomography for 3D structural details. The correlation between two sets of imaging data, optical imaging and electron imaging, was achieved using a home-built cryo-fluorescence light microscopy stage. The approach detailed here will be valuable, not only for study of virus-host cell interactions, but also for broader applications in cell biology, such as cell signaling, membrane receptor trafficking, and many other dynamic cellular processes.

We describe a correlative microscopy method that combines high-speed 3D live-cell fluorescent light microscopy and high-resolution cryo-electron tomography. We demonstrate the capability of the correlative method by imaging dynamic, small HIV-1 particles interacting with host HeLa cells.

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, ...

生物工程

Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using widefield fluorescence light microscopy and transmission electron microscopy (TEM). To demonstrate the protocol we have immunolabeled ultrathin epoxy sections of human somatostatinoma tumor using a primary antibody to somatostatin, followed by a biotinylated secondary antibody and visualization with streptavidin conjugated 585 nm cadmium-selenium (CdSe) quantum dots (QDs). The sections are mounted on a TEM specimen grid then placed on a glass slide for observation by widefield fluorescence light microscopy. Light microscopy reveals 585 nm QD labeling as bright orange fluorescence forming a granular pattern within the tumor cell cytoplasm. At low to mid-range magnification by light microscopy the labeling pattern can be easily recognized and the level of non-specific or background labeling assessed. This is a critical step for subsequent interpretation of the immunolabeling pattern by TEM and evaluation of the morphological context. The same section is then blotted dry and viewed by TEM. QD probes are seen to be attached to amorphous material contained in individual secretory granules. Images are acquired from the same region of interest (ROI) seen by light microscopy for correlative analysis. Corresponding images from each modality may then be blended to overlay fluorescence data on TEM ultrastructure of the corresponding region.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of epoxy embedded human pathology tissue. We employ commercial antibody fragment conjugated QDs that are visualized by widefield fluorescence light microscopy and transmission electron microscopy.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using ...

Quantitative 3D In Silico Modeling (q3DISM) of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer's Disease

Neuroinflammation is now recognized as a major etiological factor in neurodegenerative disease. Mononuclear phagocytes are innate immune cells responsible for phagocytosis and clearance of debris and detritus. These cells include CNS-resident macrophages known as microglia, and mononuclear phagocytes infiltrating from the periphery. Light microscopy has generally been used to visualize phagocytosis in rodent or human brain specimens. However, qualitative methods have not provided definitive evidence of in vivo phagocytosis. Here, we describe quantitative 3D in silico modeling (q3DISM), a robust method allowing for true 3D quantitation of amyloid-&#946; (A&#946;) phagocytosis by mononuclear phagocytes in rodent Alzheimer&#39;s Disease (AD) models. The method involves fluorescently visualizing A&#946; encapsulated within phagolysosomes in rodent brain sections. Large z-dimensional confocal datasets are then 3D reconstructed for quantitation of A&#946; spatially colocalized within the phagolysosome. We demonstrate the successful application of q3DISM to mouse and rat brains, but this methodology can be extended to virtually any phagocytic event in any tissue.

Quantitative 3D In Silico Modeling q3DISM of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer's Disease

We developed a methodology for quantitative 3D in silico modeling (q3DISM) of cerebral amyloid-&#946; (A&#946;) phagocytosis by mononuclear phagocytes in rodent models of Alzheimer&#39;s disease. This method can be generalized for the quantitation of virtually any phagocytic event in vivo.

定量3D<em&gt;在硅片</em&gt;建模阿尔茨海默病的啮齿动物模型脑淀粉样蛋白-β吞噬（q3DISM）

Neuroinflammation is now recognized as a major etiological factor in neurodegenerative disease. Mononuclear phagocytes are innate immune cells responsible ...

医学

Visualizing Axonal Growth Cone Collapse and Early Amyloid &#946; Effects in Cultured Mouse Neurons

Amyloid-&#946; (A&#946;) causes memory impairments in Alzheimer's disease (AD). Although therapeutics have been shown to reduce A&#946; levels in the brains of AD patients, these do not improve memory functions. Since A&#946; aggregates in the brain before the appearance of memory impairments, targeting A&#946; may be inefficient for treating AD patients who already exhibit memory deficits. Therefore, downstream signaling due to A&#946; deposition should be blocked before AD development. A&#946; induces axonal degeneration, leading to the disruption of neuronal networks and memory impairments. Although there are many studies on the mechanisms of A&#946; toxicity, the source of A&#946; toxicity remains unknown. To help identify the source, we propose a novel protocol that uses microscopy, gene transfection, and live cell imaging to investigate early changes caused by A&#946; in axonal growth cones of cultured neurons. This protocol revealed that A&#946; induced clathrin-mediated endocytosis in axonal growth cones followed by growth cone collapse, demonstrating that inhibition of endocytosis prevents A&#946; toxicity. This protocol will be useful in studying the early effects of A&#946; and may lead to more efficient and preventative AD treatment.

