Name: optical clearing of the mouse central nervous system using passive clarity
Uploaded: 2016-06-30T13:00:00.000+00:00
Duration: 10 min 28 s
Description: Watch this Scientific Journal Video about Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY at JoVE.com

这项工作是慷慨神经疾病和中风（NIH / NINDS）资助1R01NS086981和康拉德希尔顿N.基金会健康/国立全国学院的支持。我们感谢洛朗邦托利拉博士和马修SCHIBLER博士及其与激光共聚焦显微镜宝贵的援助。我们感谢那些给CLARITY论坛（http://forum.claritytechniques.org）作出了贡献。我们特别感谢卡尔戴瑟罗特博士打开了他的实验室，教这个迷人的技术。笔者是从脑成像医学研究组织，脑成像支持基金会皮尔森 - 洛夫莱斯基金会，基金会阿曼森，资本集团慈善基金会，威廉·M和琳达R. Dietel慈善基金，以及基金北极星的慷慨支持表示感谢。研究本出版物中报也部分由国家研究资源中心和民族的主任办公室的支持在奖项数量C06RR012169，C06RR015431和S10OD011939卫生研究院人。内容完全是作者的责任，并不一定代表美国国立卫生研究院的官方意见。

所有实验均按照机构动物护理和使用委员会（IACUC）的指导方针进行。 THY1-YFP，PLP-EGFP和PV-TdTomato小鼠用于这些实验，但表达的荧光蛋白的任何小鼠可以成功地清除并成像。 1.组织准备注意:多聚甲醛（PFA）和丙烯酰胺是有毒的和刺激物。执行利用这些试剂在通风橱，并配备适当的个人防护装备（实验室外套，手套和护目镜）的实验。 <ol><li>牺牲安乐死腔动物提供〜1毫升异氟烷，直到呼吸停止（〜2 - 3分钟）。 </li><li>使用蠕动泵设置为〜5毫升/分钟，以心内，在4℃下灌注灌注溶液（ 表1）的鼠标，直到血液从组织中清除（5 - 10分钟）。 </li><li>改变灌注到固定液（ 见表1）在4℃，直至颈部和尾部已经显著硬化（5 - 10分钟）。 </li><li>结束灌注和杀头的动物。通过首先除去围绕颅骨结缔组织，然后从颅骨的腹侧表面去除骨和提起脑出（ 图1a），如在20中所述，小心解剖出从头骨大脑。 注:优抚周围小脑flocculonodular叶和嗅球切除骨。 <ol><li>轻轻插入脊髓和脊椎之间的精，尖剪刀尖和切割个别脊柱段走，如在描述21从解剖脊柱脊髓。 </li></ol></li><li>通过将它放置在一个塑料小鼠大脑基质并沿与基体的刀片的中线切割Hemisect大脑。 </li><li>将每个半球和脊髓分离的50ml离心桶与ES〜40毫升的水凝胶溶液（ 表1）。从这点出发盖用铝箔管，以在所有后续步骤保护荧光。 </li><li>轻微振荡（〜30转）7天孵育含有在水凝胶中的样品的管中，在4℃。 </li></ol> 2.聚合<ol><li>丢弃约25mL水凝胶，保存样品和约25mL的水凝胶中的离心管中。 </li><li>通过去除帽和放置离心管在干燥器罐和罐连接到真空脱氧样品。施加真空，至少10分钟，以允许溶解的气体出来溶液和形式气泡。轻轻摇动赶走气泡。 </li><li> 3分钟 - 与100％的氮气罐干燥器超过2更换真空。一旦压力均衡（一旦干燥器罐的顶部可以打开），迅速帽管，以防止氧气的放归。 </li><li>聚合物通过将管与样品中的37℃水浴中3小时，直至聚合反应完成IZE水凝胶。聚合水凝胶将采取的凝胶状稠度（ 图1b）。注意:在顶部未聚合水凝胶的少量是可接受的。 </li><li>用刮刀以除去由离心管聚合样品，然后切去从样品中多余的水凝胶。 </li><li>通过将样品在软绒布擦拭，并轻轻揉并在其上滚动的组织，留下的样品聚合水凝胶的只有薄薄的一层去除剩余的水凝胶。 </li></ol> 3.脂质去除注:脂质清除的组织是脆弱的，小心处理。引流管时，使用细胞过滤器，以避免丢失样本。此外，该样品将膨胀整个结算处理，但是，肿胀可以在折射率匹配过程颠倒。 注意:SODI嗯十二烷基硫酸钠（SDS）和硼酸是刺激物。执行利用它们在通风橱，并配备适当的个人防护装备（实验室外套，手套和护目镜）的实验。 <ol><li>转移聚合样品到一个干净的50ml离心管中，加入清除缓冲液（ 表1）45毫升孵育在45℃下轻轻摇动（〜30转）。通过浇灭了用于清除缓存为化学废物和添加〜45毫升新鲜缓冲结算日更改缓冲区最初7天。保持含在铝箔覆盖，以保护荧光团的样品的离心管中。 </li><li>最初7天后，在40℃和交换结算缓冲器孵育样品每2 - 3天。继续结算，直到所有的脂类是溶解和组织达到透明度（ 图1c）。这将是4 - 6周的小鼠大脑半球2 - 4周为一个脊髓。 </li><li>一旦组织被清除，转移样品到一个新的50ml离心管中并在40℃轻微振荡（〜30转）在PBST中（ 表1）清洗4次超过24小时，以除去残余的SDS。 </li></ol> 4.染色分子注意:4&#39;，6-二脒基-2-苯二盐酸盐（DAPI）是一种刺激性和利用它应该在通风橱，并配备适当的个人防护装备（实验室外套，手套和护目镜）进行任何实验。 <ol><li>样品转移到一个干净的15ml离心管，用1微克/毫升的DAPI在1X PBST，pH值7.5填补。在室温下孵育（RT）下24小时。保持含在铝箔覆盖，以保护荧光团的样品的离心管中。 </li><li>在室温洗PBST 3次，每次至少6小时/洗。 </li></ol> 5.折射率匹配注意:2,2&#39;-硫代二乙醇（TDE）是一种刺激和利用它的任何实验SH乌尔德在通风橱，并配备适当的个人防护装备（实验室外套，手套和护目镜）来完成。 <ol><li>将样品转移到一个干净的15ml离心管中，用30％在1×PBS（体积/体积）TDE，pH值7.5填。孵育24小时40℃轻轻摇动（〜30转）。 </li><li>交换30％TDE用63％（V / V）的1×PBS中，pH值7.5 TDE。在40℃孵育轻微振荡24小时。 注意:示例将退缩到约原来的大小。 </li><li>在平坦，清洁的表面滚动的一块可重复使用的粘合剂的与均匀的直径只比所述样品的厚度稍大的圆筒。 </li><li>莱可重复使用的胶粘剂缸开放式圆（〜25内径在顶部一个小口）上有40毫米的玻璃底菜。如果修正需要进行，从玻璃移除筒和用刀片切割。 </li><li>使用P1000枪头通过创建可重复使用的粘合剂和玻璃之间的密封滚动它周围的筒的外缘。 </li><li>地方〜100微升63％TDE到玻璃菜，传遍覆盖可重复使用的胶粘剂的圆内的玻璃表面。 </li><li>使用刮刀，小心地在圆圈的中间样品，注意不要形成泡沫。 </li><li>移液器另外100微升63％TDE直接到样品，然后立即放置40mm的圆形玻璃盖在可重复使用的粘合剂。 </li><li>使用索引，中间和两手的无名指，小心地按左右的圆的周长，直到盖玻璃已经取得与样品接触。 </li><li>缓慢将玻璃底培养皿到直立位置靠在一个表面上，然后添加63％TDE用吸管直到溶液到达在上面的小开口。使用无尘擦拭擦干枪头使得溶液不会对玻璃滴落。以避免在该室的气泡，从未空吸管一路。 </li><li>至小开，加硅弹性体包围的圈子，并创建一个封闭室。等待5分钟，它的成像之前干燥。 </li></ol> 6.显微镜注意:在激光共聚焦，单一平面照明和光片显微镜使用的激光器是非常强大的，可以导致失明。任何利用激光器实验后应广泛的培训和十分谨慎和适当的保护眼睛进行。 <ol><li>放置成像室里面上的激光扫描共聚焦显微镜的阶段样品。 </li><li>打开成像软件（见材料表）。要打开氩激发激光，单击该程序，然后激光图标的上方配置选项卡。勾选旁边氩供电激光移动滑块规模的30％。观察指针慢慢温热至所选择的位置。 <ol><li>返回到获取标签的顶部。在大盒子"光束路径设置＆＃34;进入加载/保存设置框，然后选择下拉菜单中选择一个合适的波长信道图像YFP（527纳米）。 </li></ol></li><li>下的通道设置框:通过定位标记为"当前对象客观"红色超链接选择适当的成像目标。点击选项卡上打开所有可用的目标和选择会自动旋转炮塔。 <ol><li>使用的目标（ 例如 ，10X）具有大的工作距离（大于组织的厚度大要成像， 例如，第11毫米）和大数值孔径（ 例如 ，0.3），以最大限度地提高面内的图像分辨率和最小化光学切片厚度。 </li></ol></li><li>在下拉菜单，打开"XY:解析"的左栏窗口设置采集矩阵，所谓的"格式"，1024×1024或适当的目标的横向分辨率极限值。设置缩放因子尽可能低（ 例如 ，10.7）。 </li><li>在同一窗口，通过相应地在下降了单独标记菜单中设置8行平均1帧的平均参数。使用较大的值要高信噪比和小的值的快速采集。 注:8号线均线和1架平均将获得小鼠大脑半球的高品质的图像约4小时。 </li><li>找到使用阶段控制样本。一旦有代表性的领域是可见的，设置增益和偏移。 注意:增益和偏移基于所述组织类型，荧光团将基本上不同，和/或正被成像的解剖特征。对YFP，从760至780为增益和从-1.0到-1.5的偏移设定值。 </li><li>选择"瓷砖扫描"，并选择感兴趣的区域的边界。要获取设置的Z堆叠的上限和下限。设定基于所述物镜的光学部分分辨率极限的光学部分的厚度。 注:客观的光学部分分辨率极限主要由其数值孔径限定。所使用的大多数显微镜控制软件会自动计算出适当的值的目标。要10X物镜具有0.3的数值孔径，这将是大约11微米。 </li><li>开始采集。 注意:这可能需要多个小时。 </li><li>使用高倍目标从感兴趣的特定区域收集更高分辨率的图像。 </li></ol>

