The authors thank Masaki Yoda and Elise Lamar for critical reading of the manuscript, Kazumasa Takenouchi for help with histology, Mari Fujiwara for help with microscopy and Makoto Morikawa for help in photographing human and mouse auditory ossicles.

在这项研究中进行的所有动物的程序是由庆应义塾大学机构动物护理和使用委员会（IACUC - 批准文号:09221），并按照庆应大学对动物实验的机构准则在研究中使用动物。人体标本是从捐献给解剖学，医学庆应义塾大学医学院系的尸体中分离，并按照规定的机构中使用。 1.听泡和胶囊隔离<ol><li>安乐死小鼠含有上述浸泡在异氟醚或七氟烷，直到呼吸停止通风超过一分钟以上纸巾一个平台，一个罐子，然后执行颈椎脱位。要小心，以避免浸泡过的纸巾小鼠的直接接触。 </li><li>做一个小横切口在颈部的背侧和拉开皮肤朝头部和用双手尾以暴露底层的颈部亩SCLE组织。 </li><li>在使用14厘米锋利手术剪颈椎区斩首老鼠。 </li><li>剥离皮肤完全对鼻子。用鼻子和门牙一起切断所有的皮肤。 </li><li>将剪刀放进嘴里，切两侧咬肌肌肉。 </li><li>小心打开下颚和去除舌头和下颌一起。 </li><li>用锋利的剪刀，分裂颅骨和颅底入沿矢状切面（ 图1A，B）两部分。 </li><li>使用镊子，取出大脑和小脑半球及脑干。听觉泡和胶囊的位于外侧小脑和脑干。注意，听觉泡是进一步横向于听觉胶囊（ 图1C，D）。 </li><li>解剖出泡和胶囊与周围颅骨（ 图1E）。 </li><li>转移样品至含有磷酸盐缓冲盐水（PBS）pH 7.4的在RT一个菜。 </li><li> ü一个升气管双目解剖显微镜，使用镊子拉开周围的骨骼和剪刀剪开绕泡及胶囊（ 图1F）松动的边界。除去周围的骨骼是basioccipital（腹边框），exoccipital（腹，后缘），枕骨（后缘），interparietal，顶叶（背境），鳞（后背成前缘），alisphenoid（前缘）和基蝶骨（安特罗腹境）的骨头。注意，styliform过程（SP），它支持耳咽管41的鼓膜开口，是从颞骨茎突不同。 </li></ol>锤骨，砧骨和镫骨:2听骨的分离<ol><li> 锤骨 <ol><li>同时使用小剪刀和镊子，取出外耳道横向于沟tympanicus的部分，以使鼓膜是可见的（ 图2A，B）。 </li><li>在腹侧（虚线）和后（＃）的壁（ 图2C）除去鼓膜和鼓室骨附近malleal突短（圆形隆起，见讨论），两者的一部分。锤骨和鼓膜张肌现在应该接触（ 图2D，E）。 </li><li>提起锤（ 图2F）和切断鼓膜张肌与27克针（ 图2G）的斜边。注意，malleal柄牢牢附着于鼓膜，如在其他哺乳动物中看到。 </li><li>从胸骨柄，这是脆弱的仔细分离鼓膜。取下鼓膜骨露出三个听小骨。 </li><li>脱臼从砧骨锤骨的听小骨关节（ 图2H）。 </li><li>通过在goni​​ale压裂前突隔离锤骨。 </li></ol></li><li> 砧骨和镫骨 <ol><li>隔离ŧ他砧通过切断砧骨的后韧带在短脚（ 图3A）。 </li><li>隔离通过切断镫骨附近的镫骨动脉用27克针（ 图3B，C）的斜边镫骨。如果有必要，切镫骨肌肌腱在与针镫骨肌过程。 </li><li>插入一个缝纫针（或标记针）到镫骨闭孔并提起镫骨。