This work was supported in part by a grant from the Office of Naval Research (N000141310569). The Southern Illinois University School of Medicine Research Imaging Facility equipment was supported by the National Center for Research Resources-Health (S10RR027716).

伦理声明:涉及动物主题程序已经批准的机构动物护理和使用委员会在南伊利诺伊大学医学院。 1.提取颞骨的<ol><li>识别颞骨在小鼠头骨13的基极和刮掉使用标准图案镊子脑神经。 </li><li>放置标准图案镊子在听囊的前端与具有相反手压机的拇指向下后半规管撞出包封耳蜗。 </li><li>从头骨用拇指和食指或用10.5厘米细剪刀手工释放底部的颞骨的一半。 </li></ol> 2.定位后颞骨<ol><li>放置颞骨到含有250 2 ml微量离心管 - 加入500μl的4％多聚甲醛（PFA）稀释在10mM磷酸盐缓冲盐水（PBS）pH 7.4的和incub吃在RT 2 - 20小时。 注:没有开放的顶帽疫区或注射入圆形或椭圆形窗是必要的。推荐使用甲醇自由，超纯水，电子显微镜（EM）级的PFA，可以在玻璃小瓶中16％的浓度被购买。一旦小瓶打开，稀释1:在PBS中的4，制备4％的溶液，它可用于长达2周贮存在 4℃时（一些抗体需要短固定和其他人可以容忍O / N（O / N）固定）。 </li></ol> 3.脱钙颞骨<ol><li>固定后，用移液管除去PFA，并替换为120毫乙二胺四乙酸（EDTA），略超过2毫升确保以填充整个2 ml离心管，以防止当管闭合气泡。 <ol><li>如果不立 ​​即脱钙，用10毫米的PBS pH值7.4并确保样品更换PFA在4℃。 注:固定后，颞骨可存放variab乐大量的时间脱钙取决于抗原被检测之前。 </li></ol></li><li>在最终的过肩年底将管和旋转在4转速在室温。用吸管每日更换EDTA溶液。脱钙长度取决于样品的年龄和科学家做夹层的偏好。 <ol><li>使用孵化倍以下准则孵育颞骨的EDTA: 对于样品产后天（P）8〜P15:2 - 4小时;如需样品P15到P21:O / N;如需样品P21到P30:2 O / N;对于年龄大于P30样品:3以上的O / N。 注:脱钙时间都受到用户的喜好，并可以根据需要进行扩展。脱钙时间可以由天改变EDTA溶液两次，大约8小时，相隔降低。一些实验室加1％PFA中于EDTA溶液脱钙时为长于3天，以防止污染。 </li></ol></li><li>要确定样品充分脱钙，将颞骨Ø呐硅弹性体涂层剥离菜，轻轻按下钳到蜗形耳蜗。如果组织是spongey，然后脱钙完成。 </li><li>一旦脱钙完成后，用移液管除去EDTA和添加10mM的pH 7.4的PBS 500 -1,000微升。样品储存在摄氏 4度，直到准备解剖。 </li></ol> 4.创建有机硅弹性体涂层夹层菜<ol><li>结合的基极和有机硅弹性体密封剂的试剂盒，根据制造商的说明固化剂并充分混合。 </li><li>添加约2 - 3汤匙木炭粉，直到解决的办法是黑色的，拌匀。 注:液体油墨或液体木炭不会与溶液混合，并且不能使用。有机硅弹性体密封袋可以在黑色购买，允许步骤4.2省略。 </li><li>将溶液倒入60毫米的玻璃或塑料培养皿，充足的外套的底部。如果BUbbles都存在，粉扑与空气的表面上。静置至少24小时，在RT下凝固。 注:硅酮弹性体涂覆的菜肴，可反复使用多年。然而不使用乙醇进行清洗，因为这将导致有机硅弹性体产生裂纹。 </li></ol>耳蜗的5整装夹层（为P7和较旧的示例） <ol><li>分离底转由中间/顶圈<ol><li>放置在硅氧烷弹性体涂覆的解剖菜填充三分之二全用10mM pH 7.4的PBS 1脱钙颞骨和使用立体声解剖显微镜以下步骤。 </li><li>持在前庭区域的颞骨与＃4或＃5直珠宝商的镊子，并使用5毫米Vannas-杜宾根弹簧剪刀，剪去多余的听囊组织沿着侧面和顶点的上方。 </li><li>使用2.5毫米Vannas弹簧剪刀，将一个刀片插入椭圆形窗口，并沿着螺旋韧带/横向几个小伤口壁的底转。 </li><li>使用5毫米Vannas，蒂宾根弹簧剪刀，将一个刀片插入只是削减了区域并将另一个刀片上的颞骨外，内侧卵圆窗。这种切割分离底转由中部和心尖圈。 </li></ol></li><li>的底转完成清扫。 <ol><li>使用2.5毫米Vannas弹簧剪，连接到蜗轴来释放在底转的张力，切下底转，以从前庭器官分离螺旋神经节神经纤维。 </li><li>使用2.5毫米Vannas弹簧剪刀，使一系列小切口，以从上方和下方Corti器除去螺旋韧带/侧壁。使用4号或5＃直珠宝商的镊子来指导组织。引脚上的螺旋神经节神经纤维的有机硅弹性体涂层剥离菜，但不要抱到本地区的组织会撕裂。 </li><li>在前面的步骤，一些赖斯纳的MEMBRAN的则e会经常被移除。如果任何仍然存在，把握赖斯纳的膜＃4或5＃直珠宝商的镊子和Corti器的绝尘而去。 注:盖膜很少是可见的，通常浮离开，而无需去除特定的步骤。 </li><li>终于使几个切口以减小螺旋神经节轴突的厚度，以使转尽可能平整并使用＃4或＃5直珠宝商的镊子转移解剖底转，通过抓住螺旋神经节的剩余轴突，到一个48孔板（或腔室滑动）含有〜500微升的10mM pH 7.4的PBS。 </li></ol></li><li>独立的中部和顶端圈<ol><li>将剩余的三分之二的耳蜗，顶面朝下的。 </li><li>使用2.5毫米Vannas弹簧剪刀，插入一个刀片成当所用的中间转连接到底转中阶，并且使沿第的螺旋韧带/侧壁几个小伤口Ë中间转。 </li><li>用5毫米Vannas-杜宾根弹簧剪刀，插入一个刀片成刚切断与中间转放置在叶片的顶部的区域中，并把其他刀片上的骨迷路外，在从顶端末端的90°角。这种分离顶圈中间转。 </li></ol></li><li>中间转以类似的方式，以底转完整解剖（见步骤5.2.2至5.2.4节）。 </li><li>的顶圈完成清扫。 <ol><li>使用2.5毫米Vannas弹簧剪刀，打开盖顶转帽。以类似的方式，以底转完整解剖（见步骤5.2.2至5.2.4节）。 注:解剖耳蜗匝免疫染色之前存储在10毫米的PBS pH值7.4在48孔板（或玻片） 在 4℃数周。然而PBS将蒸发，需要定期补充和存储长于2 - 3个星期可能会导致对细菌或真菌生长组织，可以降低图像的质量。我们建议长期存放的样品作为undissected颞骨的。 </li></ol></li></ol> 6.免疫染色与荧光标记的抗体<ol><li>解剖后，存储每个耳蜗轮流在一个单独的井在48孔板（或玻片），淹没在〜500微升10毫PBS pH值7.4。对于每一个执行以下步骤的耳蜗转弯应该浸没在液体中，而不是浮在上面或粘到井的侧面。 注:从每个去除液体很好，很容易失去耳蜗转弯或吸起来的枪头。更改使用解剖范围200微升枪头，解决方案将帮助防止这一点。 </li><li>使用移液管，从每个孔除去的PBS，并替换为〜200 - 每阻断/透化溶液（井300微升1％的Triton X-100，1％牛血清白蛋白（BSA）和10％正常山羊血清（NGS）稀释在10mM PBS中p^ h 7.4）。孵育1小时，在室温上的三维旋转。 