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The authors have nothing to disclose.

水凝胶包埋组织（无源净度）的被动结算是用于清除大块组织的一个简单和廉价的方法。这种方法不要求专用设备，并且可以很容易地在一个温度控制的振荡器进行。在几个星期的跨度，即使是大的，高度髓组织，例如整个脑或脊髓，将成为透明，适用于显微镜。尽管这份报告集中在CNS组织中的清除，被动净度可应用于任何组织。 的光透明的技术的相对优点和弱点已在文献<s...

The ability to image complete neuroanatomical structures is immensely valuable for understanding the brain in health and disease. Traditionally, 3D imaging has required tissue sectioning to provide the axial resolution and to visualize deep anatomical structures. This approach can produce high-resolution data sets, but requires sophisticated image reconstruction techniques and is very labor intensive. As a result, it has been limited to the imaging of small volumes of tissue1-3. Optical sectioning, on the other hand, is well suited for the creation of high-resolution 3D images of fluorescently labeled tissues. Since optical sectioning is inherently three dimensional, it does not require extensive computation to produce a 3D image volume. However, light scattering and tissue opacity limit the depth at which tissues can be optically sectioned. The depth of imaging is limited to about 150 &#181;m in laser scanning confocal microscopy and to less than 800 &#181;m using two-photon excitation microscopy4-8.
In order to overcome these limitations, several optical clearing techniques have been recently developed and then further refined to permit the deep microscopic imaging of intact tissues. The use of benzyl alcohol and benzyl benzoate (BABB) to render fixed tissues transparent was among one of the earliest approaches9. However, this approach was limited by the quenching of fluorescence in the samples and incomplete clearing of highly myelinated structures10. Refinements of this technique, such as 3D Imaging of Solvent Cleared Organs (3DISCO), have led to very rapid and complete tissue clearing, but still suffer from rapid loss of fluorescent signals, especially yellow fluorescent protein (YFP)10. Water-based clearing solutions, such as Sca/e11 and SeeDB12, preserve fluorescent signals, but do not completely clear highly myelinated tissues. Clear, Unobstructed Brain Imaging Cocktails and Computational analysis (CUBIC) is a promising new optical clearing technology that appears to overcome the limitations of previously developed water-based clearing solutions13. In contrast with other optical clearing techniques, Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY)14 embeds the brain in a porous matrix that provides structural integrity to proteins, nucleic acids and small molecules, while leaving lipids unbound. The lipids are then removed electrophoretically, culminating in an optically cleared brain that can be easily visualized and equally easily probed using commonly available techniques.
Electrophoretic tissue clearing (ETC) is used to remove lipids from hydrogel embedded tissues in CLARITY, however, ETC can be difficult to implement consistently and tissues subject to electrophoresis can exhibit tissue distortion, browning, loss of fluorescence and antibody reactivity. Passive lipid clearing avoids these limitations, but requires increased clearing times15-17. Passive clearing also permits the clearing of large numbers of samples in parallel, since it is not limited by the number of ETC apparatuses available. Decreasing the concentration of paraformaldehyde and excluding bis-acrylamide from the hydrogel have led to great increases in the speed of clearing at the expense of greater tissue expansion16. Less expensive alternatives to the refractive index matching solution used in the original CLARITY technique have been developed17-19. 2,2'-thiodiethanol (TDE) is an inexpensive and rapid brain clearing agent that reverses some of the tissue expansion that occurs during lipid removal18. This report incorporates passive clearing of hydrogel embedded tissue and the use of TDE as a refractive index matching solution to the original CLARITY technique to produce a highly reproducible and inexpensive protocol for the optical clearing of the mouse central nervous system.