除去镫骨后，卵圆窗开口应清晰可见（ 图3D）。 </li></ol></li></ol> 3.听泡和胶囊包埋<ol><li> 在石蜡包埋块准备 <ol><li>如第1节隔离肺大泡和胶囊。 </li><li>切割泡（在styliform处理）关闭，用剪刀的前端，浸泡在PBS中的4％多聚甲醛（PFA）的泡和胶囊，在4℃C和允许固定剂进入到大疱。如果空气变得在泡捕集，用针和注射器将其删除。留在固定剂的泡和胶囊，在4℃的CO / N于管旋转。 注意:PFA是有毒的，应谨慎处理。 </li><li>用PBS洗一次。 </li><li>在10％的乙二胺四乙酸二钠盐二水合物脱钙泡和胶囊为一个星期，在4℃（EDTA-2Na盐），100毫摩尔Tris碱，pH 7.0，在2毫升管中。更改缓冲区隔日1次。 </li><li>用PBS洗一次。标本可在4℃储存在水中70％的乙醇。任选地，通过分级醇系列（30％，50％，在水中的70％）转移到70％的乙醇。 </li><li>上的组织处理器，脱水在等级系列的乙醇溶液的试样（70％，2×95％，3×100％，每1小时），清除在二甲苯（4倍，在40℃下各1小时），并渗入与标本熔化的石蜡42。或者，替代二甲苯与商业组织clearin摹解决方案（ 例如 ，组织相容-清除）。 </li><li>从处理器卸载标本，并从他们的磁带中删除。 </li><li>在一个组织包埋控制台系统，地方标本放入盛有熔化的石蜡模具。继续嵌入（第4节）。 </li></ol></li><li> 在冷冻块嵌入（川本的电影法）制备43 <ol><li>如第1节隔离肺大泡和胶囊。 </li><li>切割泡（在styliform处理）关闭，用剪刀的前端，浸泡在固定液大疱和胶囊，在4℃（2％或1％的PFA而非PBS中的4％至保留抗原性）。如果空气被截留在大疱，使用针和注射器将其删除。留在固定剂的泡和胶囊，在4℃的CO / N于管旋转。 </li><li>在PBS快速洗涤泡和胶囊以及液体冷冻包埋化合物在4℃立即浸入。 </li><li>重要提示:除去气泡，如果任何中间并通过抽吸外耳通过针，并通过添加用钳子嵌入化合物。继续嵌入（第4节）。 </li></ol></li></ol> 4.样品方向和嵌入注:全部泡和胶囊必须嵌入到切期望段期间被布置在一个特定的方向。下面概述的过程用于部分中不同取向的锤骨。 <ol><li> 锤骨纵向（矢状窦旁）切片 <ol><li>把泡或外耳道的侧面向下在温暖石蜡（或冷冻包埋化合物）。调整方向，使得锤骨的颈部和横向薄片平行于嵌入盘（ 图4A - C）的水平底部。需要注意的是鼓膜在小鼠头部（ 图4A以大约30°的角度倾斜于垂直;图59中的Kampen <suP&gt; 44）。 </li></ol></li><li> 锤骨水平切片 <ol><li>水平放置背嵴在温暖的石蜡（或低温嵌入化合物）。调整泡和胶囊的取向，使得锤骨的颈部和横向薄层垂直于嵌入碟（ 图4D - F）的底部。 </li></ol></li><li> 柄及鼓膜5的正面切片（横截面） <ol><li>放置在温暖石蜡（或冷冻包埋化合物）的malleal柄，使得其垂直于嵌入盘的底部。 </li></ol></li><li>冷却块到相应于一个组织灌封控制台系统上硬化石蜡温度（可替代地，使用冷冻包埋化合物在干冰/己烷浴）。 </li><li>流程组织块，用常规方法切段。例如，用苏木精和曙红（H＆＃染色石蜡切片38; E），番红O（软骨），或抗酒石酸酸性磷酸酶（TRAP）活性（为破骨细胞）3，或者通过免疫组化。脱钙冰冻切片都适合使用荧光染料14骨标签，茜素红染色钙和免疫42。 </li></ol>