注意:如果使用的任何第一抗体是在山羊宿主，然后一个第二抗羊抗体将需要和NGS 不应该被用于任何的步骤。正常马血清可以用作替代NGS。 </li><li>使用移液管除去阻断/透化溶液，并替换为每一次抗体溶液以及〜100微升（0.1％的Triton X-100，稀释在10mM pH 7.4的PBS的1％BSA和5％NGS）。每个初级抗体的稀释因子而变化。孵育O / N（最小14小时），在摄氏 4度的三维旋转。 注意:如果一个以上的一级抗体时，所有可组合成孵育相同的溶液，只要确保每个主抗体具有不同的主机。 </li><li>使用移液管除去第一抗体溶液，并以每孔〜500微升执行的10mM pH 7.4的PBS洗涤3次。每次洗涤孵化上的3D旋转器最少5分钟，在室温。 </li><li>删除后PBS洗涤利用移液管，并替换为〜100μl的每二次抗体溶液以及（0.1％的Triton X-100，稀释在10mM pH 7.4的PBS的1％BSA和5％NGS）。每个稀释因子荧光标记的第二抗体，通常为1:500，或1:1000。将48孔板在一个黑盒子，以保护荧光从光标记二级抗体。孵育2 - 3小时，在室温上的三维旋转。 注意:如果一个以上的第二抗体是需要的，它们可以组合成用于孵育的相同溶液。确保有交叉标记的可能性（例如，使用山羊抗兔和鸡抗山羊二抗） </li><li>使用移液管除去二级抗体溶液，并在每孔〜500微升执行的10mM pH 7.4的PBS洗涤3次。孵育每次洗涤为至少5分钟，在室温上的三维旋转。保持48孔板的一个黑盒子避光。 </li><li>用吸管取出最后的PBS洗涤和更换，每Hoec的嘛〜100微升HST 33342（稀释1:2000在10mM的PBS pH 7.4）中来标记细胞核。孵育15 - 在3D肩在室温下20分钟。保持48孔板的一个黑盒子避光。不要孵育时间超过20分钟。 </li><li>使用移液管除去Hoechst的溶液，并在每孔〜500微升执行的10mM pH 7.4的PBS洗涤3次。孵育每次洗涤为至少5分钟，在室温上的三维旋转。保持48孔板的一个黑盒子避光。 </li><li>如果需要的话所有步骤可以延长，除Hoechst的孵化，由几个小时。要暂停反应在任何阶段，只是淹没在〜500微升10毫PBS pH7.4的样品，并在 4℃，直到准备在48 -嗯板（或玻片）存储恢复协议小时或1 -后2天。 </li></ol> 7.安装人工耳蜗打开幻灯片<ol><li>标签幻灯片关于所使用的样品和抗体的相关信息。 </li><li>吸取〜50微升安装介质到每张幻灯片，小心以防止泡沫。离心管中的安装介质，以消除任何气泡。 </li><li>使用＃4或＃5直珠宝商的镊子，把握螺旋神经节的轴突从48孔板在安装介质轻轻传送一个耳蜗转于滑动和地点。每滑动摩1耳蜗转，以防止在成像过程中曝光和光漂白。 </li><li>使用立体声解剖显微镜，以确保耳蜗转弯不折叠，扭曲或附近的气泡。如果这些情况发生，使用＃4或5＃直珠宝商的镊子重新定位耳蜗转。 </li><li>放置在幻灯片上的盖玻片的一端，轻轻松开，让盖玻片下降。 </li><li>使用立体声解剖显微镜，以确保耳蜗转弯没有被折叠，扭曲或附近的气泡。如果这些情况发生时，轻轻将盖玻片来回重新定位耳蜗转。 </li><li>将幻灯片在一个幻灯片文件夹，以便他们放平。让安装媒体治愈O / N在室温（保持在黑暗中） </li><li>密封盖玻片透明指甲油和储存在室温或-20℃，直到成像。玻片可以存储在一个文件夹中滑动或滑动箱。 注:幻灯片可以在-20℃或-80℃，荧光保存长期将维持数月。 </li><li>使用共聚焦显微镜与基于免疫染色过程中所用的第二抗体的合适的波长的图像的幻灯片。亮度和对比度调整可以使用由共焦供应商提供的成像软件来执行。 </li></ol>

