Name: whole mount dissection and immunofluorescence of the adult mouse cochlea
Uploaded: 2016-01-01T15:00:00.000+00:00
Duration: 12 min 2 s
Description: Watch this Scientific Journal Video about Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea at JoVE.com

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。

<table><tbody><tr><td>Standard&amp;nbsp; pattern forceps</td><td>Fine Science Tools</td><td>11000-12</td><td>can be purchased from other vendors</td></tr><tr><td>10.5 cm fine scissors</td><td>Fine Science Tools</td><td>14060-11</td><td>can be purchased from other vendors</td></tr><tr><td>2 ml microcentrifuge tubes</td><td>MidSci</td><td>AVSS2000</td><td>can be purchased from other vendors</td></tr><tr><td>16% formaldehyde, methanol free, ultra pure, EM grade</td><td>Polysciences</td><td>18814</td><td>TOXIC --wear gloves and cannot be disposed of in the sink. Can be purchased from other vendors.&amp;nbsp;</td></tr><tr><td>PBS pH 7.4&amp;nbsp;</td><td>Sigma</td><td>P3813-10PAK</td><td>can be purchased from other vendors</td></tr><tr><td>EDTA</td><td>Fisher</td><td>BP118-500</td><td>can be purchased from other vendors</td></tr><tr><td>end-over-end tube rotator</td><td>Fisher</td><td>05-450-127</td><td>can be purchased from other vendors</td></tr><tr><td>60 mm petri dish</td><td>Fisher</td><td>50-202-037</td><td>can be purchased from other vendors</td></tr><tr><td>Dow Corning Sylgard 184 silicone encapsulant kit</td><td>Ellsworth Adhesives</td><td>184 SIL ELAST KIT 0.5KG</td><td></td></tr><tr><td>activated charcoal</td><td>Fisher</td><td>AC134372500</td><td>can be purchased from other vendors</td></tr><tr><td>stereo dissection microscope</td><td>Zeiss</td><td>Stemi 2000</td><td>can be purchased from other vendors</td></tr><tr><td>Dumont #4 jeweler&#039;s forceps</td><td>Fine Science Tools</td><td>11241-30</td><td></td></tr><tr><td>Dumont #5 jeweler&#039;s forceps</td><td>Fine Science Tools</td><td>11251-20</td><td></td></tr><tr><td>2.5 mm Vannas spring scissors</td><td>Fine Science Tools</td><td>15001-08</td><td>curved&amp;nbsp;</td></tr><tr><td>5 mm Vannas-Tubingen spring scissors</td><td>Fine Science Tools</td><td>15003-08</td><td>straight</td></tr><tr><td>48-well plates</td><td>Fisher</td><td>08-772-52</td><td>can be purchased from other vendors</td></tr><tr><td>8-well chamber slides</td><td>Fisher</td><td>1256518</td><td>can be purchased from other vendors</td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>X100-500</td><td>can be purchased from other vendors</td></tr><tr><td>BSA, fraction V</td><td>Fisher</td><td>BP1605</td><td>can be purchased from other vendors</td></tr><tr><td>NGS</td><td>Vector labs</td><td>S-1000</td><td>can be purchased from other vendors</td></tr><tr><td>NHS</td><td>Vector labs</td><td>S-2000</td><td>can be purchased from other vendors</td></tr><tr><td>3D rotator</td><td>Midsci</td><td>R3D-710</td><td>can be purchased from other vendors</td></tr><tr><td>Western blot incubation box XL</td><td>Licor</td><td>929-97401</td><td></td></tr><tr><td>Hoechst 33342</td><td>Life Technologies</td><td>H3570</td><td>can be purchased from other vendors</td></tr><tr><td>Prolong gold antifade mounting media</td><td>Life Technologies</td><td>P36930</td><td>can be purchased from other vendors,but mounting medias vary in their ability to protect against photobleaching</td></tr><tr><td>Superfrost Plus Slides</td><td>Fisher</td><td>12-550-15</td><td>can be purchased from other vendors</td></tr><tr><td>coverslips 22 x 22 x 1</td><td>Fisher</td><td>12-548-B</td><td>can be purchased from other vendors</td></tr><tr><td>clear nail polish</td><td>Local drug store</td><td></td><td>can be purchased from other vendors</td></tr><tr><td>cardboard slide folder</td><td>Fisher</td><td>12-587-10</td><td>can be purchased from other vendors</td></tr><tr><td>plastic slide box</td><td>Fisher</td><td>03-448-10</td><td>can be purchased from other vendors</td></tr></tbody></table>

