我们感谢整个Koshy实验室进行有益的讨论。我们感谢侯佩岑Jansma和亚利桑那州神经科学系的大学征求意见，并与成像帮助。我们也感谢波雷卡实验室使用他们的vibratome的。这项研究是由健康的美国国家研究院​​（NIH NS065116，AAK）的支持。

注:小鼠饲养，并保持在一个温度和湿度控制的房间12小时逆转食物和水可用自由采食光/暗周期在亚利桑那大学。实验下的指引和亚利桑那大学的机构动物护理和使用委员会批准进行的。所作的所有努力，尽量减少痛苦。该Cre的报告小鼠是在C57BL / 6背景11和可商购。 1.鼠标感染注:小鼠感染弓形虫下面所描述的方法已被用于在先前公布的10项研究- 12。 <ol style="margin-left: 40px;"><li>生长弓形虫菌株在人包皮成纤维细胞（HFFS）的Dulbecco氏高糖培养的改良的Eagles培养基（DMEM），补充有10％FBS，100U / ml青霉素，100mg / ml的链霉霉素，和2mM L-谷氨酰胺（cDMEM）的T-25烧瓶内的5％的CO 2培养箱中，直到寄生虫已形成大虫空泡。 </li><li>通过从烧瓶底部刮的细胞和使所得到的溶液通过一个25号针头连接到3毫升注射器的2倍的烧瓶内，则所有溶液转移至5ml注射器壳体连接到27号针头的注射器释放寄生虫已被置于一个15毫升的锥形管内上下颠倒。柱塞连接到注射器，并通过该寄生虫溶液进入锥形管。 </li><li>离心管在300×g下10分钟。吸出上清液，然后重悬沉淀在4-6毫升无菌USP级1×磷酸盐缓冲盐水（PBS）pH 7.4的（原液）。 </li><li>装入10微升原液血球，等待5-10分钟的寄生虫定居然后计算寄生虫的数目，中间大正方形的四角和中心方块。 </li><li>第稀网站的100K / 200微升合适的接种物大小的1X PBS，然后用200微升的总体积注射小鼠腹膜内（IP）。 注:在此过程中，小鼠接种100K型III（CEP）13速殖子200μl的1X PBS。当感染小鼠弓形虫的其它菌株，接种体浓度可能需要进行调整以考虑毒力的水平。 </li></ol> 2.灌注和脑收获注意:此协议是第21天感染后（dpi）的进行，因为这代表了时间点被发现在小鼠大脑囊肿峰的数字，但也可以使用其它的时间点。大小，数量，位置，以及存在包囊的存在与否，取决于很多因素，包括小鼠品系，寄生虫菌株类型以及接种量7,14 - 17。研究者将需要单独确定合适的接种物为鼠标和Toxoplas马应变，他/她使用。 <ol style="margin-left: 40px;"><li>设置所有手术收获的工具（ 图1）。 </li><li>对于每个小鼠，制备并保持在冰上:20ml注射器填充有0.9％的氯化钠（NaCl）中+ 10单位/毫升肝素在1×无菌USP级的PBS（肝素/盐水）; 20毫升注射器填充有4％多聚甲醛（PFA）;闪烁小瓶用15毫升的4％的PFA。对于多个小鼠，制备填充有所需的所有小鼠，然后制定一个新鲜注射器充满每个灌注溶液各溶​​液的全部量的烧杯中。 </li><li>麻醉小鼠IP用200μl/ 25克体重的氯胺酮/甲苯​​噻嗪混合物（24毫克/毫升与4.8毫克/毫升，分别）在感兴趣的日子。通过检查脚趾捏反射监视鼠标麻醉水平。继续协议只有一次鼠标显示了一个坚定的脚趾捏没有反应。 注:如之前达到麻醉的适当水平的麻醉的其它方法也可以使用，只要在继续进行协议。 </li><li>鼠标放置在有盖的泡沫平台上仰卧位。 PIN所有四肢出在脚上，然后喷洒胸部和腹部用70％乙醇（乙醇）（ 图2A）。另外的鼠标下几个无菌纸巾可以用来帮助吸收灌注流体的溢出。 </li><li>正下方用钳子胸骨抬起皮肤，然后用剪刀做一个切口。拉回来的皮肤暴露胸部（ 图2B）。 </li><li>提起剑突，使刚刚膈下腹部切口，暴露出这是钝性分离离膈肌肝脏。使两个切口，一个在左，一个在通过隔膜和右肋的底部。延伸的左，右的切口大约1 / 2-2 / 3的方式向上胸腔，然后将其反射回以打开胸腔（ 图2C）。 注意:小心不要削减任何器官或主要血管杜响此过程，因为这样做会损害灌注质量。 </li><li>用10-20毫升肝素/盐其次是10-20毫升4％PFA灌注transcardially。 注意:如果灌注得当，肝脏会从红色变成棕褐色（ 图2D）。如果没有正确完成，血管将可见（ 图2E）。 </li><li>把鼠标放在其上腹部，重脚脚再喷头部和颈部用70％乙醇。从颈部的底座抬起皮肤，使一个切口。去除皮肤暴露的头骨（ 图2F）。斩首头在肩的水平。取出颅骨，轻轻取出大脑，并放入闪烁瓶中装满4％PFA（ 图2G-H）。 注意:如果灌注良好，大脑会出现白色无可见的血管（ 图2I）。如果大脑没有得到很好的灌注，血管就会可见（ 图2J）。 </li><li>将火花化小瓶大脑在4％PFA在4℃过夜，涵盖了从光。冲洗脑部非USP级1×PBS中，并转移到一个新的闪烁小瓶填充有冷却的1×PBS。存储大脑在1×PBS中，在4℃，涵盖了从光。 