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The authors have nothing to disclose.

<html>有成功的整装解剖和免疫组化的几个关键步骤。但是前两种方法之一被执行，需要耳蜗组织的正确固定。我们建议使用甲醇免费，超纯水，EM级PFA。 PFA从粉末制成的可以有甲醇和不稳定的pH值从而降低免疫荧光的质量的痕迹。其他组也表明类似夹层是可能使用的固定剂不含有甲醛14-16。固定的长度也很重要，是抗体特异性的。一些抗体能够容忍的O / N固定，而另一些人不只是1小时正常?...

内耳的螺旋形耳蜗，包含在颞骨内，容纳尔蒂，在哺乳动物听觉感官端器官的器官。耳蜗是tonotopically组织并通常分为顶，中间，和基底轮流对应于不同的频率范围具有高频率的声音的检测，在碱和低频检测在顶点1。毛细胞，Corti器的mechanosensory细胞，运行耳蜗，这大约是6毫米长的小鼠2,3的长度。这些细胞将声波转换，这是通过填充流体的膜迷路发送，进入通过中央听觉结构处理的神经信号的机械能。此处所描述的技术提供了制备柯蒂氏器的全样载耳蜗后钙化的方法完成（为样本范围从年龄一周成年）。我们还提出了immunosta的方法都进不去，整个安装耳蜗组织。人工耳蜗整个坐骑是所有毛细胞的可视化和周边配套细胞在自然空间布局的关键，并允许进行分析的三个维度与使用共焦显微镜。 博士。汉斯恩斯特龙和哈洛阿德斯最初描述整个支架耳蜗解剖法在1966年他们详述的技术迅速修复和解剖钙化耳蜗浸没在液体中从各种哺乳动物，保留柯蒂氏器的短完好分段微观分析4。不固定，钙化大鼠耳蜗的解剖也已在教学视频5所示 。博士。芭芭拉Bohne和加里·史密斯在华盛顿大学取得了一些重要的修改这个方法。在他们的耳蜗整装方法的版本，颞骨是脱钙，嵌入塑料，和五个半圈，十四分之一圈是dissec特德6,7。查尔斯·利伯曼博士，并在伊顿皮博迪实验室，麻省眼耳医院的同事，修改了此技术使塑料包埋是不需要8。该技术的进一步修改发生在践祚博士的实验室在圣裘德儿童研究医院9-12该通知这里介绍的清扫方法。我们使用了不同的策略来访问科尔蒂比Bohne和利伯曼，的器官，它允许完全根尖，中间，和基底匝隔离。因此，解剖组织较大且较不可能在解剖或免疫染色过程丢失或损坏。此外，目前的方法有利于从心尖尖端或基底钩，以确定的频率区域的距离的测量。 尽管许多实验室进行耳蜗组织免疫，目前还不清楚其中这些方法起源。其结果是有各种配方用于阻断buffeRS和抗体孵育缓冲液可能影响个体初级抗体的性能。这里，我们提出的一种方法使用本身是适用于听觉领域最常用的抗体荧光标记的抗体的免疫染色。 形状复杂的耳蜗，柯蒂氏器的精巧结构，和骨包套提供用于组织学和生物化学分析的一个挑战。多种技术的听证会现场，目前用于克服这些困难的特点和尔蒂，每种技术都有自己的优点和缺点的器官内可视化的细胞。这里介绍的协议允许成年小鼠耳蜗的整装解剖和，稍加修改，可潜在地用于检查耳蜗内的关键结构从各种在该领域中使用的其他模型生物。

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whole mount dissection and immunofluorescence of the adult mouse cochlea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.

我们提出了一个方法，以分离Corti器的三个完整的耳蜗匝（先端，中间，和碱）从耳蜗组织即钙化， 与图1中呈现键清扫步骤。在第一产后周的发展，钙化小鼠耳蜗是不完整的，更简单的夹层方法可用于13。使用新生儿整装解剖法耳蜗Corti器从P7和小鼠的结果流泪老年人和碎纸。螺旋韧带/侧壁现在更牢固地附着，并且不能剥离远离感觉上皮?...

Watch this Scientific Journal Video about Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea at JoVE.com

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

我们提出了一个方法，以分离科尔蒂的成年器官三个完整耳蜗匝（先端，中间，和碱）。我们还演示了一个程序，用荧光标记的抗体免疫染色。连同这些技术允许毛细胞，支持细胞，以及其他细胞类型中的耳蜗研究发现。

全山解剖和成年小鼠耳蜗的免疫

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a ...

whole-mount-dissection-immunofluorescence-adult-mouse

Research

JoVE Journal

Neuroscience

54.6K 次观看.  Southern Illinois University, School of Medicine. 我们提出了一个方法，以分离科尔蒂的成年器官三个完整耳蜗匝（先端，中间，和碱）。我们还演示了一个程序，用荧光标记的抗体免疫染色。连同这些技术允许毛细胞，支持细胞，以及其他细胞类型中的耳蜗研究发现。 