The authors have nothing to disclose.

在这里，我们提出来隔离在出生后小鼠听泡和胶囊有用的方法。在此之前P12，组织是脆弱和孤立过程中可能会损坏。 P12之后，听觉泡和胶囊可以容易地从周围组织中分离。切片之前解剖从头部泡具有几个优点。首先，发生汽蚀产后和听泡的增长率从P6起最积极，是由P14 50完成。鼓膜和耳蜗壁之间的间充质组织是由空气通过气蚀过程所取代。在中耳腔所得空气可阻碍期间固定，脱钙和嵌入组织和液体之间的接触。这是比较容易通过切断的前端（styliform处理），而不是试图在未分离大疱这样做从分离的听觉泡除去空气。第二，锤骨（和鼓膜）的方向不垂直在头部。它是一节中所希望的平面内的锤骨嵌入分离的听觉泡和胶囊在给定的取向，因此更容易。 一旦分离，听泡和胶囊可用于多种分析非常有用。例如，高清晰度的X射线显微CT可以揭示骨微结构的形态，例如在锤骨14成骨毛细管。所述stereofluorescence解剖显微镜是可视化中评价表达在中耳或内耳33荧光蛋白报告小鼠结构的有力工具。此外，各种在体内或离体荧光标记的方法和整装免疫检测可以进行。光片荧光显微镜也用于三维分析51是有用的。虽然这里没有描述，与听觉泡和胶囊如外周神经，血管相关多样的解剖结构，并在中耳的鼓膜也可使用此协议进行评价。 注意，石蜡切片需要嵌入前骨组织脱钙，因此不允许矿化的分析。与此相反，可以在不脱钙进行用于制备冰冻切片的川本膜的方法43，适合使用体内骨标记技术或特殊染色，如茜素染色矿化研究。冰冻切片条件应根据基于鼠标的年龄进行优化。例如，在低温恒温室内的较少凉爽温度推荐为老年小鼠的标本尽量减少部分的损坏。 在鼠标的锤骨的突出的半球形突起正确的术语是"圆形隆起"。尽管如此，术语"突短"已被广泛地用于指示更多次的圆形隆起一个二十年，特别是在小鼠的发育生物学家16,20,22-25。 "短突"原指横向过程（突外侧），它从圆形隆起不同。在人类中，横向处理类似的轻微锥形突起形成附着于鼓膜总路线，从柄（未示于图6B中 ，内侧视图）延伸。在小鼠中，横向过程也是在相对端的壳顶48上的柄的投影。鼓膜的鼓膜松弛部是上述锤骨的横向过程。圆形隆起是不被人类锤骨显而易见。

中耳的三个听小骨，即锤骨，砧骨和镫骨，形成从鼓膜到内耳或耳蜗1,2-发送声音的哺乳动物特异性听觉链。听力功能可在小鼠中通过测量听性脑干反应（ABR）阈值3-6，和鼓膜可使用激光多普勒Vibrometry（LDV）7监视后面锤骨的振动来评价。通过结合ABR，LDV和畸变产物耳声发射（DPOAE）的测量，传导性听力损失可以从感减值8歧视。 耳条件动物模型都需要，给予听觉和耳部健康的患者的青睐福祉的重要性。例如，中耳炎是人类婴儿和儿童，以及严重，急性中耳炎看到一种极为常见的耳部感染，如果康迪其并发症可能会出现化是不是适当的抗生素治疗9。中耳炎小鼠模型可以证明在了解发病机制和开发治疗10,11有用。 鼠小骨，其中（除了锤骨的goniale部分）由软骨内骨化12,13形成的，是对骨代谢和形态发生的研究高度相关。首先，它们的小尺寸允许骨头高分辨率分析用X射线或荧光显微镜14一个完整的骨膜。其次，异常骨代谢，如过度或不足骨吸收或骨细胞15名受损的相互作用，可以分析作为一个潜在的贡献者听力损失3,4,7。第三，异常小骨形态发生报道在几个基因缺陷型小鼠，如缺乏HOXA2 16-19，MSX1 20-22 Prrx1 23，Goosecoid动物（GSC）24,25，Bapx1 13，Tshz1 26，DUSP6（Mkp3）27， 头蛋白 （ 木栓 ）28，29 FGFR1，甲状腺激素受体（THRA，THRB）5，Bcl2的 30和其他1,31或过表达的小鼠HOXA2 32。最后，尽管其体积小，听小骨与诸如肌肉33和关节34,35相关的结构进行访问。 鼠标小骨比人听小骨小，但值得注意的是，鼠标中耳不是其人力对应的微缩版。例如，在小鼠中，镫骨动脉，穿过镫骨的环，持续整个生命36，而在人类中，该胚胎镫骨动脉妊娠期间消失。此外，鼠标锤骨的形态不同于第的不同Ë人骨（ 见图6）。在小鼠中，听觉（鼓膜）泡包围充满空气的中耳腔，而在人类中，在颞骨小梁骨组成乳突气房容纳小骨，而不是一个大泡37。在这两个物种中，听觉胶囊（耳囊，骨迷路）包围耳蜗及内耳半规管。中耳的比较与进化生物学已经被广泛的审查38-40。 下面首先提供的协议描述了如何解剖分别出听泡和胶囊，其主要包括中耳和内耳。该协议还演示了如何隔离听觉泡锤骨，砧骨和镫骨。最后，它显示了如何定向听泡和胶囊制剂中嵌入的听骨组织切片。