<table><tbody><tr><td>10x phosphate buffered saline (PBS)</td><td>Fisher</td><td>BP399-1</td><td>buffers</td></tr><tr><td>32% paraformaldehyde (PFA)</td><td>Electron Microscopy Sciences</td><td>15714-5</td><td>perfusion and hydrogel</td></tr><tr><td>2,2&#039;-thiodiethanol (TDE)</td><td>Sigma-Aldrich</td><td>166782-500G</td><td>refractive index matching solution</td></tr><tr><td>40% acrylamide</td><td>Bio-Rad</td><td>161-0140</td><td>hydrogel</td></tr><tr><td>2% bis-acrylamide</td><td>Bio-Rad</td><td>161-0142</td><td>hydrogel</td></tr><tr><td>VA-044 initiator</td><td>Wako Pure Chemical Industries, Ltd.</td><td>VA044</td><td>hydrogel</td></tr><tr><td>Boric acid</td><td>Sigma-Aldrich</td><td>B7901</td><td>clearing buffer</td></tr><tr><td>Sodium dodecyl sulfate (SDS)</td><td>Fisher</td><td>BP166-5</td><td>clearing buffer</td></tr><tr><td>Sodium hydroxide</td><td>Fisher</td><td>55255-1</td><td>buffers</td></tr><tr><td>Sodium azide</td><td>Sigma-Aldrich</td><td>52002-100G</td><td>preservative</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>X100-500G</td><td>buffers</td></tr><tr><td>Heparin</td><td>Sigma-Aldrich</td><td>H3393-50KU</td><td>perfusion</td></tr><tr><td>Sodium nitrate</td><td>Fisher</td><td>BP360-500</td><td>perfusion</td></tr><tr><td>DAPI</td><td>Molecular Probes</td><td>D1306</td><td>nuclear stain</td></tr><tr><td>99.9% isoflurane</td><td>Phoenix</td><td>57319-559-06</td><td>anesthetic</td></tr><tr><td>Hydrocholoric acid</td><td>Fisher</td><td>A1445-500</td><td>buffers</td></tr><tr><td>Glass bottom dish</td><td>Willco</td><td>HBSB-5040</td><td>Willco dishes</td></tr><tr><td>Reusable adhesive</td><td>Bostick</td><td>371351</td><td>Blu-Tack</td></tr><tr><td>Silicon elastomer</td><td>World Precision Instruments</td><td>KWIK-CAST</td><td>Kwik-Cast</td></tr><tr><td>Lint-free wipe</td><td>Kimberly-Clark</td><td>34120</td><td>KimWipe</td></tr><tr><td>Mouse brain matrix</td><td>Roboz</td><td>SA-2175</td><td>sectioning tissue</td></tr><tr><td>Matrix blades</td><td>Roboz</td><td>RS-9887</td><td>sectioning tissue</td></tr><tr><td>Peristaltic pump</td><td>Cole-Parmer</td><td>77122-24</td><td>pefusion</td></tr><tr><td>Laser scanning confocal microscope</td><td>Leica</td><td>SP5</td><td>microscopy</td></tr><tr><td>Imaging software</td><td>Leica</td><td>LAS AF</td><td>microscopy</td></tr></tbody></table>

optical clearing of the mouse central nervous system using passive clarity

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique non-linear deformations, dramatically hampering one's ability to evaluate cellular morphology, distribution and connectivity in the central nervous system (CNS). However, optical clearing techniques are changing the way tissues are visualized. These approaches permit one to probe deeply into intact organ preparations, providing tremendous insight into the structural organization of tissues in health and disease. Techniques such as Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) achieve this goal by providing a matrix that binds important biomolecules while permitting light-scattering lipids to freely diffuse out. Lipid removal, followed by refractive index matching, renders the tissue transparent and readily imaged in 3 dimensions (3D). Nevertheless, the electrophoretic tissue clearing (ETC) used in the original CLARITY protocol can be challenging to implement successfully and the use of a proprietary refraction index matching solution makes it expensive to use the technique routinely. This report demonstrates the implementation of a simple and inexpensive optical clearing protocol that combines passive CLARITY for improved tissue integrity and 2,2&#8242;-thiodiethanol (TDE), a previously described refractive index matching solution.