视频: Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the procedure for the visualization of Drosophila embryonic nervous system using antibodies to neuronal proteins. Since the entire embryonic peripheral nervous and central nervous systems are well characterized at the level of individual cells (Dambly-Chaudi&egrave;re et al., 1986; Bodmer et al., 1987; Bodmer et al., 1989), any aberrations to these systems can be easily identified using antibodies to different neuronal proteins. The developing embryos are collected at certain times to ensure that the embryos are in the proper developmental stages for visualization. After collection, the outer layers of the embryo, the chorion membrane and the vitelline envelope that surrounds the embryo, are removed before fixation. Embryos are then incubated with neuronal antibodies and visualized using fluorescently labeled secondary antibodies. Embryos at stages 12-17 are visualized to access the embryonic nervous system. At stage 12 the CNS germ band starts shortening and by stage 15 the definitive pattern of the commissure has been achieved. By stage 17 the CNS contracts and the PNS is fully developed (Campos-Ortega et al. 1985). Thus changes in the pattern of the PNS and CNS can be easily observed during these developmental stages.

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

We describe the procedure to prepare staged Drosophila embryos for the visualization of the embryonic nervous system during embryogenesis.

在全安装的胚胎神经系统的可视化果蝇胚胎

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the ...

科研

神经科学

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and cones). The RPE is a monolayer of pigmented cuboidal cells and associates closely with the neural retina just above it. This association makes the RPE of great interest to researchers studying retinal diseases. The RPE is also the site of an in vivo assay of homology-directed DNA repair, the pun assay. The mouse eye is particularly difficult to dissect due to its small size (about 3.5mm in diameter) and its spherical shape. This article demonstrates in detail a procedure for dissection of the eye resulting in a whole mount of the RPE. In this procedure, we show how to work with, rather than against, the spherical structure of the eye. Briefly, the connective tissue, muscle, and optic nerve are removed from the back of the eye. Then, the cornea and lens are removed. Next, strategic cuts are made that result in significant flattening of the remaining tissue. Finally, the neural retina is gently lifted off, revealing an intact RPE, which is still attached to the underlying choroid and sclera. This whole mount can be used to perform the pun assay or for immunohistochemistry or immunofluorescent assessment of the RPE tissue.

A formal demonstration of the dissection of a mouse eye, resulting in a whole mount of the retinal pigment epithelium.

解剖鼠标眼的视网膜色素上皮的全山

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and ...

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled samples with little to no tissue damage enables us to analyze cell morphology and to compare individual samples to each other, hence allowing the identification of mutant anomalies.
In the demonstrated dissection method the nervous system remains mostly inside the adult fly. Through a dorsal incision, the abdomen and thorax are opened and most of the internal organs are removed. Only the dorsal side of the ventral nerve cord (VNC) and the cervical connective 
(CvC) containing the big axons of the giant fibers (GFs)1 are exposed, while the brain containing the GF cell body and dendrites remains2 in the 
intact head. In this preparation most nerves of the VNC should remain attached to their muscles. 
Following the dissection, the intracellular filling of the giant fiber (GF) with a fluorescent dye is demonstrated. In the CvC the GF axons are located at the dorsal surface and thus can be easily visualized under a microscope with differential interference contrast (DIC) optics. This allows the injection of the GF axons with dye at this site to label the entire GF including the axons and their terminals in the VNC. This method results in reliable and strong staining of the GFs allowing the neurons to be imaged immediately after filling with an epifluorescent microscope. Alternatively, the fluorescent signal can be enhanced using standard immunohistochemistry procedures3 suitable for high resolution confocal microscopy.

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

An in vivo dissection of the adult Drosophila ventral nerve cord (VNC) is demonstrated. This particular dissection method causes little damage to the VNC allowing the subsequent labeling of the giant fiber neurons with fluorescent dye for high resolution imaging.

成人整装准备果蝇巨纤维染料注射腹侧神经索

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

重复性小鼠坐骨神经挤压和再生的后续评估整装肌肉分析

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

Dissection of Adult Mouse Utricle and Adenovirus-mediated Supporting-cell Infection