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

54.9K 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

Vibratome Sectioning for Enhanced Preservation of the Cytoarchitecture of the Mammalian Organ of Corti

The mammalian organ of Corti is a highly ordered cellular mosaic of mechanosensory hair and nonsensory supporting cells (reviewed in 1,2).Visualization of this cellular mosaic often requires that the organ of Corti is cross-sectioned. In particular, the nonsensory pillar and Deiters' cells, whose nuclei are located basally with respect to the hair cells, cannot be visualized without cross-sectioning the organ of Corti. However, the delicate cytoarchitecture of the mammalian organ of Corti, including the fine cytoplasmic processes of the pillar and Deiters' cells, is difficult to preserve by routine histological procedures such as paraffin and cryo-sectioning, which are compatible with standard immunohistochemical staining techniques.
Here I describe a simple and robust procedure consisting of vibratome sectioning of the cochlea, immunohistochemical staining of these vibratome sections in whole mount, followed by confocal microscopy. This procedure has been used widely for immunhistochemical analysis of multiple organs, including the mouse limb bud, zebrafish gut, liver, pancreas, and heart (see 3-6 for selected examples). In addition, this procedure was sucessful for both imaging and quantitificaton of pillar cell number in mutant and control organs of Corti in both embryos and adult mice 7. This method, however, is currently not widely used to examine the mammalian organ of Corti. The potential for this procedure to both provide enhanced preservation of the fine cytoarchitecture of the adult organ of Corti and allow for quantification of various cell types is described.

A simple procedure of vibratome sectioning the organ of Corti, followed by immunohistochemistry and confocal microscopy is described. This procedure allows for improved preservation of the fine cytoarchitecture of the mammalian organ of Corti, and consequently allows for accurate quantification of cell types.

Vibratome切片增强保尔蒂哺乳动物器官的细胞构筑

The mammalian organ of Corti is a highly ordered cellular mosaic of mechanosensory hair and nonsensory supporting cells (reviewed in 1,2).Visualization of ...

科研

神经科学

Whole-mount Confocal Microscopy for Adult Ear Skin: A Model System to Study Neuro-vascular Branching Morphogenesis and Immune Cell Distribution

Here, we present a protocol of a whole-mount adult ear skin imaging technique to study comprehensive three-dimensional neuro-vascular branching morphogenesis and patterning, as well as immune cell distribution at a cellular level. The analysis of peripheral nerve and blood vessel anatomical structures in adult tissues provides some insights into the understanding of functional neuro-vascular wiring and neuro-vascular degeneration in pathological conditions such as wound healing. As a highly informative model system, we have focused our studies on adult ear skin, which is readily accessible for dissection. Our simple and reproducible protocol provides an accurate depiction of the cellular components in the entire skin, such as peripheral nerves (sensory axons, sympathetic axons, and Schwann cells), blood vessels (endothelial cells and vascular smooth muscle cells), and inflammatory cells. We believe this protocol will pave the way to investigate morphological abnormalities in peripheral nerves and blood vessels as well as the inflammation in the adult ear skin under different pathological conditions.

Here, we describe a high resolution whole-mount imaging method in the entire adult mouse ear skin, which enables us to visualize branching morphogenesis and patterning of peripheral nerves and blood vessels, as well as immune cell distribution.

成人耳皮肤全安装共焦显微术: 一种研究神经血管分支形态发生和免疫细胞分布的模型系统

Here, we present a protocol of a whole-mount adult ear skin imaging technique to study comprehensive three-dimensional neuro-vascular branching ...

发育生物学

Evaluation of Planar-Cell-Polarity Phenotypes in Ciliopathy Mouse Mutant Cochlea

In recent years, primary cilia have emerged as key regulators in development and disease by influencing numerous signaling pathways. One of the earliest signaling pathways shown to be associated with ciliary function was the non-canonical Wnt signaling pathway, also referred to as planar cell polarity (PCP) signaling. One of the best places in which to study the effects of planar cell polarity (PCP) signaling during vertebrate development is the mammalian cochlea. PCP signaling disruption in the mouse cochlea disrupts cochlear outgrowth, cellular patterning and hair cell orientation, all of which are affected by cilia dysfunction. The goal of this protocol is to describe the analysis of PCP signaling in the developing mammalian cochlea via phenotypic analysis, immunohistochemistry and scanning electron microscopy. Defects in convergence and extension are manifested as a shortening of the cochlear duct and/or changes in cellular patterning, which can be quantified following dissection from developing mouse mutants. Changes in stereociliary bundle orientation and kinocilia length or positioning can be observed and quantitated using either immunofluorescence or scanning electron microscopy (SEM). A deeper insight into the role of ciliary proteins in cellular signaling pathways and other biological phenomena is crucial for our understanding of cellular and developmental biology, as well as for the development of targeted treatment strategies.