可视化脑的结构组织为理解形态学和连通如何影响在健康和疾病的脑功能的关键。光透明的技术使得有可能在三维图像的细胞群在完整组织，使我们能够研究形态学和连接内聚。 小鼠表达荧光蛋白通过特异于亚群的启动子驱动的细胞允许一个梳理开神经连接的大脑中的复杂的图案。 THY1-YFP的转基因小鼠进行了广泛的光透明文?...

Watch this Scientific Journal Video about Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY at JoVE.com

Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY

Optical clearing techniques are revolutionizing the way tissues are visualized. In this report we describe modifications of the original Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) protocol that yields more consistent and less expensive results.

鼠标中枢神经系统的光透明使用被动CLARITY

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique ...

optical-clearing-mouse-central-nervous-system-using-passive

Research

JoVE Journal

Neuroscience

13.4K 次观看.  University of California, Los Angeles. Optical clearing techniques are revolutionizing the way tissues are visualized. In this report we describe modifications of the original Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) protocol that yields more consistent and less expensive results.

视频: Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY

Multiphoton Microscopy of Cleared Mouse Brain Expressing YFP

Multiphoton microscopy of intrinsic fluorescence and second harmonic generation (SHG) of whole mouse organs is made possible by optically clearing the organ before imaging.1,2 However, for organs that contain fluorescent proteins such as GFP and YFP, optical clearing protocols that use methanol dehydration and clear using benzyl alcohol:benzyl benzoate (BABB) while unprotected from light3 do not preserve the fluorescent signal. The protocol presented here is a novel way in which to perform whole organ optical clearing on mouse brain while preserving the fluorescence signal of YFP expressed in neurons. Altering the optical clearing protocol such that the organ is dehydrated using an ethanol graded series has been found to reduce the damage to the fluorescent proteins and preserve their fluorescent signal for multiphoton imaging.4 Using an optimized method of optical clearing with ethanol-based dehydration and clearing by BABB while shielded from light, we show high-resolution multiphoton images of yellow fluorescent protein (YFP) expression in the neurons of a mouse brain more than 2 mm beneath the tissue surface.

Multiphoton microscopy of whole mouse organs is possible by optically clearing the organ before imaging, but not all protocols preserve the fluorescent signal of fluorescent proteins. Using an optical clearing method with ethanol-based dehydration and benzyl alcohol:benzyl benzoate clearing, we show high-resolution multiphoton images of whole mouse brain expressing YFP.

多光子显微镜的清除小鼠脑组织表达YFP

Multiphoton microscopy of intrinsic fluorescence and second harmonic generation (SHG) of whole mouse organs is made possible by optically clearing the ...

科研

神经科学

Micron-scale Resolution Optical Tomography of Entire Mouse Brains with Confocal Light Sheet Microscopy

Understanding the architecture of mammalian brain at single-cell resolution is one of the key issues of neuroscience. However, mapping neuronal soma and projections throughout the whole brain is still challenging for imaging and data management technologies. Indeed, macroscopic volumes need to be reconstructed with high resolution and contrast in a reasonable time, producing datasets in the TeraByte range. We recently demonstrated an optical method (confocal light sheet microscopy, CLSM) capable of obtaining micron-scale reconstruction of entire mouse brains labeled with enhanced green fluorescent protein (EGFP). Combining light sheet illumination and confocal detection, CLSM allows deep imaging inside macroscopic cleared specimens with high contrast and speed. Here we describe the complete experimental pipeline to obtain comprehensive and human-readable images of entire mouse brains labeled with fluorescent proteins. The clearing and the mounting procedures are described, together with the steps to perform an optical tomography on its whole volume by acquiring many parallel adjacent stacks. We showed the usage of open-source custom-made software tools enabling stitching of the multiple stacks and multi-resolution data navigation. Finally, we illustrated some example of brain maps: the cerebellum from an L7-GFP transgenic mouse, in which all Purkinje cells are selectively labeled, and the whole brain from a thy1-GFP-M mouse, characterized by a random sparse neuronal labeling.