Hearing loss and balance disturbances are often caused by death of mechanosensory hair cells, which are the receptor cells of the inner ear. Since there is no cell line that satisfactorily represents mammalian hair cells, research on hair cells relies on primary organ cultures. The best-characterized in vitro model system of mature mammalian hair cells utilizes organ cultures of utricles from adult mice (Figure 1) 1-6. The utricle is a vestibular organ, and the hair cells of the utricle are similar in both structure and function to the hair cells in the auditory organ, the organ of Corti. The adult mouse utricle preparation represents a mature sensory epithelium for studies of the molecular signals that regulate the survival, homeostasis, and death of these cells.
Mammalian cochlear hair cells are terminally differentiated and are not regenerated when they are lost. In non-mammalian vertebrates, auditory or vestibular hair cell death is followed by robust regeneration which restores hearing and balance functions 7, 8. Hair cell regeneration is mediated by glia-like supporting cells, which contact the basolateral surfaces of hair cells in the sensory epithelium 9, 10. Supporting cells are also important mediators of hair cell survival and death 11. We have recently developed a technique for infection of supporting cells in cultured utricles using adenovirus. Using adenovirus type 5 (dE1/E3) to deliver a transgene containing GFP under the control of the CMV promoter, we find that adenovirus specifically and efficiently infects supporting cells. Supporting cell infection efficiency is approximately 25-50%, and hair cells are not infected (Figure 2). Importantly, we find that adenoviral infection of supporting cells does not result in toxicity to hair cells or supporting cells, as cell counts in Ad-GFP infected utricles are equivalent to those in non-infected utricles (Figure 3). Thus adenovirus-mediated gene expression in supporting cells of cultured utricles provides a powerful tool to study the roles of supporting cells as mediators of hair cell survival, death, and regeneration.

Mechanosensory hair cells are the receptor cells of the inner ear. The best-characterized in vitro model system of mature mammalian hair cells utilizes organ cultures of utricles from adult mice. We present the dissection of the adult mouse utricle, and we demonstrate adenovirus-mediated infection of supporting cells in cultured utricles.

解剖成年小鼠的椭圆囊和腺病毒介导的支撑细胞的感染

Hearing loss and balance disturbances are often caused by death of mechanosensory hair cells, which are the receptor cells of the inner ear.  Since there ...

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Angiogenesis is the complex process of new blood vessel formation defined by the sprouting of new blood vessels from a pre-existing vessel network. Angiogenesis plays a key role not only in normal development of organs and tissues, but also in many diseases in which blood vessel formation is dysregulated, such as cancer, blindness and ischemic diseases. In adult life, blood vessels are generally quiescent so angiogenesis is an important target for novel drug development to try and regulate new vessel formation specifically in disease. In order to better understand angiogenesis and to develop appropriate strategies to regulate it, models are required that accurately reflect the different biological steps that are involved. The mouse neonatal retina provides an excellent model of angiogenesis because arteries, veins and capillaries develop to form a vascular plexus during the first week after birth. This model also has the advantage of having a two-dimensional (2D) structure making analysis straightforward compared with the complex 3D anatomy of other vascular networks. By analyzing the retinal vascular plexus at different times after birth, it is possible to observe the various stages of angiogenesis under the microscope. This article demonstrates a straightforward procedure for analyzing the vasculature of a mouse retina using fluorescent staining with isolectin and vascular specific antibodies.

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

The neonatal murine retina provides a well characterized physiological model of angiogenesis, which permits investigations of the roles of different genes or drugs that modulate angiogenesis in an in vivo context. Immunofluorescent staining to accurately visualize the vascular plexus is pivotal to the success of these types of studies.

整装免疫荧光染色新生小鼠视网膜血管生成调查<em&gt;在体内</em

Angiogenesis  is the complex process of new blood vessel formation defined by the sprouting  of new blood vessels from a pre-existing vessel network. ...

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat muscles can offer significant advantage over traditionally used thicker muscles, such as those from the hind limb (e.g. gastrocnemius). Thin muscles allow for comprehensive overview of the entire innervation pattern for a given muscle, which in turn permits identification of selectively vulnerable pools of motor neurons. These muscles also allow analysis of parameters such as motor unit size, axonal branching, and terminal/nodal sprouting. A common obstacle in using such muscles is gaining the technical expertise to dissect them. In this video, we detail the protocol for dissecting the transversus abdominis (TVA) muscle from young mice and performing immunofluorescence to visualize axons and neuromuscular junctions (NMJs). We demonstrate that this technique gives a complete overview of the innervation pattern of the TVA muscle&#160;and can be used to investigate NMJ pathology in a mouse model of the childhood motor neuron disease, spinal muscular atrophy.

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

In this video we demonstrate a protocol for dissection of the transversus abdominis muscle of the mouse and use immunofluorescence and microscopy to visualize neuromuscular junctions.