Here a protocol to investigate the early effects of amyloid-&#946; (A&#946;) in the brain is presented. This shows that A&#946; induces clathrin-mediated endocytosis and collapse of axonal growth cones. The protocol is useful in studying early effects of A&#946; on axonal growth cones and may facilitate prevention of Alzheimer's disease.

在培养的小鼠神经元中可视化轴突生长锥塌陷和早期淀粉样β效应

Amyloid-&#946; (A&#946;) causes memory impairments in Alzheimer's disease (AD). Although therapeutics have been shown to reduce A&#946; levels in the ...

神经科学

Immunofluorescence Staining Using IBA1 and TMEM119 for Microglial Density, Morphology and Peripheral Myeloid Cell Infiltration Analysis in Mouse Brain

This is a protocol for the dual visualization of microglia and infiltrating macrophages in mouse brain tissue. TMEM119 (which labels microglia selectively), when combined with IBA1 (which provides an exceptional visualization of their morphology), allows investigation of changes in density, distribution, and morphology. Quantifying these parameters is important in providing insights into the roles exerted by microglia, the resident macrophages of the brain. Under normal physiological conditions, microglia are regularly distributed in a mosaic-like pattern and present a small soma with ramified processes. Nevertheless, as a response to environmental factors (i.e., trauma, infection, disease, or injury), microglial density, distribution, and morphology are altered in various manners, depending on the insult. Additionally, the described double-staining method allows visualization of infiltrating macrophages in the brain based on their expression of IBA1 and without colocalization with TMEM119. This approach thus allows discrimination between microglia and infiltrating macrophages, which is required to provide functional insights into their distinct involvement in brain homeostasis across various contexts of health and disease. This protocol integrates the latest findings in neuroimmunology that pertain to the identification of selective markers. It also serves as a useful tool for both experienced neuroimmunologists and researchers seeking to integrate neuroimmunology into projects.

This protocol describes a step-by-step workflow for immunofluorescent costaining of IBA1 and TMEM119, in addition to analysis of microglial density, distribution, and morphology, as well as peripheral myeloid cell infiltration in mouse brain tissue.

使用IBA1和TMEM119进行小鼠脑微胶质密度、形态学和周围骨髓细胞渗透分析的免疫荧光染色

This is a protocol for the dual visualization of microglia and infiltrating macrophages in mouse brain tissue. TMEM119 (which labels microglia ...

免疫与感染

Detecting Amyloid-&#946; Accumulation via Immunofluorescent Staining in a Mouse Model of Alzheimer's Disease

Alzheimer's disease (AD) is a neurodegenerative disease that contributes to 60-70% dementia around the world. One of the hallmarks of AD undoubtedly lies on accumulation of amyloid-&#946; (A&#946;) in the brain. A&#946; is produced from the proteolytic cleavage of the beta-amyloid precursor protein (APP) by &#946;-secretase and &#947;-secretase. In pathological circumstances, the increased &#946;-cleavage of APP leads to overproduction of A&#946;, which aggregates into A&#946; plaques. Since A&#946; plaques are a characteristic of AD pathology, detecting the amount of A&#946; is very important in AD research. In this protocol, we introduce the immunofluorescent staining method to visualize A&#946; deposition. The mouse model used in our experiments is 5&#215;FAD, which carries five mutations found in human familial AD. The neuropathological and behavioral deficits of 5xFAD mice are well-documented, which makes it a good animal model to study A&#946; pathology. We will introduce the procedure including transcardial perfusion, cryosectioning, immunofluorescent staining and quantification to detect A&#946; accumulation in 5&#215;FAD mice. With this protocol, researchers can investigate A&#946; pathology in an AD mouse model.

In the neuropathology of Alzheimer's disease, one of the most crucial characteristics is the deposition of amyloid-&#946;. In this protocol, we describe the method of immunofluorescent staining in 5&#215;FAD transgenic mouse to detect amyloid-&#946; accumulation in plaques. The process of perfusion, cryosectioning, staining and quantification will be described in detail.

Alzheimer's disease (AD) is a neurodegenerative disease that contributes to 60-70% dementia around the world. One of the hallmarks of AD undoubtedly lies ...