<table border="0" cellpadding="0" cellspacing="0" width="823">
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			<td height="20" style="height:20px;">Paper towel</td>
			<td>DAIO PAPER CORPORATION</td>
			<td>703347</td>
			<td>can be purchased from other vendors</td>
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			<td>Various</td>
			<td></td>
			<td>can be purchased from other vendors</td>
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			<td height="20" style="height:20px;">14cm surgical scissors</td>
			<td>Fine Science Tools (F.S.T.)</td>
			<td>91400-14</td>
			<td>can be purchased from other vendors</td>
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			<td height="20" style="height:20px;">Extra fine scissors-straight</td>
			<td>Fine Science Tools (F.S.T.)</td>
			<td>14084-08</td>
			<td>can be purchased from other vendors</td>
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			<td height="20" style="height:20px;">Fine Forceps Angled 45&#176;</td>
			<td>Fine Science Tools (F.S.T.)</td>
			<td>11063-07</td>
			<td>can be purchased from other vendors</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Dissecting microscope</td>
			<td>Nikon</td>
			<td>SMZ800N</td>
			<td>for routine dissection</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Dissecting microscope</td>
			<td>Nikon</td>
			<td>SMZ18</td>
			<td>for movies&#160;</td>
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			<td height="20" style="height:20px;">Injection needle 27G</td>
			<td>TERUMO</td>
			<td>NN-2719S</td>
			<td></td>
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			<td height="20" style="height:20px;">Syringe (1ml)</td>
			<td>TERUMO</td>
			<td>SS-01T</td>
			<td></td>
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			<td>Various</td>
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			<td height="20" style="height:20px;">Tube rotator RT-50</td>
			<td>TAITEC</td>
			<td>0000165-000</td>
			<td>can be purchased from other vendors</td>
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			<td height="67" style="height:67px;">Cryostat</td>
			<td>Leica</td>
			<td>CM3050S</td>
			<td style="width:259px;">http://www.leicabiosystems.com/histology-equipment/cryostats/details/product/leica-cm3050-s/</td>
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			<td height="20" style="height:20px;">TC-65 Tungsten blade</td>
			<td>Leica</td>
			<td>14021626379</td>
			<td>for Kawamoto&#39;s firm method</td>
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			<td height="20" style="height:20px;">Stainless containers</td>
			<td>Leica</td>
			<td></td>
			<td>for Kawamoto&#39;s firm method</td>
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			<td height="20" style="height:20px;">Cryofilm type IIC</td>
			<td>Leica</td>
			<td></td>
			<td>for Kawamoto&#39;s firm method</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Silane coated slide (New Silane II)</td>
			<td>Muto Pure Chemicals</td>
			<td>511617</td>
			<td>can be purchased from other vendors</td>
		</tr>
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			<td height="20" style="height:20px;">Cover glass</td>
			<td>Matsunami</td>
			<td></td>
			<td>can be purchased from other vendors</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Tissue processor</td>
			<td>Sakura Finetek</td>
			<td style="width:124px;">VIP-5</td>
			<td>can be purchased from other vendors</td>
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			<td height="20" style="height:20px;">Tissue Embedding Console System</td>
			<td>Sakura Finetek</td>
			<td style="width:124px;">Tissue-Tek TEC 5&#160;</td>
			<td>can be purchased from other vendors</td>
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			<td height="20" style="height:20px;">Sliding microtome for paraffin</td>
			<td>Yamato Kohki Industrial</td>
			<td>REM-710</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Path Blade+pro for hard tissue</td>
			<td>Matsunami</td>
			<td>PB3503C</td>
			<td>for paraffin section</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Micro-CT</td>
			<td>RIGAKU</td>
			<td>R_mCT2</td>
			<td>http://www.rigaku.com/en</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluorescence microscope</td>
			<td>KEYENCE</td>
			<td>BZ-9000</td>
			<td></td>
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			<td height="20" style="height:20px;">Name</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Isoflurane</td>
			<td>Maruishi pharmaceutical Co. Ltd</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaCl</td>
			<td>wako</td>
			<td>191-01665</td>
			<td>for PBS</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KCl</td>
			<td>wako</td>
			<td>285-14</td>
			<td>for PBS</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Na2HPO4 12H2O</td>
			<td>wako</td>
			<td>196-02835</td>
			<td>for PBS</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">KH2PO4</td>
			<td>wako</td>
			<td>287-21</td>
			<td>for PBS</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Paraformaldehyde(EM Grade)</td>
			<td>TAAB</td>
			<td>P001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EDTA-2Na</td>
			<td>wako</td>
			<td>15111-45</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trizma base</td>
			<td>Sigma</td>
			<td>T1503-1KG</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Super Cryoembedding Medium</td>
			<td>Leica</td>
			<td></td>
			<td>for Kawamoto&#39;s firm method</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dry Ice</td>
			<td>Various</td>
			<td></td>
			<td>for Kawamoto&#39;s firm method</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hexane</td>
			<td>wako</td>
			<td>080-03423</td>
			<td>for Kawamoto&#39;s firm method</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Super Cryomouting Medium type R2</td>
			<td>Leica</td>
			<td></td>
			<td>for Kawamoto&#39;s firm method</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Paraffin</td>
			<td>Sakura Finetek</td>
			<td>781001A0107</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Histo-Clear</td>
			<td>NDS</td>
			<td>HS-200</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Calcein</td>
			<td>DOJINDO</td>
			<td>340-00433</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hematoxylin&#160;</td>
			<td>wako</td>
			<td>131-09665</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Eosin</td>
			<td>wako</td>
			<td>051-06515</td>
			<td></td>
		</tr>
	</tbody>
</table>