全山神经肌肉交界分析的 横向解剖阿布多米尼斯 肌肉

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

嗅觉受体神经元在全山免疫标记<em&gt;果蝇</em&gt;天线

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we describe a method to label in situ a subset of dorsal root ganglion (DRG) axon projections to assess their phenotypic characteristics using several genetically manipulated mouse lines. The TrkA-positive neurons are nociceptor neurons, dedicated to the transmission of pain signals. We utilize a TrkAtaulacZ mouse line to label the trajectories of all TrkA-positive peripheral axons in the intact mouse embryo. We further breed the TrkAtaulacZ line onto a Bax null background, which essentially abolishes neuronal apoptosis, in order to assess growth-related questions independently of possible effects of genetic manipulations on neuronal survival. Subsequently, genetically modified mice of interest are bred with the TrkAtaulacZ/Bax null line and are then ready for study using the techniques described herein. This presentation includes detailed information on mouse breeding plans, genotyping at the time of dissection, tissue preparation, staining and clearing to allow for visualization of full-length axonal trajectories in whole-mount preparation.

We present here an optimized protocol to genotype, stain and prepare fetal mice for the imaging of peripheral nociceptor axon projections in the whole animal, as an effective method to assess sensory axon growth phenotypes in developing genetically engineered mice.

小鼠胚胎感觉轴突预测整个安装成像

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we ...

Whole Mount Labeling of Cilia in the Main Olfactory System of Mice

The mouse olfactory system comprises 6-10 million olfactory sensory neurons in the epithelium lining the nasal cavity. Olfactory neurons extend a single dendrite to the surface of the epithelium, ending in a structure called dendritic knob. Cilia emanate from this knob into the mucus covering the epithelial surface. The proteins of the olfactory signal transduction cascade are mainly localized in the ciliary membrane, being in direct contact with volatile substances in the environment. For a detailed understanding of olfactory signal transduction, one important aspect is the exact morphological analysis of signaling protein distribution. Using light microscopical approaches in conventional cryosections, protein localization in olfactory cilia is difficult to determine due to the density of ciliary structures. To overcome this problem, we optimized an approach for whole mount labeling of cilia, leading to improved visualization of their morphology and the distribution of signaling proteins. We demonstrate the power of this approach by comparing whole mount and conventional cryosection labeling of Kirrel2. This axon-guidance adhesion molecule is known to localize in a subset of sensory neurons and their axons in an activity-dependent manner. Whole mount cilia labeling revealed an additional and novel picture of the localization of this protein.

Cilia of olfactory sensory neurons contain proteins of the signal transduction cascade, but a detailed spatial analysis of their distribution is difficult in cryosections. We describe here an optimized approach for whole mount labeling and en face visualization of ciliary proteins.

纤毛对小鼠的主要嗅觉系统整装标签

The mouse olfactory system comprises 6-10 million olfactory sensory neurons in the epithelium lining the nasal cavity. Olfactory neurons extend a single ...

全山解剖和成年小鼠耳蜗的免疫

摘要

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此视频中的章节

在全安装的胚胎神经系统的可视化果蝇胚胎

解剖鼠标眼的视网膜色素上皮的全山

成人整装准备果蝇巨纤维染料注射腹侧神经索

重复性小鼠坐骨神经挤压和再生的后续评估整装肌肉分析

解剖成年小鼠的椭圆囊和腺病毒介导的支撑细胞的感染

整装免疫荧光染色新生小鼠视网膜血管生成调查

全山神经肌肉交界分析的横向解剖阿布多米尼斯肌肉

嗅觉受体神经元在全山免疫标记

小鼠胚胎感觉轴突预测整个安装成像

纤毛对小鼠的主要嗅觉系统整装标签

全山解剖和成年小鼠耳蜗的免疫

摘要

探索更多视频

此视频中的章节

在全安装的胚胎神经系统的可视化果蝇胚胎

解剖鼠标眼的视网膜色素上皮的全山

成人整装准备果蝇巨纤维染料注射腹侧神经索

重复性小鼠坐骨神经挤压和再生的后续评估整装肌肉分析

解剖成年小鼠的椭圆囊和腺病毒介导的支撑细胞的感染

整装免疫荧光染色新生小鼠视网膜血管生成调查

全山神经肌肉交界分析的 横向解剖阿布多米尼斯 肌肉

嗅觉受体神经元在全山免疫标记

小鼠胚胎感觉轴突预测整个安装成像

纤毛对小鼠的主要嗅觉系统整装标签

全山神经肌肉交界分析的横向解剖阿布多米尼斯肌肉