In this article we describe the full experimental procedure to reconstruct, with high resolution, the fine brain anatomy of fluorescently labeled mouse brains. The described protocol includes sample preparation and clearing, specimen mounting for imaging, data post-processing and multi-scale visualization.

整个老鼠大脑与光共聚焦显微镜板微米级分辨率光学层析成像

Understanding the architecture of mammalian  brain at single-cell resolution is one of the key issues of neuroscience.  However, mapping neuronal soma and ...

Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal cord of the majority of these fiber systems have not been thoroughly investigated in any species. Serotonergic fibers, which project to the spinal cord, have been studied in rats and opossums on histological sections and their functional significance has been deduced based on their fiber termination pattern in the spinal cord. With the development of CLARITY and CUBIC techniques, it is possible to investigate this fiber system and its distribution in the spinal cord, which is likely to reveal previously unknown features of serotonergic supraspinal pathways. Here, we provide a detailed protocol for imaging the serotonergic fibers in the mouse spinal cord using the combined CLARITY and CUBIC techniques. The method involves perfusion of a mouse with a hydrogel solution and clarification of the tissue with a combination of clearing reagents. Spinal cord tissue was cleared in just under two weeks, and the subsequent immunofluorescent staining against serotonin was completed in less than ten days. With a multi-photon fluorescent microscope, the tissue was scanned and a 3D image was reconstructed using Osirix software.

Supraspinal projections are important for pain perception and other behaviors, and serotonergic fibers are one of these fiber systems. The present study focused on the application of the combined CLARITY/CUBIC protocol to the mouse spinal cord in order to investigate the termination of these serotonergic fibers.

影像学检查HT能纤维在小鼠脊髓使用CLARITY / CUBIC技术

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal ...

Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human central nervous system (CNS) autoimmune demyelinating disease, creation of an AQP4-targeted model with both clinical and histologic manifestations of CNS autoimmunity has proven challenging. Immunization of wild-type (WT) mice with AQP4 peptides elicited T cell proliferation, although those T cells could not transfer disease to na&#239;ve recipient mice. Recently, two novel AQP4 T cell epitopes, peptide (p) 135-153 and p201-220, were identified when studying immune responses to AQP4 in AQP4-deficient (AQP4-/-) mice, suggesting T cell reactivity to these epitopes is normally controlled by thymic negative selection. AQP4-/- Th17 polarized T cells primed to either p135-153 or p201-220 induced paralysis in recipient WT mice, that was associated with predominantly leptomeningeal inflammation of the spinal cord and optic nerves. Inflammation surrounding optic nerves and involvement of the inner retinal layers (IRL) were manifested by changes in serial optical coherence tomography (OCT). Here, we illustrate the approaches used to create this new in vivo model of AQP4-targeted CNS autoimmunity (ATCA), which can now be employed to study mechanisms that permit development of pathogenic AQP4-specific T cells and how they may cooperate with B cells in NMO pathogenesis.

Here, we present a protocol to induce paralysis and opticospinal inflammation by transfer of aquaporin-4 (AQP4)-specific T cells from AQP4-/- mice into WT mice. In addition, we demonstrate how to use serial optical coherence tomography to monitor visual system dysfunction.

诱导麻痹及视觉系统损伤小鼠 T 细胞特异的视神经脊髓炎抗原水通道蛋白-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human ...

免疫与感染

Novel Passive Clearing Methods for the Rapid Production of Optical Transparency in Whole CNS Tissue

Since the development of CLARITY, a bioelectrochemical clearing technique that allows for three-dimensional phenotype mapping within transparent tissues, a multitude of novel clearing methodologies including CUBIC (clear, unobstructed brain imaging cocktails and computational analysis), SWITCH (system-wide control of interaction time and kinetics of chemicals), MAP (magnified analysis of the proteome), and PACT (passive clarity technique), have been established to further expand the existing toolkit for the microscopic analysis of biological tissues. The present study aims to improve upon and optimize the original PACT procedure for an array of intact rodent tissues, including the whole central nervous system (CNS), kidneys, spleen, and whole mouse embryos. Termed psPACT (process-separate PACT) and mPACT (modified PACT), these novel techniques provide highly efficacious means of mapping cell circuitry and visualizing subcellular structures in intact normal and pathological tissues. In the following protocol, we provide a detailed, step-by-step outline on how to achieve maximal tissue clearance with minimal invasion of their structural integrity via psPACT and mPACT.

Here, we present two novel methodologies, psPACT and mPACT, for achieving maximal optical transparency and subsequent microscopic analysis of tissue vasculature in the intact rodent whole CNS.