注:成功清算和成像已经完成与存储在1×PBS中进行长达一个月的部分，但最好是部分在几天之内为长时间储存​​在PBS将扭转PFA固定和大脑可能导致低于最佳的图像。自体荧光和增加的模糊已注意到在一些路段储存后成像在1×PBS中一个月或更长的时间。 </li></ol> 3.脑切片<ol style="margin-left: 40px;"><li>从1 PBS取出大脑，并把它变成一个大脑基质（ 图3A），以便更精确，通过大脑甚至削减。剪掉小脑和嗅球，如果存在的话，然后切成冠脑部成2个或3段。 <b转/&gt;注:切边和脑切割可以不经大脑基质的帮助完成。正在使用的切割冠将取决于的vibratome的切割距离段数。大脑可以分段在任何方向，虽然矢状面和冠状节最常用于神经解剖鉴定。 </li><li>贴上各脑切片上与氰基丙烯酸酯样品安装块。加盖的背后支持的脑切片（ 图3B）的5％琼脂糖凝胶的小块。 注:其它支持机制都可以使用，但不提供支持，所述的vibratome可推动对脑造成不均匀的切片。 </li><li>切割组织成厚的切片用的vibratome设置为4的速度和9振幅为160μm（用于成像物镜的工作距离）。 注:在速度和幅度的设置可以根据特定的机器上进行调整所使用，以获得均匀，平滑的部分。如果设置不正确，结果可能是不均匀的部分，组织或振痕撕裂。 </li><li>收集组织切片成闪烁小瓶填充有新鲜，冷冻的1×PBS，如果他们打算一个星期内，以被清除。如果段不打算在一周内被清除，放置部分成1.5ml微量管中填充有在-20℃冷冻保存液溶液和存储。 </li></ol>脑组织切片4.光学结算注:此协议已被修改从先前公布的方法8。 <ol style="margin-left: 40px;"><li>在1×PBS + 2％吐温-20（GL / PBST）中制备25％，50％，75％和90％体积/体积甘油。 </li><li>从1×PBS中移除部分，或者，如果在冷冻保存液，冲洗在1×PBS中并转移到含有10毫升25％的GL / PBST闪烁瓶中。地方小瓶在振荡器上在4℃下，盖从暴露于光，12小时。 </li><li>确认所有部分已经沉没到微博TTOM小瓶。吸出25％的GL / PBST中，只留下足以覆盖部分然后填充在振荡器上在小瓶用10毫升50％的GL / PBST和地点在4℃下，盖从暴露于光，12小时。对于75％GL / PBST那么90％GL / PBST重复此过程。留在90％GL / PBST路段24小时达到最高结算。在结算过程中的过程中，逐步观察光透明（ 图4）。 注意:如果解决方案更改后立即路段已经沉没到小瓶的底部这个过程可能会加快。在75％与90％的溶液，部分不会下沉一路小瓶的底部，但将在中间浮动。 </li></ol> 5.安装脑组织切片<ol style="margin-left: 40px;"><li>切1.5号盖玻片到5个创建间隔。用尺子和金刚石尖铅笔得分和打破盖玻片沿图5A中所示的虚线。丢弃吨他围绕一块，安排4件外的一个普通的载玻片，以创建一个窗口，然后刷透明指甲油到接缝坚持间隔的碎片在正确的配置（ 图5B）的幻灯片。 注:该垫片将被用于创建载玻片和盖玻片用于安装清除部分之间的必要的空间。盖玻片可被切割以不同的方式来创建所需的任何尺寸的窗口。预切割泡沫衬垫也可商购。为了让指甲油完全干燥，最好是安装部分，至少提前一天进行幻灯片与垫片。 </li><li>使用具有尖端的塑料移液管转移到节幻灯片切断。请务必填写周边地区有90％GL / PBST。小心地放下盖玻片在确保不引入任何气泡的部分。多余的GL / PBST可吸走了无绒擦拭。 </li><li>图像GFP +含RFP元+ <em&gt;弓形虫包囊用共聚焦显微镜立即（ 图7A-B），然后存储幻灯片平坦和覆盖从光在4℃。 注意:可替换地，密封滑动完全围绕所有边缘来存储和成像在以后的时间。使用的透明指甲油密封滑动是可选的，并且可以使之更难以除去盖玻片如果一个人以除去从滑动部分在稍后的日期，以便进一步处理。 </li></ol> 6.成像注意:任何显微镜，可以使用具有获得高解析度的z栈的能力。 <ol style="margin-left: 40px;"><li>在最初发现的焦平面的10倍，变为20倍的目标，并通过部分扫描识别位于GFP +细胞内囊肿。在视场中心的感兴趣区域（ROI）的区域。 </li><li>更改为40X的目标。注重上下贯通整个投资回报率，以确保整个细胞和CYST是可见的。 注:图片均使用共焦显微镜用40X / 1.30石油DIC M27目标获得。 </li><li>使用安装的成像软件，得到Z堆叠包括与囊肿整个单元。用于获取图像的平均设置示于图6中 。 注:设置可能需要根据每个研究者的组织切片和显微镜的能力进行调整。 </li><li>由突起的数目设置为1。由突起的数目设定为64获得完整的旋转视图获取Z堆叠的最大投影图像。 注:其它软件包可用，并且可以被用于获得最大突出，旋转以及Z堆叠图像的其它视图。 </li><li>分析图像与图像分析软件。 </li></ol>