Primary cilia influence various signaling pathways. The mammalian cochlea is ideal for examining planar cell polarity (PCP) signaling. Cilia dysfunction affects cochlear outgrowth, cellular patterning and hair cell orientation, readouts of PCP. Our goal is to analyze PCP signaling in mouse cochlea via phenotypic analysis, immunohistochemistry and scanning electron microscopy.

在Ciliopathy鼠标突变耳蜗平面 - 细胞极性表型的评价

In recent years, primary cilia have emerged as key regulators in development and disease by influencing numerous signaling pathways. One of the earliest ...

医学

Cryosectioning and Immunostaining Mouse Inner Ear Tissue: From Embryonic to Adult Stages

This protocol details general histology methods for preparing inner ear samples from embryonic, neonatal, and adult mice. The purpose of this protocol is to provide a straightforward and standardized method for inner ear tissue processing for researchers, possibly new to the field, interested in working with the mouse cochlea. Included here are protocols for dissection, fixation, embedding, cryosectioning, and immunostaining. Section immunostaining is one of the best methods for examining individual cells within the context of the entire cochlea.
Key steps include dissecting the inner ear away from the skull, fixing the tissue using 4% paraformaldehyde, embedding with Optimal Cutting Temperature compound, cryosectioning, and immunostaining with antibodies targeting specific proteins expressed by the cochlea.
Included in our methods are special considerations made for the cochlea given its 
shape and structure.&#160;Reasonably good focus and dissection skills are needed, as tissue damage can affect the quality of immunostaining and consistency among samples. However, with the procedures we outline, most users can be trained in a few weeks to make beautiful preparations. Overall, this protocol offers a valuable way to promote research to understand auditory development, function, and disease in mouse models.

This protocol, intended for novice users of mouse inner ear tissue, comprehensively details steps for processing mouse inner ears at different developmental stages for section immunostaining.

小鼠内耳组织的冷冻切片和免疫染色：从胚胎阶段到成人阶段

This protocol details general histology methods for preparing inner ear samples from embryonic, neonatal, and adult mice. The purpose of this protocol is ...

Morphological and Functional Evaluation of Ribbon Synapses at Specific Frequency Regions of the Mouse Cochlea

Cochlear inner hair cells (IHCs) transmit acoustic signals to spiral ganglion neurons (SGNs) through ribbon synapses. Several experimental studies have indicated that hair cell synapses may be the initial targets in sensorineural hearing loss (SNHL). Such studies have proposed the concept of cochlear &#34;synaptopathy&#34;, which refers to alterations in ribbon synapse number, structure, or function that result in abnormal synaptic transmission between IHCs and SGNs. While cochlear synaptopathy is irreversible, it does not affect the hearing threshold. In noise-induced experimental models, restricted damage to IHC synapses in select frequency regions is employed to identify the environmental factors that specifically cause synaptopathy, as well as the physiological consequences of disturbing this inner ear circuit. Here, we present a protocol for analyzing cochlear synaptic morphology and function at a specific frequency region in adult mice. In this protocol, cochlear localization of specific frequency regions is performed using place-frequency maps in conjunction with cochleogram data, following which the morphological characteristics of ribbon synapses are evaluated via synaptic immunostaining. The functional status of ribbon synapses is then determined based on the amplitudes of auditory brainstem response (ABR) wave I. The present report demonstrates that this approach can be used to deepen our understanding of the pathogenesis and mechanisms of synaptic dysfunction in the cochlea, which may aid in the development of novel therapeutic interventions.

This manuscript describes an experimental protocol for evaluating the morphological characteristics and functional status of ribbon synapses in normal mice. The present model is also suitable for noise-induced and age-related cochlear synaptopathy-restricted models. The correlative results of previous mouse studies are also discussed.

小鼠耳蜗特定频率区域带状带突起的形态和功能评估

Cochlear inner hair cells (IHCs) transmit acoustic signals to spiral ganglion neurons (SGNs) through ribbon synapses. Several experimental studies have ...