dissection of the auditory bulla in postnatal mice: isolation of the middle ear bones and histological analysis

在大多数哺乳动物中，在中耳听小骨，包括锤骨，砧骨和镫骨，是最小的骨头。在小鼠中，骨性结构称为听觉泡容纳小骨，而听觉胶囊包封内耳，即耳蜗和半规管。鼠小骨是极大的兴趣在耳鼻喉科领域的研究人员和听觉因此，至关重要，但它们的代谢，发育和进化是其他领域密切相关。改变的骨代谢能影响听力功能在成年小鼠和各种基因缺陷小鼠显示在子宫内听小骨的形态发生变化。尽管鼠听小骨是微小的，它们的操作是可行的，如果人理解他们的解剖定位和三维结构。在这里，我们将介绍如何剖析听泡和出生后的小鼠胶囊，然后通过去除肺大泡的一部分分离单个听小骨。我们还讨论了如何EM床在不同的方向的泡和胶囊，以产生石蜡或冰冻切片适于制备锤骨的纵向，横向，或正面部的。最后，我们列举小鼠和人类听小骨之间的解剖学差异。这些方法将在分析听小骨的病理，发育和进化方面和小鼠中耳有用。

这个协议呈现分离从小鼠听泡小骨的方法。首先，将泡和胶囊被解剖出作为从头骨（ 图1）的单件。然后解剖泡用于制备锤骨（ 图2）和砧骨及镫骨（ 图3）。听泡和胶囊的地标在肺大泡，背嵴，前半规管，以及subarcuate窝（ 图1F）的前端的styliform过程。 Microcomputed断层扫描（CT）成像揭示听觉泡小骨，以及对那些听小骨的纵向和横向切片（ 图4）的最佳定向。 对于锤骨的纵向石蜡切片中，泡和胶囊是在EDTA中，在4℃脱钙一周，嵌入在巴在图4所示的一个方向拉芬块- C，切片在4微米，然后用H＆E染色。附着在听觉泡鼓膜锤骨揭示在P14（ 图5A）正在进行的软骨内骨化。为了形象化新骨形成，钙黄绿素（30微克/克体重），腹腔注入P20鼠标和大疱和胶囊均在P21隔离24小时后。未经脱钙样品包埋冷冻，然后在使用基于川本43的方法的粘合膜为6μm冰冻切片。用DAPI核染色（4&#39;，6-二脒基-2-苯基）后，在荧光显微镜下观察到的部分。钙黄绿素信号（绿色）揭示了在锤骨（米）的新骨形成，泡和胶囊（ 图5B）。对于锤骨的横切片，从5周龄小鼠中分离听觉泡未经脱钙嵌入冷冻（对于定向见图4D - F），在使用方法川本6微米冰冻切片，用H＆E染色。所述malleal突短（MPB）的横切片还示出了耳蜗（ 图5C）。 从P31小鼠中分离合适的听小骨的内侧视图显示鼠标锤的典型特征，即"滑翔，海鸥翼状"（或波斯剑状45）柄，一个突出的突短（圆形隆起见讨论），以及横向薄片（ 图6）。请注意，前突（前突）是围绕goniale解剖过程中的断裂，并从鼓膜环（ectotympanic）分离。这种代表性的样本表现出锤砧骨和之间的完整incudomalleolar关节，而砧镫关节脱臼。腱插入到malleal和镫骨肌过程是可检测的（ 图6A，星号）。 图6B以相同放大倍率比较小鼠和人的听小骨。品种不同，大小比其他的，包括以下内容。该malleal柄是翼状的小鼠，但俱乐部般的人类。解剖轴线（或旋转轴，通过锤骨和砧骨的短过程的前过程中的线）和柄之间的角度是在小鼠中小得多，在两个几乎平行的，而不是接近垂直在人类中6,46-48。在人类的听小骨，vibrometric研究揭示，incudo，踝关节移动，而不是固定的功能49。鼠标锤展品种类，薄，平整横向叶片在人类47并不明显。在小鼠中，突前保险丝膜质骨，即goniale和定音鼓C环，而在人类中突前减小到骨41的小毛刺。小鼠和人类的镫骨也不同:在小鼠中，前小腿是弯曲的，并且后小腿更直的，而在人类中，前小腿更直比后部小腿。