一种新的用于全中枢神经系统光透明快速生成的无源清除方法

Since the development of CLARITY, a bioelectrochemical clearing technique that allows for three-dimensional phenotype mapping within transparent tissues, ...

Immunostaining of Whole-Mount Retinas with the CLARITY Tissue Clearing Method

The tissue hydrogel delipidation method (CLARITY), originally developed by the Deisseroth laboratory, has been modified and widely used for immunostaining and imaging of thick brain samples. However, this advanced technology has not yet been used for whole-mount retinas. Although the retina is partially transparent, its thickness of approximately 200 &#181;m (in mice) still limits the penetration of antibodies into the deep tissue as well as reducing light penetration for high-resolution imaging. Here, we adapted the CLARITY method for whole-mount mouse retinas by polymerizing them with an acrylamide monomer to form a nanoporous hydrogel and then clearing them in sodium dodecyl sulfate to minimize protein loss and avoid tissue damage. CLARITY-processed retinas were immunostained with antibodies for retinal neurons, glial cells, and synaptic proteins, mounted in a refractive index matching solution, and imaged. Our data demonstrate that CLARITY&#160;can improve the quality of standard immunohistochemical staining and imaging for retinal neurons and glial cells in whole-mount preparation. For instance, 3D resolution of fine axon-like and dendritic structures of dopaminergic amacrine cells were much improved by CLARITY. Compared to non-processed whole-mount retinas, CLARITY can reveal immunostaining for synaptic proteins such as postsynaptic density protein 95. Our results show that CLARITY renders the retina more optically transparent after the removal of lipids and preserves fine structures of retinal neurons and their proteins, which can be routinely used for obtaining high-resolution imaging of retinal neurons and their subcellular structures in whole-mount preparation.

Here we present a protocol to adapt the CLARITY method of the brain tissues for whole-mount retinas to improve the quality of standard immunohistochemical staining and high-resolution imaging of retinal neurons and their subcellular structures.

使用清晰组织清除方法对全山视网膜进行免疫染色

The tissue hydrogel delipidation method (CLARITY), originally developed by the Deisseroth laboratory, has been modified and widely used for immunostaining ...

Imaging and Quantification of Intact Neuronal Dendrites via CLARITY Tissue Clearing

Brain activity, the electrochemical signals passed between neurons, is determined by the connectivity patterns of neuronal networks, and from the morphology of processes and substructures within these neurons. As such, much of what is known about brain function has arisen alongside developments in imaging technologies that allow further insight into how neurons are organized and connected in the brain. Improvements in tissue clearing have allowed for high-resolution imaging of thick brain slices, facilitating morphological reconstruction and analyses of neuronal substructures, such as dendritic arbors and spines. In tandem, advances in image processing software provide methods of quickly analyzing large imaging datasets. This work presents a relatively rapid method of processing, visualizing, and analyzing thick slices of labeled neural tissue at high-resolution using CLARITY tissue clearing, confocal microscopy, and image analysis. This protocol will facilitate efforts toward understanding the connectivity patterns and neuronal morphologies that characterize healthy brains, and the changes in these characteristics that arise in diseased brain states.

Neuronal dendritic morphology often underlies function. Indeed, many disease processes that affect the development of neurons manifest with a morphological phenotype. This protocol describes a simple and powerful method for analyzing intact dendritic arbors and their associated spines.

通过 CLARITY 组织清除对完整神经元树突进行成像和定量

Brain activity, the electrochemical signals passed between neurons, is determined by the connectivity patterns of neuronal networks, and from the ...

A Tissue Clearing Method for Neuronal Imaging from Mesoscopic to Microscopic Scales

A detailed protocol is provided here to visualize neuronal structures from mesoscopic to microscopic levels in brain tissues. Neuronal structures ranging from neural circuits to subcellular neuronal structures are visualized in mouse brain slices optically cleared with ScaleSF. This clearing method is a modified version of ScaleS and is a hydrophilic tissue clearing method for tissue slices that achieves potent clearing capability as well as a high-level of preservation of fluorescence signals and structural integrity. A customizable three dimensional (3D)-printed imaging chamber is designed for reliable mounting of cleared brain tissues. Mouse brains injected with an adeno-associated virus vector carrying enhanced green fluorescent protein gene were fixed with 4% paraformaldehyde and cut into slices of 1-mm thickness with a vibrating tissue slicer. The brain slices were cleared by following the clearing protocol, which include sequential incubations in three solutions, namely, ScaleS0 solution, phosphate buffer saline (&#8211;), and ScaleS4 solution, for a total of 10.5&#8211;14.5 h. The cleared brain slices were mounted on the imaging chamber and embedded in 1.5% agarose gel dissolved in ScaleS4D25(0) solution. The 3D image acquisition of the slices was carried out using a confocal laser scanning microscope equipped with a multi-immersion objective lens of a long working distance. Beginning with mesoscopic neuronal imaging, we succeeded in visualizing fine subcellular neuronal structures, such as dendritic spines and axonal boutons, in the optically cleared brain slices. This protocol would facilitate understanding of neuronal structures from circuit to subcellular component scales.