Cochlear Surface Preparation in the Adult Mouse

Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous etiologies such as exposure to excessive noise, the use of ototoxic medications, bacterial or viral ear infections, head injuries, and the aging process. Loss of sensory hair cells is a common pathological feature of the varieties of acquired hearing loss. Additionally, the inner hair cell synapse can be damaged by mild insults. Therefore, surface preparations of cochlear epithelia, in combination with immunolabeling techniques and confocal imagery, are a very useful tool for the investigation of cochlear pathologies, including losses of ribbon synapses and sensory hair cells, changes in protein levels in hair cells and supporting cells, hair cell regeneration, and determination of report gene expression (i.e., GFP) for verification of successful transduction and identification of transduced cell types. The cochlea, a bony spiral-shaped structure in the inner ear, holds the auditory sensory end organ, the organ of Corti (OC). Sensory hair cells and surrounding supporting cells in the OC are contained in the cochlear duct and rest on the basilar membrane, organized in a tonotopic fashion with high-frequency detection occurring in the base and low-frequency in the apex. With the availability of molecular and genetic information and the ability to manipulate genes by knockout and knock-in techniques, mice have been widely used in biological research, including in hearing science. However, the adult mouse cochlea is miniscule, and the cochlear epithelium is encapsulated in a bony labyrinth, making microdissection difficult. Although dissection techniques have been developed and used in many laboratories, this modified microdissection method using cell and tissue adhesive is easier and more convenient. It can be used in all types of adult mouse cochleae following decalcification.

This article presents a modified cochlear surface preparation method that requires decalcification and use of a cell and tissue adhesive to adhere the pieces of cochlear epithelia to 10 mm round cover slips for immunohistochemistry in adult mouse cochleae.

成年小鼠中的科利耳表面制备

Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous ...

In vitro Time-lapse Live-Cell Imaging to Explore Cell Migration toward the Organ of Corti

To study the effects of mesenchymal stem cells (MSCs) on cell regeneration and treatment, this method tracks MSC migration and morphological changes after co-culture with cochlear epithelium. The organ of Corti was immobilized on a plastic coverslip by pressing a portion of the Reissner's membrane generated during the dissection. MSCs confined by a glass cylinder migrated toward cochlear epithelium when the cylinder was removed. Their predominant localization was observed in the modiolus of the organ of Corti, aligned in a direction similarly to that of the nerve fibers. However, some MSCs were localized in the limbus area and showed a horizontally elongated shape. In addition, migration into the hair cell area was increased, and the morphology of the MSCs changed to various forms after kanamycin treatment. In conclusion, the results of this study indicate that the coculture of MSCs with cochlear epithelium will be useful for the development of therapeutics via cell transplantation and for studies of cell regeneration that can examine various conditions and factors.

In this study, we present a real-time imaging method using confocal microscopy to observe cells moving toward damaged tissue by ex vivo incubation with the cochlear epithelium containing the organ of Corti.

体外延时活细胞成像探索细胞向科尔蒂器官迁移

To study the effects of mesenchymal stem cells (MSCs) on cell regeneration and treatment, this method tracks MSC migration and morphological changes after ...

Immunolabeling and Counting Ribbon Synapses in Young Adult and Aged Gerbil Cochleae

The loss of ribbon synapses connecting inner hair cells and afferent auditory nerve fibers is assumed to be one cause of age-related hearing loss. The most common method for detecting the loss of ribbon synapses is immunolabeling because it allows for quantitative sampling from several tonotopic locations in an individual cochlea. However, the structures of interest are buried deep inside the bony cochlea. Gerbils are used as an animal model for age-related hearing loss. Here, routine protocols for fixation, immunolabeling gerbil cochlear whole mounts, confocal imaging, and quantifying ribbon synapse numbers and volumes are described. Furthermore, the particular challenges associated with obtaining good material from valuable aging individuals are highlighted. 
Gerbils are euthanized and either perfused cardiovascularly, or their tympanic bullae are carefully dissected out of the skull. The cochleae are opened at the apex and base and directly transferred to the fixative. Irrespective of the initial method, the cochleae are postfixed and subsequently decalcified. The tissue is then labeled with primary antibodies against pre- and postsynaptic structures and hair cells. Next, the cochleae are incubated with secondary fluorescence-tagged antibodies that are specific against their respective primary ones. The cochleae of aged gerbils are then treated with an autofluorescence quencher to reduce the typically substantial background fluorescence of older animals' tissues. 
Finally, cochleae are dissected into 6-11 segments. The entire cochlear length is reconstructed such that specific cochlear locations can be reliably determined between individuals. Confocal image stacks, acquired sequentially, help visualize hair cells and synapses at the chosen locations. The confocal stacks are deconvolved, and the synapses are either counted manually using ImageJ, or more extensive quantification of synaptic structures is carried out with image analysis procedures custom-written in Matlab.