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</ol>

作者什么都没有透露。

由于在感染宿主细胞的细胞变化都与疾病结果在感染细胞内的其他生物如HIV，狂犬病，和衣原体18,19，我们开发出一种技术，使我们研究CNS之间发生亲密互动宿主细胞和弓形虫 。这里介绍的方法实现这一目标，通过使慢性感染神经细胞的有效成像。在此之前的方法的发展，这样的成像是耗时的，昂贵的，或者是不可能的。 现在，我们可以使用这种技术来回答有关弓形虫 -neuron互动的基本问题，比如是由寄生虫针对特定的神经元的地方？是否弓形虫株之间的这种差异？做在其中驻留囊肿从细胞的其余部分不同（ 即不感染，并且从相同的神经元未感染的树突有棘相同数量的蜂窝区）？什么是被感染的神经元共享蜂窝特点？回答这些问题的能力是必不可少的理解如何弓形体感染改变中枢神经系统，包括在行为水平。 以获得最佳的图像成为可能，这是非常重要的，以确保组织被切割成连部分。如果部分是不均匀的，盖玻片不正确地导致与导致亚最佳成像的目标不均匀接触的部分坐。在这个方案的最优化，一些改动做了。原来的结算过程中重现称为SeeDB 20的技术显示的结果，企图用果糖做的。这种方法是用这种模式不兼容，因为它淬火RFP（特别mCherry）。另一个关键的一步，可能是最重要的是，允许组织切片最大结算。如果没有正确清零，背景光衍射会更高和图像噪点。我们发现，加入吐温20至甘油给予了比单独甘油稍微清晰的图像。目前的技术允许我们通过160μm厚的脑组织切片，以获得Z堆栈的图像，但在未来，我们希望增加这个厚度500微米以上，以便更深入的分析。 只有通过观察整个小区及其连接，我们就可以开始开发大脑如何在全球范围受到弓形虫一种机械的想法。这个新的协议提供了研究弓形虫 -neuron相互作用在单细胞水平和这样做以快速，经济的方式提供了机会。我们还没有探讨所有的潜在用途的这个协议，以及它如何可能转化到其他系统，但我们预计将有多个应用程序。

A correction was made to <a href="http://www.jove.com/video/52237">"3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii"</a>. The ordering of the authors was inverted. The order has been updated from:
 Anita A. Koshy, Carla M. Cabral
to:
Carla M. Cabral, Anita A. Koshy

A correction was made to <a href="http://www.jove.com/video/52237">"3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii"</a>.

Erratum: 3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii

该方法的总的目标是要获得高的分辨率，被感染的专性细胞内寄生虫弓形虫单个神经元的三维图像。 弓形体通常被认为是由于其较大的中间宿主范围，包括人类和啮齿类动物中最成功的寄生虫之一。在人类和啮齿类动物，通过污染的食物或水摄入的急性感染后， 弓形虫能够通过从它的快速复制的形式（将速殖子）转化成其慢复制和encysting形式引起的中枢神经系统的持续性感染（在缓殖子）。在免疫活性个体，这种潜中枢神经系统感染被认为是相对无症状的，但在免疫受损的个体，如爱滋病患者或移植者，寄生虫的复发可导致致命弓形虫脑炎1,2。此外，最近的研究公顷已经表明，隐性感染弓形虫会导致行为的变化在啮齿类动物3,4，尽管机制仍不清楚。 令人惊讶的是，尽管这些数据突出了CNS- 弓形虫相互作用的重要性，相对知之甚少这种关系，特别是在细胞和分子水平。研究脑寄生虫互动，甚至简单的方面的能力受到了阻碍部分由TECHNOLOGIC限制。例如，大多数的工作表明神经元的细胞，其中包囊坚持已经完成，电子显微镜（EM）5,6。虽然EM提供高分辨率，这是费时，劳动强度大，而且价格昂贵。免疫荧光（IF）测定法最近已结合使用共聚焦显微镜来确认由EM 7所做的工作。中频测定在技术上易于实施和相对便宜的，但使用这些技术来underst和囊肿和感染的神经元之间的空间关系，需要串行重建，这是耗时的，在技术上困难，并且可能会导致有价值的信息丢失。因此，我们开发了可与中枢神经系统弓形虫病的小鼠模型中使用，并允许我们图像感染的神经元的全部未经EM或免疫组织化学（IHC）的方法。通过开发这样的技术，我们可以开始探索以相对快速和廉价的方式感染细胞和囊肿之间的蜂窝式关系。 我们开发的方法，结合了用于光学结算和成像厚脑切片通过共聚焦显微镜8较新的技术与系统，它标志着已经注射了寄生虫蛋白9,10 体内细胞。在这个系统中，我们感染表达绿色荧光蛋白（GFP）的Cre-报告小鼠仅后的Cre介导的重组11 弓形虫 </em&gt;表达红色荧光蛋白（RFP）和Cre重组酶注入到宿主细胞中的9株。这种组合可以让我们收获了感染小鼠的大脑后，中枢神经系统感染建立后，切成厚脑切片，并迅速查明相关领域的图像通过查找RFP +囊肿。要注意，作为绿色荧光蛋白的宿主细胞中表达完全取决于注射的Cre的寄生虫，而不是在感染，一些将GFP +细胞不含有寄生虫10是很重要的。作为该协议的目的是为了能够将图像的整体感染的神经元，重点是仅在GFP +神经元还含有RFP +囊肿，但该协议也可以用于图像的GFP + / RFP -神经元。 一旦感染脑收获并切片，该切片是由甘油结算呈现透明。部的适当的区域，然后用共焦显微镜，人成像感染的宿主细胞，并在其整体的成囊寄生虫降脂前所未有的可视化。在这里，我们提供了一个完整的协议识别，光学结算和成像感染神经元。