The protocol provides a detailed method of neuronal imaging in brain slice using a tissue clearing method, ScaleSF. The protocol includes brain tissue preparation, tissue clarification, handling of cleared slices and confocal laser scanning microscopy imaging of neuronal structures from mesoscopic to microscopic levels.

一种从介观尺度到微观尺度的神经元成像组织清除方法

A detailed protocol is provided here to visualize neuronal structures from mesoscopic to microscopic levels in brain tissues. Neuronal structures ranging ...

Three-dimensional Reconstruction of the Vascular Architecture of the Passive CLARITY-cleared Mouse Ovary

The ovary is the main organ of the female reproductive system and is essential for the production of female gametes and for controlling the endocrine system, but the complex structural relationships and three-dimensional (3D) vasculature architectures of the ovary are not well described. In order to visualize the 3D connections and architecture of blood vessels in the intact ovary, the first important step is to make the ovary optically clear. In order to avoid tissue shrinkage, we used the hydrogel fixation-based passive CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/ Immunostaining/In situ-hybridization-compatible Tissue Hydrogel) protocol method to clear an intact ovary. Immunostaining, advanced multiphoton confocal microscopy, and 3D image-reconstructions were then used for the visualization of ovarian vessels and follicular capillaries. Using this approach, we showed a significant positive correlation (P &#60;0.01) between the length of the follicular capillaries and volume of the follicular wall.

Here we present an adaptation of the passive CLARITY and 3D reconstruction method for visualization of the ovarian vasculature and follicular capillaries in intact mouse ovaries.

无源清清除小鼠卵巢血管构筑的三维重建

The ovary is the main organ of the female reproductive system and is essential for the production of female gametes and for controlling the endocrine ...

生物学

Imaging the Gut with "CLARITY"

CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging compatible Tissue hYdrogel) has recently evolved as a valuable technique involving acrylamide embedding to delipidate tissue (without sectioning) and to preserve the 3-D tissue structure for immunostaining. The technique is highly relevant in imaging the dynamic gut environment where different cell types interact during homeostasis and disease states. This method optimized for the mouse gut is described here, which helps to trace cell types like epithelia, enteroendocrine, neurons, glia, and the neuronal projections into the epithelia or enteroendocrine cells that mediate microbial sensing or nutrient chemo sensing respectively. The gut tissue (1-1.5 cm) is fixed in 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) at 4 &#176;C overnight on day 1. On day 2, PFA is discarded, and the tissue is washed thrice with PBS. The tissue is hydrogel embedded to preserve its integrity by incubation in 4% hydrogel (acrylamide) solution in PBS (diluted from 30% ProtoGel) overnight at 4 &#176;C. On day 3, the tissue-hydrogel solution is incubated at 37&#160;&#176;C for 1 h to allow hydrogel polymerization. Tissue is then washed thrice gently with PBS to remove excess hydrogel. The subsequent step of delipidation (clearing) involves tissue incubation in sodium dodecyl sulfate (8% SDS in PBS) at 37 &#176;C for 2 days (days 4 &#38; 5) on a shaker at room temperature (RT). On day 6, the cleared tissue is thoroughly washed with PBS to remove SDS. Tissue can be immunostained by incubation in primary antibodies (diluted in 0.5% normal donkey serum in PBS containing 0.3% Triton X-100), overnight at 4&#176;C, and subsequent incubation in appropriate secondary Alexa Fluor antibodies for 1.5 h at RT, and nuclear staining with DAPI (1: 10000). The tissue is transferred onto a clean glass slide and mounted using VectaShield for confocal imaging.

We describe the protocol for passive CLARITY (PACT) staining of mouse intestine to enable visualization of subepithelial tissues, including neurons, glia and enteroendocrine cells (EEC) without tissue sectioning. The protocol involves hydrogel embedding of formaldehyde-fixed tissue, and subsequent delipidation using an anionic detergent to "clear" the tissue.

CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging compatible Tissue hYdrogel) has recently evolved as a valuable technique involving ...