A protocol for processing young adult and aged gerbil cochleae by immunolabeling the afferent synaptic structures and hair cells, quenching autofluorescence in aged tissue, dissecting and estimating the length of the cochleae, and quantifying the synapses in image stacks obtained with confocal imaging is presented.

年轻成人和老年沙鼠耳蜗的免疫标记和计数带状突触

The loss of ribbon synapses connecting inner hair cells and afferent auditory nerve fibers is assumed to be one cause of age-related hearing loss. The ...

Imaging the Aging Cochlea with Light-Sheet Fluorescence Microscopy

Deafness is the most common sensory impairment, affecting approximately 5% or 430 million people worldwide as per the World Health Organization1. Aging or presbycusis is a primary cause of sensorineural hearing loss and is characterized by damage to hair cells, spiral ganglion neurons (SGNs), and the stria vascularis. These structures reside within the cochlea, which has a complex, spiral-shaped anatomy of membranous tissues suspended in fluid and surrounded by bone. These properties make it technically difficult to investigate and quantify histopathological changes. To address this need, we developed a light-sheet microscope (TSLIM) that can image and digitize the whole cochlea to facilitate the study of structure-function relationships in the inner ear. Well-aligned serial sections of the whole cochlea result in a stack of images for three-dimensional (3D) volume rendering and segmentation of individual structures for 3D visualization and quantitative analysis (i.e., length, width, surface, volume, and number). Cochleae require minimal processing steps (fixation, decalcification, dehydration, staining, and optical clearing), all of which are compatible with subsequent high-resolution imaging by scanning and transmission electron microscopy. Since all the tissues are present in the stacks, each structure can be assessed individually or relative to other structures. In addition, since imaging uses fluorescent probes, immunohistochemistry and ligand binding can be used to identify specific structures and their 3D volume or distribution within the cochlea. Here we used TSLIM to examine cochleae from aged mice to quantify the loss of hair cells and spiral ganglion neurons. In addition, advanced analyses (e.g., cluster analysis) were used to visualize local reductions of spiral ganglion neurons in Rosenthal&#39;s canal along its 3D volume. These approaches demonstrate TSLIM microscopy&#39;s ability to quantify structure-function relationships within and between cochleae.

A light-sheet microscope was developed to image and digitize whole cochlea.

使用光片荧光显微镜对老化耳蜗进行成像

Deafness is the most common sensory impairment, affecting approximately 5% or 430 million people worldwide as per the World Health Organization1. Aging or ...

Modified Experimental Conditions for Noise-Induced Hearing Loss in Mice and Assessment of Hearing Function and Outer Hair Cell Damage

An animal model of noise-induced hearing loss (NIHL) is useful for pathologists, therapists, pharmacologists, and hearing researchers to thoroughly understand the mechanism of NIHL, and subsequently optimize the corresponding treatment strategies. This study aims to create an improved protocol for developing a mouse model of NIHL. Male C57BL/6J mice were used in this study. Unanesthetized mice were exposed to loud noises (1 and 6 kHz, presented simultaneously at 115-125 dB SPL-A) continuously for 6 h per day for 5 consecutive days. Auditory function was assessed 1 day and 1 week after noise exposure, using auditory brainstem response (ABR). After the ABR measurement, the mice were sacrificed, and their organs of Corti were collected for immunofluorescence staining. From the auditory brainstem response (ABR) measurements, significant hearing loss was observed 1 day after noise exposure. After 1 week, the hearing thresholds of the experimental mice decreased to ~80 dB SPL, which was still a significantly higher level than the control mice (~40 dB SPL). From the results of immunofluorescence imaging, outer hair cells (OHCs) were shown to be damaged. In summary, we created a model of NIHL using male C57BL/6J mice. A new and simple device for generating and delivering pure-tone noise was developed and then employed. Quantitative measurements of hearing thresholds and morphological confirmation of OHC damage both demonstrated that the applied noise successfully induced an expected hearing loss.

Here, we present a protocol for a mouse model of noise-induced hearing loss (NIHL). To induce NIHL, we developed a new and simple device using corrugated plastic, a rat trap cage, and a speaker. Auditory brainstem response and immunofluorescence imaging were employed to assess the hearing function and outer hair cell damage, respectively.

改进小鼠噪声性听力损失的实验条件及听力功能和外毛细胞损伤的评估

An animal model of noise-induced hearing loss (NIHL) is useful for pathologists, therapists, pharmacologists, and hearing researchers to thoroughly ...