<table><tbody><tr><td>Vibratome Series 1000 Sectioning System</td><td>Technical Products International, Inc.</td><td></td><td>Other vibratomes are compatible</td></tr><tr><td>Glycerol</td><td>Fisher Scientific</td><td>BP229-1</td><td></td></tr><tr><td>Tween-20</td><td>Fisher Scientific</td><td>BP337-500</td><td></td></tr><tr><td>Premium Slides</td><td>Fisher Scientific</td><td>12-544-2</td><td></td></tr><tr><td>#1.5 Coverslips</td><td>VWR</td><td>48393 251</td><td></td></tr><tr><td>Diamond Scriber</td><td>VWR</td><td>52865-005</td><td></td></tr><tr><td>Zeiss LSM 510 Meta confocal microscope</td><td>Zeiss</td><td>LSM 510</td><td></td></tr><tr><td>Ketaject&amp;reg; Ketamine HCl Inj., USP 100 mg/ml</td><td>Western Medical Supply, Inc.</td><td>4165</td><td></td></tr><tr><td>AnaSed&amp;reg; Injection Xylazine 20 mg/ml</td><td>Lloyd Inc.</td><td></td><td></td></tr><tr><td>ZsGreen Mice</td><td>Jackson Laboratories</td><td>7906</td><td>B6.Cg-Gt(ROSA)26Sortm6(CAG-ZsGreen1)Hze/J</td></tr><tr><td>Surgical equipment</td><td></td><td></td><td>Thumb forceps; Fine scissors-angled to side, sharp-sharp; Sharp-sharp scissors; Kelly hemostats; Mayo scissors; Micro spatula.</td></tr><tr><td>Human Foreskin Fibroblasts (HFF) cells</td><td></td><td></td><td>These are primary cells from human foreskins.&amp;nbsp; We make these in-house but they may be purchased from outside vendors.</td></tr><tr><td>Dulbecco&#039;s High Glucose Modified Eagles Medium (DMEM)</td><td>HyClone</td><td>SH30081.01</td><td></td></tr><tr><td>Penicillin Streptomycin Solution, 100x</td><td>Corning</td><td>30-002-Cl</td><td></td></tr><tr><td>200 mM L-alanyl-L-glutamine</td><td>Corning</td><td>25-015-Cl</td><td></td></tr><tr><td>25 cm&lt;sup&gt;2&lt;/sup&gt; Canted neck flask</td><td>Fisher Scientific</td><td>1012639</td><td></td></tr><tr><td>Phosphate-Buffered Saline, 1x Without Calcium and Magnesium</td><td>VWR</td><td>45000-446</td><td></td></tr><tr><td>Phosphate-Buffered Saline, 10x, USP Sterile Ultra Pure Grade</td><td>amresco</td><td>K813-500ml</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Gibco</td><td>26140-079</td><td></td></tr><tr><td>Bright-Line Hemocytometer</td><td>Sigma-aldrich</td><td>Z359629-1EA</td><td></td></tr><tr><td>Mouse Brain Slicer Matrix</td><td>Zivic Instruments</td><td>BSMAS005-1</td><td></td></tr><tr><td>Sodium Chloride</td><td>Fisher Scientific</td><td>BP358-1</td><td></td></tr><tr><td>Heparin sodium salt from porcine intestinal mucosa</td><td>Sigma-aldrich</td><td>H3393-100KU</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Fisher Scientific</td><td>O4042-500</td><td></td></tr><tr><td>20 ml Disposable Scintillation Vials</td><td>Fisher Scientific</td><td>FS74500-20</td><td></td></tr><tr><td>Alcohol, Ethyl, 95%, 190 Proof</td><td>In-house</td><td>17212945</td><td>This product is purchased from an in-house stockroom.&amp;nbsp; Other companies are compatible.</td></tr><tr><td>Imaris Software</td><td>Bitplane</td><td></td><td></td></tr><tr><td>Clear nail polish</td><td></td><td></td><td>Other brands are compatible</td></tr><tr><td>10 ml Syringe with Luer-Lok</td><td>VWR</td><td>BD309604</td><td>Other syringes are compatible</td></tr><tr><td>Three-way Stopcock</td><td></td><td></td><td>Any brand is compatible</td></tr><tr><td>Hypodermic needle</td><td></td><td></td><td>Any brand is compatible - used to pin down mouse.</td></tr><tr><td>Cell Scraper</td><td></td><td></td><td>Any brand is compatible</td></tr><tr><td>25 G x 12&quot; Tubing, Safety Blood Collection Set, with Luer Adapter</td><td>Greiner Bio-One</td><td>450099</td><td>Other brands are compatible</td></tr></tbody></table>

3-d imaging and analysis of neurons infected in vivo with toxoplasma gondii

弓形虫是一种专性细胞内寄生虫具有广泛的宿主范围，包括人类和啮齿类动物。在人类和啮齿类动物， 弓形虫建立在大脑终身持续感染。虽然这种脑部感染是无症状的，在大多数人的免疫功能，在发育中的胎儿或免疫功能低下的个体如获得性免疫缺陷综合征（AIDS）患者，这种偏爱与执着在大脑中可能会导致灾难性的神经系统疾病。因此，很显然，献计弓形虫相互作用是由弓形虫产生的症状的疾病的关键，但我们对中枢神经系统（CNS）和寄生虫的细胞之间的细胞或分子的相互作用的了解甚少。在中枢神经系统的小鼠模型弓形体病已经知道了30多年的神经元中的寄生虫仍然存在的细胞，但信息很少哪些神经元的一部分，通常感染（体细胞，树枝状，轴突），如果菌株之间的这种蜂窝关系变化。在某种程度上，这是缺乏二次成像的难度，从动物整体可视化感染神经元。这些图像通常需要连续切片和组织通过电子显微镜或免疫染色后，激光共聚焦显微镜成像的拼接。通过组合多种技术，这里所描述的方法使得能够使用厚的切片（160微米），以确定和包含包囊图像的完整细胞，允许三维可视化和个人，慢性感染的神经元的分析，而不需要免疫染色，电子显微镜，或连续切片和拼接。使用这种技术，我们可以开始理解寄生虫和感染的神经元之间的蜂窝式关系。