值得一提的是，相对于身体大小的锤骨头在物种大量扩大诸如金摩尔，以"最小"筋骨48异速生长关系证明显著变性。 <img alt="图1" src="/files/ftp_upload/55054/55054fig1.jpg" /> 图1.听泡和胶囊的解剖。 （A）一P31鼠标的头骨被分成左右两半。 A，前壁; P，后路; L，左; R，右。 （B）中的等分的，皮肤的头部的右半的内侧表面上。 CX，大脑皮层; CB，小脑;烧烤，brainstem。 D，背;五，腹。大脑的（C）去除用钳子。 （D）在右侧颅骨听觉胶囊内侧视图。背嵴（箭头）位于颅中窝（MCF），后颅窝（PCF）与分离后背成前，听囊腹，后表面。比例尺，2毫米。 （五）听泡和胶囊（内侧视图）的高倍。有限公司耳蜗;七，面神经;八，听神经; AC，前（上级）半规管; SF，subarcuate窝，里面有小脑paraflocculus。比例尺，1毫米。孤立听泡和胶囊（内侧视图）（F）显微照片。 SP，styliform过程。比例尺，1毫米。 （A - E），P31鼠标。 （F），P33鼠标。 <a href="http://ecsource.jove.com/files/ftp_upload/55054/55054fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55054/55054fig2.jpg" /> 图2.解剖锤骨。 （A）右侧听泡和胶囊的腹外侧视图。沟tympanicus（ST，虚线箭头）是鼓膜的附着位点。骨横向于ST是外耳的一部分，和骨内侧到ST形成中耳腔的楼层。 A，前壁; P，后路; D，背;五，腹。清除外耳道后（B）查看揭示鼓膜（TM），包括鼓膜松弛部（PF）和收杆tensa（PT）。 （C）去除鼓室骨（虚线和＃）附近的malleal短突（MPB）的部件。男，锤骨; MM，malleal柄。箭头，气泡在通过鼓膜看出中耳空腔。 （D）裸露锤。马力士头表示。带点线表示砧骨的关节面。 （E）的张量鼓肌肌腱（TT）连接到锤骨。当锤骨被抬起（F）的张鼓被拉动。 *，肌肉发达的过程。 （G）的张量鼓被使用针切断。 （H）去除鼓膜后3听小骨。该incudo，踝关节脱臼。男，锤骨;我，砧骨; S，镫骨;去吧，goniale（融合到锤骨和鼓膜环，TR）。所有比例尺，0.5毫米。 （A，H），P33鼠标。 （B - G），P31鼠标。 <a href="http://ecsource.jove.com/files/ftp_upload/55054/55054fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/55054/55054fig3.jpg" /> 图3.砧骨和镫骨解剖。 （A）和砧骨除去锤骨的后镫骨。的镫骨动脉（SA）穿过镫骨（多个）。虚线表示砧骨的关节面。注意，该短脚砧骨（ⅰ）的（ICB，小腿杆菌）由后韧带固定（未示出）。星号，镫骨肌过程。 （B）去除砧骨镫骨后。针尖用于切割镫骨动脉（SA）。箭头，血流方向。虚线表示镫骨的关节面。 （C）中镫骨动脉从镫骨除去。 X表示镫骨动脉（SA）的切口处。 （D）卵圆窗（嗷， 卵圆窗或前庭膜孔 ）的去除镫骨后可见。 RW，圆窗（ 窗孔圆形或窗孔耳蜗 ）。比例尺，0.5毫米。 <a href="http://ecsource.jove.com/files/ftp_upload/55054/55054fig3large.jpg" target="_blank">请点击此处查看这个大版本</a>数字。 <img alt="图4" src="/files/ftp_upload/55054/55054fig4.jpg" /> 图4 为嵌入在纵向定向听泡和胶囊（矢状窦旁，A - C）和水平（D - E） 切片 锤骨。 （A - C）的颈部和横向锤骨层被平行放置嵌入盘的底部。 （A）侧视图:显微CT图像显示在泡（伪彩色蓝色）右侧锤骨嵌入。锤骨和砧骨的伪彩色绿色。虚线，所希望的切割平面。实线，嵌入菜底部。男，锤骨;箭头，背嵴。男，内侧; L，横向; D，背;五，腹。 （B）顶视图:显微CT图像。需要注意的是，除去泡（styliform过程）的前端。我，砧骨。 （C）俯视图:显微照片（与拍摄滤色器）。 AC，前（上级）半规管; SF，subarcuate窝; SP，styliform过程。 A，前壁; P，后路; D，背;五，腹。 （D - F）的锤骨的突短置于垂直于嵌入盘的底部。 （D）侧视图:显微CT图像显示正确的锤骨嵌入。虚线，所希望的切割平面。实线，嵌入菜底部。 （E）顶视图:显微CT图像。 MM，malleal柄。 （F）顶视图:显微照片（用彩色滤光片拍摄）。比例尺1毫米。在5微米的体素分辨率获得显微CT图像，如前所述7。 <a href="http://ecsource.jove.com/files/ftp_upload/55054/55054fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/55054/55054fig5.jpg" /> Figure 5.组织学。 （一）H＆E染色。在P14听觉泡（虚线）的石蜡包埋右锤（M），纵向（矢状窦旁）一节。 TM，鼓膜。 （二）骨骼钙黄绿标志。在P21听觉泡冷冻，脱钙左锤（米）的纵切面。染液，DAPI。 （C）H＆E染色。在听泡和胶囊（5周龄鼠）冷冻，脱钙离开malleal短突（MPB）的水平段。有限公司，耳蜗。比例尺1毫米。 <a href="http://ecsource.jove.com/files/ftp_upload/55054/55054fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/55054/55054fig6.jpg" /> 图6. 听骨内侧观。 （a）芘的右听小骨31鼠标。 A，前壁; P，后路; D，背;五，腹。比例尺，1毫米。锤骨头（卡普特鼻疽，头状花序鼻疽）;颈部（科勒姆鼻疽）;叶片（横向叶片）;毫米（柄鼻疽）;黑色星号（锤骨肌工艺）; MPA（前突，突纤细）; MPB（短突）;砧骨体（科珀斯克里incudis）; ICB（小腿短双歧杆菌，短脚，短流程）; ICL（小腿茇，小腿长，漫长的过程）; IPL（荚状突，透镜状过程中，西尔隆起）;镫头（卡普特stapedis）;白星号（镫肌肉工艺）; SCA（小腿前段，前小腿）; SCP（小腿后韧带，后小腿）;基地（基础stapedis，踏板）; SOF（闭孔，intercrural孔）。 （B）一个76岁的人类女性的权利听骨（解剖学教研室，庆应义塾大学医学部提供）。 P31小鼠的听小骨（右下）在相同的放大倍率成像作为用于人类听小骨。 CURV编箭头指示的解剖轴线和柄（虚线）之间的角度。比例尺，2毫米。 <a href="http://ecsource.jove.com/files/ftp_upload/55054/55054fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Dissection of the Auditory Bulla in Postnatal Mice: Isolation of the Middle Ear Bones and Histological Analysis at JoVE.com