Spatiotemporal Analysis of Microglial Ca2+ Activity at Single-Cell Resolution

Microglia are the sole resident immune cells in the central nervous system. Their morphology is highly plastic, changing depending on their activity. Under homeostatic conditions, microglia possess a highly ramified morphology. This facilitates their monitoring of the surrounding environment through the continuous extending and retracting of their processes. During brain injury and inflammation, however, microglia become activated and undergo dramatic morphological changes, retracting their ramified processes and swelling their cell body. This facilitates activities such as migration and phagocytosis, which microglia undertake to navigate the brain environment to a less pathological state.
This close relationship between microglial morphology and changes in their activity have enabled considerable insights into various microglial functions. However, such morphological and activity changes are themselves phenomena that can result from any number of intracellular signaling pathways. Moreover, the time-lag between stimulus and response, as well as the highly compartmentalized morphology of microglia, make it difficult to isolate the causative mechanisms that underpin function. To solve this problem, we developed a genetically modified mouse line in which a highly sensitive fluorescent Ca2+-indicator protein is specifically expressed in microglia.
After describing methods for in vivo microglial Ca2+ imaging, this paper presents a structured analysis approach that classifies this Ca2+ activity to rationally defined subcellular regions, thus ensuring that the spatial and temporal dimensions of the encoded information are meaningfully extracted. We believe that this approach will provide a detailed understanding of the intracellular signaling rules that govern the diverse array of microglial activities associated with both higher brain functions and pathological conditions.

Spatiotemporal Analysis of Microglial Ca2+ Activity at Single-Cell Resolution

In this paper, we describe a protocol for in vivo imaging of microglial Ca2+ activity and subsequent analysis of its spatiotemporal dynamics. This method enables thorough characterization of how microglia respond to changes in the brain environment, appropriately capturing the fine spatiotemporal scales at which such events occur.

Microglia are the sole resident immune cells in the central nervous system. Their morphology is highly plastic, changing depending on their activity. ...

Two-Photon Microscopy for Studying Microglial Process Attraction Toward a Compound in a Mouse Brain Slice

<a href='https://appjove.unicartagenaproxy.elogim.com/v/58788/two-photon-imaging-microglial-processes-attraction-toward-atp-or'>Source: Etienne, F. et al. Two-photon Imaging of Microglial Processes' Attraction Toward ATP or Serotonin in Acute Brain Slices.&#160;J. Vis. Exp. (2019)</a>This video demonstrates the use of two-photon microscopy to study the attraction of microglial processes toward an injected compound in brain slices. It provides an overview of preparing mouse brain slices, injecting a chemoattractant test compound, and imaging fluorescently labeled microglia to visualize their responses.

Source: Etienne, F. et al. Two-photon Imaging of Microglial Processes' Attraction Toward ATP or Serotonin in Acute Brain Slices.&#160;J. Vis. Exp. ...

JoVE Encyclopedia of Experiments

Neuroimaging

Live Imaging & In Vivo Tracking

Analyzing Amyloid Structures in a Tissue Section Using Fluorescence Lifetime Imaging Microscopy

On the day of staining, immerse the sections in consecutive baths of 99% ethanol, 70% ethanol, dH_2O, and PBS for 10 minutes each time, then allow the tissue sections to dry under ambient conditions. While the tissue dries, prepare a working solution of HFTAA by diluting the stock 1 to 10,000 in PBS. When the tissue is dry, add droplets of the HFTAA working solution to each tissue section to cover it. Incubate for 30 minutes at room temperature.Rinse off the staining solution with 500 microliters of PBS, and then immerse the slide in the PBS bath for 10 minutes. After allowing the section to dry under ambient conditions, mount using fluorescence mounting medium. Allow the mounting medium to settle overnight.Amyloid detection can be performed directly after mounting or even without mounting. However, if the goal of the experiment is to collect high-quality spectral information, overnight incubation is preferred.Switch the microscope to FLIM mode, set the pinhole to 20, the excitation wavelength to 490 nanometers, and the laser intensity to 0.5%. Use pulsed lasers at 40 megahertz. In the FLIM software, set up photon counting over 550 nanometers. In the Display Parameters window, follow the photon counting until the max count is around 4,000 photon counts. Save the file and export it as SPC image.

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

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用高光谱共聚焦显微镜和荧光寿命成像对发光共轭吩染色的淀粉样组织的成像

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定量3D

在培养的小鼠神经元中可视化轴突生长锥塌陷和早期淀粉样β效应

使用IBA1和TMEM119进行小鼠脑微胶质密度、形态学和周围骨髓细胞渗透分析的免疫荧光染色

Detecting Amyloid-β Accumulation via Immunofluorescent Staining in a Mouse Model of Alzheimer's Disease

Spatiotemporal Analysis of Microglial Ca²⁺ Activity at Single-Cell Resolution

Two-Photon Microscopy for Studying Microglial Process Attraction Toward a Compound in a Mouse Brain Slice

Analyzing Amyloid Structures in a Tissue Section Using Fluorescence Lifetime Imaging Microscopy