图7包括两个GFP +神经元从两个不同的160微米厚的切片，以及来自囊肿至细胞体为图7B的距离的代表测量结果。 图含有囊肿-7的A和 B示出了这个新的代表图像协议允许在其全部被感染的神经元的可视化。 图7C示出了与该成像技术，现在有可能量化囊肿和胞体（了Imaris 7.7）之间的距离。 图7D是图的一个完整旋转视图7B。的z栈以这种方式的可视化允许核查细胞和孢囊的完全捕获的。 图7E是一个3D旋转动画显示如何从7D的图像可以通过了Imaris 7.7软件进行分析，以测量来自囊肿的距离，以胞体无论是从中间到中间Øř边缘到边缘。虽然使用下列实际的GFP +进程到细胞体囊肿和细胞体，测定之间的直线做图示的测量，也可产生（数据未显示）。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/52237/52237fig1highres.jpg" width="500" /> 图1:用于去除小鼠脑的外科设备。 （A）吸收垫。（B）泡沫块。（C）针牵制脚。（D）25克血收集组。（E）3路阀。（F）20毫升注射器肝素/盐。（ G）20毫升注射器4％PFA。（H）拇指钳。（I）精细剪刀，角度的摆动，尖锐犀利。（J）夏普锋利的剪刀。（K）凯利止血。（L），梅奥剪刀。 （M）的 </STRONG&gt;微锅铲。（N）闪烁小瓶的4％PFA。比例尺= 8公分。 <img alt="图2" src="/files/ftp_upload/52237/52237fig2highres.jpg" /> 图2:收获小鼠大脑。不当灌注后（A）鼠标牵制和胸部喷以70％乙醇。（B）裸露的胸部。（C）胸腔打开的。适当的灌注后，（D）肝。（E）肝。（F）裸露的头骨。（ G）与脑颅骨的一半删除。（H）小瓶装有4％PFA（I）适当灌注后，脑收获的大脑。（J）脑灌注不当后。 <img alt="图3" src="/files/ftp_upload/52237/52237fig3highres.jpg" /> 图3:切片鼠脑。 （A）鼠脑马特里<html> X（B）小鼠大脑和琼脂糖凝胶贴安装块与氰基丙烯酸酯的vibratome室洋溢着冷1X PBS内。 <img alt="图4" fo:content-width="6in" src="/files/ftp_upload/52237/52237fig4highres.jpg" width="600" /> 图4:清除处理的小鼠脑切片的过程中清算过程的每个步骤代表性图像。 <img alt="图5" src="/files/ftp_upload/52237/52237fig5highres.jpg" /> 图5:切割和固定间隔滑动。一个号1.5盖玻片有代表性的标志切割盖玻片到5个; B。每一块切盖玻片普通载玻片上涨到位透明指甲油来创建一个窗口。 <html> 2237 / 52237fig6highres.jpg"/&gt; 图6:屏幕快照用于图像采集常规参数。 <img alt="图7" src="/files/ftp_upload/52237/52237fig7highres.jpg" /> 图7: 弓形虫包囊可以驻留遥远的神经元突起中（AB）最大的投影GFP的图像+含RFP +囊肿（白色箭头）神经元。比例尺= 20微米。（CE）提供的B中所示的囊肿-神经元关系的其他可视化和分析。（C）的了Imaris生成的直线距离的囊肿到细胞体的边缘上的边缘测量（149μm ）。（D） <a href="https://www.jove.com/files/ftp_upload/52237/Animated-Video Figure 7D.avi" target="_blank">3-D旋转的电影</a> 。（E）了Imaris生成www.jove.com/files/ftp_upload/52237/Animated-Video图7E.avi"目标="_空白"&gt;的3-D电影表示从囊肿到细胞体的测量两者中间到中间（163微米）和边缘到边缘（149微米）。

Watch this Scientific Journal Video about 3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii at JoVE.com

3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii

使用该协议，我们能够从感染寄生虫弓形虫 ，使可视化和的encysting寄生虫和感染的神经元之间的空间关系分析的小鼠图像160微米厚的脑切片。

神经元的3-D成像和分析感染<em&gt;在体内</em&gt;与<em&gt;弓形虫</em

Toxoplasma gondii is an obligate, intracellular parasite with a broad host range, including humans and rodents. In both humans and rodents, Toxoplasma ...

3-d-imaging-analysis-neurons-infected-vivo-with-toxoplasma

Nanyang Technological University

Research

JoVE Journal

Neuroscience

3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii

11.5K 次观看.  University of Arizona. 使用该协议，我们能够从感染寄生虫弓形虫 ，使可视化和的encysting寄生虫和感染的神经元之间的空间关系分析的小鼠图像160微米厚的脑切片。 

视频: 3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or thinned-skull 3 preparations. While the open-skull technique is very versatile, it is not optimal for studying microglia because it is invasive and can cause microglial activation. Even though the thinned-skull approach is minimally invasive, the repeated re-thinning of skull required for chronic imaging increases the risks of tissue injury and microglial activation and allows for a limited number of imaging sessions. Here we present a chronic thin-skull window method for monitoring murine microglia in vivo over an extended period of time using two-photon microscopy. We demonstrate how to prepare a stable, accessible, thinned-skull cortical window (TSCW) with an apposed glass coverslip that remains translucent over the course of three weeks of intermittent observation. This TSCW preparation is far more immunologically inert with respect to microglial activation than open craniotomy or repeated skull thinning and allows an arbitrary number of imaging sessions during a time period of weeks. We prepare TSCW in CX3CR1 GFP/+ mice 4 to visualize microglia with enhanced green fluorescent protein to &le;150 &mu;m beneath the pial surface. We also show that this preparation can be used in conjunction with stereotactic brain injections of the HIV-1 neurotoxic protein Tat, adjacent to the TSCW, which is capable of inducing durable microgliosis. Therefore, this method is extremely useful for examining changes in microglial morphology and motility over time in the living brain in models of HIV Associated Neurocognitive Disorder (HAND) and other neurodegenerative diseases with a neuroinflammatory component.

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

We describe a method for repeatedly visualizing murine microglia and circulating monocytes in vivo over hours, days or weeks using transcranial two-photon microscopy. We demonstrate how to prepare a thinned-skull window that allows intermittent observation of quiescent microglia that can be activated by adjacent stereotactic injection of the HIV-1 regulatory protein Tat.

一个慢性双光子薄颅骨窗口技术在体内 Neuroinflammation模型小鼠小胶质细胞成像

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or ...

科研

神经科学

Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-&#947;-Dependent Cell Autonomous Immunity

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan pathogen. The parasite invades and replicates within virtually any warm blooded vertebrate cell type. During parasite invasion of a host cell, the parasite creates a parasitophorous vacuole (PV) that originates from the host cell membrane independent of phagocytosis within which the parasite replicates. While IFN-dependent-innate and cell mediated immunity is important for eventual control of infection, innate immune cells, including neutrophils, monocytes and dendritic cells, can also serve as vehicles for systemic dissemination of the parasite early in infection. An approach is described that utilizes the host innate immune response, in this case macrophages, in a forward genetic screen to identify parasite mutants with a fitness defect in infected macrophages following activation but normal invasion and replication in na&#239;ve macrophages. Thus, the screen isolates parasite mutants that have a specific defect in their ability to resist the effects of macrophage activation. The paper describes two broad phenotypes of mutant parasites following activation of infected macrophages: parasite stasis versus parasite degradation, often in amorphous vacuoles. The parasite mutants are then analyzed to identify the responsible parasite genes specifically important for resistance to induced mediators of cell autonomous immunity. The paper presents a general approach for the forward genetics screen that, in theory, can be modified to target parasite genes important for resistance to specific antimicrobial mediators. It also describes an approach to evaluate the specific macrophage antimicrobial mediators to which the parasite mutant is susceptible. Activation of infected macrophages can also promote parasite differentiation from the tachyzoite to bradyzoite stage that maintains chronic infection. Therefore, methodology is presented to evaluate the importance of the identified parasite gene to establishment of chronic infection.

Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-&#947;-Dependent Cell Autonomous Immunity

Forward genetics is a powerful approach to identify genes in intracellular pathogens important for resistance to cell autonomous immunity. The current approach uses innate immune cells, specifically macrophages, to identify novel Toxoplasma gondii genes important for immune evasion.

正向遗传学屏风使用巨噬细胞识别<em&gt;弓形虫</em&gt;基因的重要的抗IFN-γ依赖性细胞免疫自主

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan pathogen. The parasite invades and replicates within ...

免疫与感染

Large-scale Three-dimensional Imaging of Cellular Organization in the Mouse Neocortex

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical properties, synaptic connections, and in vivo functions, but their basic functional and anatomical organization from cellular to network scale is poorly understood. Here we describe a method for the three-dimensional imaging of fluorescently-labeled neurons across large areas of the brain for the investigation of the cortical cellular organization. Specific types of neurons are labeled by the injection of fluorescent retrograde neuronal tracers or expression of fluorescent proteins in transgenic mice. Block brain samples, e.g., a hemisphere, are prepared after fixation, made transparent with tissue clearing methods, and subjected to fluorescent immunolabeling of the specific cell types. Large areas are scanned using confocal or two-photon microscopes equipped with large working distance objectives and motorized stages. This method can resolve the periodic organization of the cell type-specific microcolumn functional modules in the mouse neocortex. The procedure can be useful for the study of three-dimensional cellular architecture in the diverse brain areas and other complex tissues.

Here we describe a procedure for tissue clearing, fluorescent labeling, and large-scale imaging of mouse brain tissue which, thereby, enables visualization of the three-dimensional organization of cell types in the neocortex.

小鼠大脑皮层细胞组织的大尺度三维成像

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical ...

Concomitant Isolation of Primary Astrocytes and Microglia for Protozoa Parasite Infection

Astrocytes and microglia are the most abundant glial cells. They are responsible for physiological support and homeostasis maintenance in the central nervous system (CNS). The increasing evidences of their involvement in the control of infectious diseases justify the emerging interest in the improvement of methodologies to isolate primary astrocytes and microglia in order to evaluate their responses to infections that affect the CNS. Considering the impact of Trypanosoma cruzi (T. cruzi) and Toxoplasma gondii (T. gondii) infection in the CNS, here we provide a method to extract, maintain, dissociate and infect murine astrocytes and microglia cells with protozoa parasites. Extracted cells from newborn cortices are maintained in vitro for 14 days with periodic differential media replacement. Astrocytes and microglia are obtained from the same extraction protocol by mechanical dissociation. After phenotyping by flow cytometry, cells are infected with protozoa parasites. The infection rate is determined by fluorescence microscopy at different time points, thus enabling the evaluation of differential ability of glial cells to control protozoan invasion and replication. These techniques represent simple, cheap and efficient methods to study the responses of astrocytes and microglia to infections, opening the field for further neuroimmunology analysis.

The overall goal of this protocol is to instruct how to extract, maintain, and dissociate murine astrocyte and microglia cells from the central nervous system, followed by infection with protozoa parasites.

原生动物寄生虫感染原发性星细胞和微胶质的伴随分离

Astrocytes and microglia are the most abundant glial cells. They are responsible for physiological support and homeostasis maintenance in the central ...

High-Resolution 3D Imaging of Rabies Virus Infection in Solvent-Cleared Brain Tissue

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and study the distribution, tropism, and abundance of pathogens inside of organ tissues provides pivotal data on disease development and progression. Using conventional microscopy methods, immunolabeling is mostly restricted to thin sections obtained from paraffin-embedded or frozen samples. However, the limited 2D image plane of these thin sections may lead to the loss of crucial information on the complex structure of an infected organ and the cellular context of the infection. Modern multicolor, immunostaining-compatible tissue clearing techniques now provide a relatively fast and inexpensive way to study high-volume 3D image stacks of virus-infected organ tissue. By exposing the tissue to organic solvents, it becomes optically transparent. This matches the sample&#8217;s refractive indices and eventually leads to a significant reduction of light scattering. Thus, in combination with long free working distance objectives, large tissue sections up to 1 mm in size can be imaged by conventional confocal laser scanning microscopy (CLSM) at high resolution. Here, we describe a protocol to apply deep-tissue imaging after tissue clearing to visualize rabies virus distribution in infected brains in order to study topics like virus pathogenesis, spread, tropism, and neuroinvasion.

Novel, immunostaining-compatible tissue clearing techniques like the ultimate 3D imaging of solvent-cleared organs allow the 3D visualization of rabies virus brain infection and its complex cellular environment. Thick, antibody-labeled brain tissue slices are made optically transparent to increase imaging depth and to enable 3D analysis by confocal laser scanning microscopy.

溶剂清除脑组织狂犬病病毒感染的高分辨率3D成像

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and ...

Quantitative 3D Imaging of Trypanosoma cruzi-Infected Cells, Dormant Amastigotes, and T Cells in Intact Clarified Organs

Chagas disease is a neglected pathology that affects millions of people worldwide, mainly in Latin America. The Chagas disease agent, Trypanosoma cruzi (T. cruzi), is an obligate intracellular parasite with a diverse biology that infects several mammalian species, including humans, causing cardiac and digestive pathologies. Reliable detection of T. cruzi in vivo infections has long been needed to understand Chagas disease&#39;s complex biology and accurately evaluate the outcome of treatment regimens. The current protocol demonstrates an integrated pipeline for automated quantification of T. cruzi-infected cells in 3D-reconstructed, cleared organs. Light-sheet fluorescent microscopy allows for accurately visualizing and quantifying of actively proliferating and dormant T. cruzi parasites and immune effector cells in whole organs or tissues. Also, the CUBIC-HistoVision pipeline to obtain uniform labeling of cleared organs with antibodies and nuclear stains was successfully adopted. Tissue clearing coupled with 3D immunostaining provides an unbiased approach to comprehensively evaluate drug treatment protocols, improve the understanding of the cellular organization of T. cruzi-infected tissues, and is expected to advance discoveries related to anti-T. cruzi immune responses, tissue damage, and repair in Chagas disease.

Quantitative 3D Imaging of Trypanosoma cruzi-Infected Cells, Dormant Amastigotes, and T Cells in Intact Clarified Organs

The present protocol describes light-sheet fluorescent microscopy and automated software-assisted methods to visualize and precisely quantify proliferating and dormant Trypanosoma cruzi parasites and T cells in intact, cleared organs and tissues. These techniques provide a reliable way to evaluate treatment outcomes and offer new insights into parasite-host interactions.

完整澄清器官中克 氏锥虫感染细胞、休眠无鞭毛虫和 T 细胞的定量 3D 成像

Chagas disease is a neglected pathology that affects millions of people worldwide, mainly in Latin America. The Chagas disease agent, Trypanosoma cruzi ...

Live Animal Imaging and Cell Sorting Methods for Investigating Neurodegeneration in a C. elegans Excitotoxic Necrosis Model

Excitotoxic necrosis is a leading form of neurodegeneration. This process of regulated necrosis is triggered by the synaptic accumulation of the neurotransmitter glutamate, and the excessive stimulation of its postsynaptic receptors. However, information on the subsequent molecular events that culminate in the distinct neuronal swelling morphology of this type of neurodegeneration is lacking. Other aspects, such as changes in specific subcellular compartments, or the basis for the differential cellular vulnerability of distinct neuronal subtypes, remain under-explored. Furthermore, a range of factors that come into play in studies that use in vitro or ex vivo preparations might modify and distort the natural progression of this form of neurodegeneration. It is therefore important to study excitotoxic necrosis in live animals by monitoring the effects of interventions that regulate the extent of neuronal necrosis in the genetically amenable and transparent&#160;model system of the nematode Caenorhabditis elegans. This protocol describes methods of studying excitotoxic necrosis in C. elegans neurons, combining optical, genetic, and molecular analysis. To induce excitotoxic conditions in C. elegans, a&#160;knockout of a glutamate transporter gene (glt-3) is combined with a neuronal sensitizing genetic background (nuls5 [Pglr-1::G&#945;S(Q227L)]) to produce glutamate receptor hyperstimulation and neurodegeneration. Nomarski differential interference contrast (DIC), fluorescent, and confocal microscopy in live animals are methods used to quantify neurodegeneration, follow subcellular localization of fluorescently labeled proteins, and quantify mitochondrial morphology in the degenerating neurons. Neuronal Fluorescence Activated Cell Sorting (FACS) is used to distinctly sort at-risk neurons for cell-type specific transcriptomic analysis of neurodegeneration. A combination of live imaging and FACS methods as well as the benefits of the C. elegans model organism allow researchers to leverage this system to obtain reproducible data with a large sample size. Insights from these assays could translate to novel targets for therapeutic intervention in neurodegenerative diseases.

<meta charset="utf8"></head><body>用于研究秀丽隐杆线虫兴奋性中毒性坏死模型中神经退行性的活体动物成像和细胞分选方法

In a C. elegans excitotoxicity model, this protocol employs in vivo imaging to analyze the regulation of necrotic neurodegeneration, the effect of genes encoding candidate mediators, and involvement of mitochondria. Cell dissociation and sorting is used to specifically obtain at-risk neurons for cell-specific transcriptomic analysis of neurodegeneration and neuroprotection mechanisms.

用于研究 秀丽隐杆线 虫兴奋性中毒性坏死模型中神经退行性的活体动物成像和细胞分选方法

Excitotoxic necrosis is a leading form of neurodegeneration. This process of regulated necrosis is triggered by the synaptic accumulation of the ...

Utilizing In Vivo Postnatal Electroporation to Study Cerebellar Granule Neuron Morphology and Synapse Development

Neurons undergo dynamic changes in their structure and function during brain development to form appropriate connections with other cells. The rodent cerebellum is an ideal system to track the development and morphogenesis of a single cell type, the cerebellar granule neuron (CGN), across time. Here, in vivo electroporation of granule neuron progenitors in the developing mouse cerebellum was employed to sparsely label cells for subsequent morphological analyses. The efficacy of this technique is demonstrated in its ability to showcase key developmental stages of CGN maturation, with a specific focus on the formation of dendritic claws, which are specialized structures where these cells receive the majority of their synaptic inputs. In addition to providing snapshots of CGN synaptic structures&#160;throughout cerebellar development, this technique can be adapted to genetically manipulate granule neurons in a cell-autonomous manner to study the role of any gene of interest and its effect on CGN morphology, claw development, and synaptogenesis.

Utilizing In Vivo Postnatal Electroporation to Study Cerebellar Granule Neuron Morphology and Synapse Development

Here we describe a method to visualize synaptogenesis of granule neurons in the mouse cerebellum over the time course of postnatal brain development when these cells refine their synaptic structures and form synapses to integrate themselves into the overall brain circuit.

利用 体内 出生后电穿孔研究小脑颗粒神经元形态和突触发育

Neurons undergo dynamic changes in their structure and function during brain development to form appropriate connections with other cells. The rodent ...

通过荧光报告基因定量进行全器官病原体追踪

High&#45;Resolution Three&#45;Dimensional Whole&#45;Organ Tomography of Microbial Infections

Most infections take place within three-dimensional host tissues with intricate anatomy and locally varying host physiology. The positioning of pathogen cells within this diverse environment significantly affects their stress levels, responses, fate, and contribution to the overall progression of the disease and treatment failure. However, due to the technical difficulties in locating &#181;m-sized pathogen cells within cm-sized host organs, this area of research has been relatively unexplored. Here, we present a method for addressing this challenge. We employ serial two-photon tomography and AI-enhanced image analysis to locate individual Salmonella cells throughout the entire spleen, liver lobes, and whole lymph nodes of infected mice. Using fluorescent reporters and in vivo antibody administration, the replication rate of single Salmonella cells, their local interaction with specific immune cells, and bacterial responses to antibiotics can be determined. These methodologies open avenues for a comprehensive examination of infections, their prevention, and treatment within the three-dimensional tissue context.

作者聚焦:揭示细菌对组织中抗生素和免疫系统的反应

Here, we describe a procedure that enables organ-wide detection of pathogenic bacteria during infection and quantification of fluorescent reporter activities.

微生物感染的高分辨率三维全器官断层扫描

Most infections take place within three-dimensional host tissues with intricate anatomy and locally varying host physiology. The positioning of pathogen ...

神经元的3-D成像和分析感染

摘要

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一个慢性双光子薄颅骨窗口技术在体内 Neuroinflammation模型小鼠小胶质细胞成像

正向遗传学屏风使用巨噬细胞识别

小鼠大脑皮层细胞组织的大尺度三维成像

原生动物寄生虫感染原发性星细胞和微胶质的伴随分离

溶剂清除脑组织狂犬病病毒感染的高分辨率3D成像

完整澄清器官中克氏锥虫感染细胞、休眠无鞭毛虫和 T 细胞的定量 3D 成像

用于研究秀丽隐杆线虫兴奋性中毒性坏死模型中神经退行性的活体动物成像和细胞分选方法

用于研究秀丽隐杆线虫兴奋性中毒性坏死模型中神经退行性的活体动物成像和细胞分选方法

利用体内出生后电穿孔研究小脑颗粒神经元形态和突触发育

微生物感染的高分辨率三维全器官断层扫描

神经元的3-D成像和分析感染

摘要

探索更多视频

一个慢性双光子薄颅骨窗口技术在体内 Neuroinflammation模型小鼠小胶质细胞成像

正向遗传学屏风使用巨噬细胞识别

小鼠大脑皮层细胞组织的大尺度三维成像

原生动物寄生虫感染原发性星细胞和微胶质的伴随分离

溶剂清除脑组织狂犬病病毒感染的高分辨率3D成像

完整澄清器官中克 氏锥虫感染细胞、休眠无鞭毛虫和 T 细胞的定量 3D 成像

用于研究 秀丽隐杆线 虫兴奋性中毒性坏死模型中神经退行性的活体动物成像和细胞分选方法

用于研究 秀丽隐杆线 虫兴奋性中毒性坏死模型中神经退行性的活体动物成像和细胞分选方法

利用 体内 出生后电穿孔研究小脑颗粒神经元形态和突触发育

微生物感染的高分辨率三维全器官断层扫描

完整澄清器官中克氏锥虫感染细胞、休眠无鞭毛虫和 T 细胞的定量 3D 成像

用于研究秀丽隐杆线虫兴奋性中毒性坏死模型中神经退行性的活体动物成像和细胞分选方法

用于研究秀丽隐杆线虫兴奋性中毒性坏死模型中神经退行性的活体动物成像和细胞分选方法

利用体内出生后电穿孔研究小脑颗粒神经元形态和突触发育