Dissection of the Auditory Bulla in Postnatal Mice: Isolation of the Middle Ear Bones and Histological Analysis

We present a protocol to isolate the auditory bulla, capsule, and ossicles from postnatal mice for whole mount and histological analysis.

在出生后的小鼠听泡解剖:中耳骨骼和组织学分析的分离

In most mammals, auditory ossicles in the middle ear, including the malleus, incus and stapes, are the smallest bones. In mice, a bony structure called ...

dissection-auditory-bulla-postnatal-mice-isolation-middle-ear-bones

Research

JoVE Journal

Biology

29.9K 次观看.  Keio University School of Medicine. We present a protocol to isolate the auditory bulla, capsule, and ossicles from postnatal mice for whole mount and histological analysis. 

视频: Dissection of the Auditory Bulla in Postnatal Mice: Isolation of the Middle Ear Bones and Histological Analysis

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish (Danio rerio)

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the cellular and molecular features of the epithelia, which are mainly composed of two types of cells: hair cells (HCs) and supporting cells (SCs). Here we choose to study the inner ear sensory epithelia in adult zebrafish not only because the epithelial structures are highly conserved in all vertebrates studied, but also because the adult zebrafish is able to regenerate HCs, an ability that mammals lose shortly after birth. We use the inner ear of adult zebrafish as a model system to study the mechanisms of inner ear HC regeneration in adult vertebrates that could be helpful for clinical therapy of hearing/balance deficits in human as a result of HC loss.
Here we demonstrate how to do gross and fine dissections of inner ear sensory epithelia in adult zebrafish. The gross dissection removes the tissues surrounding the inner ear and is helpful for preparing tissue sections, which allows us to examine the detailed structure of the sensory epithelia. The fine dissection cleans up the non-sensory-epithelial tissues of each individual epithelium and enables us to examine the heterogeneity of the whole epithelium easily in whole-mount epithelial samples.

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish Danio rerio

The inner ear sensory epithelium of adult zebrafish is a good model system for understanding the mechanisms of hair cell regeneration in adult vertebrates. This protocol demonstrates the fine dissection of the epithelia, through which we can get tissue samples for studying the regenerative events at cellular and subcellular levels.

格罗斯和精细解剖内耳感觉上皮细胞在成年斑马鱼（斑马鱼）

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the ...

科研

生物学

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

原代培养及质粒电穿孔尔蒂小鼠器官。

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Isolation of Neonatal Extrahepatic Cholangiocytes

The intra and extrahepatic bile ducts of the liver are developmentally distinct, and may be differentially affected by certain diseases. However, differences between intra and extrahepatic cholangiocytes, and between neonatal and adult cells, are not well understood.
Methods for the isolation of cholangiocytes from intrahepatic bile ducts are well established1-4. Isolation of extrahepatic ductal cells, especially from the neonate, has not yet been described, although this would be of great benefit in understanding the differences between distinct cholangiocyte populations and in studying diseases such as biliary atresia that appear to target the extrahepatic ducts. Described here is an optimized technique to isolate both neonatal and adult mouse extrahepatic bile duct cells. This technique yields a pure cell population with minimal contamination from mesenchymal cells like fibroblasts.
This method is based on the removal of the extrahepatic ducts and gallbladder, followed by meticulous dissection and scraping to remove fat and fibroblast layers. Structures are embedded in thick layers of collagen and cultured for approximately 3 weeks to allow outgrowth of cholangiocytes in monolayers, which can then be trypsinized and re plated for experimental use.

A technique to isolate cholangiocytes from the extrahepatic bile ducts of neonatal mice is described. The ducts are meticulously dissected, and then cells are isolated by outgrowth in thick collagen gels. This method provides a useful tool for studying extrahepatic bile duct development and pathology.

新生儿肝外胆管的隔离

The intra and extrahepatic bile ducts of the liver are developmentally distinct, and may be differentially affected by certain diseases. However, ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

鼠阀门内皮细胞的分离

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

高分辨率成像定时，并在活体人脐静脉内皮细胞微管动力学自动分析

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

核酸的分离过滤:一种简单快速的DNA提取方法

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

Dissection and Lipid Droplet Staining of Oenocytes in Drosophila Larvae

Lipids are essential for animal development and physiological homeostasis. Dysregulation of lipid metabolism results in various developmental defects and diseases, such as obesity and fatty liver. Usually, lipids are stored in lipid droplets, which are the multifunctional lipid storage organelles in cells. Lipid droplets vary in size and number in different tissues and under different conditions. It has been reported that lipid droplets are tightly controlled through regulation of its biogenesis and degradation. In Drosophila melanogaster, the oenocyte is an important tissue for lipid metabolism and has been recently identified as a human liver analogue regarding lipid mobilization in response to stress. However, the mechanisms underlying the regulation of lipid droplet metabolism in oenocytes remain elusive. To solve this problem, it is of utmost importance to develop a reliable and sensitive method to directly visualize lipid droplet dynamic changes in oenocytes during development and under stressful conditions. Taking advantage of the lipophilic BODIPY 493/503, a lipid droplet-specific fluorescent dye, described here is a detailed protocol for the dissection and subsequent lipid droplet staining in the oenocytes of Drosophila larvae in response to starvation. This allows for qualitative analysis of lipid droplet dynamics under various conditions by confocal microscopy. Furthermore, this rapid and highly reproducible method can also be used in genetic screens for indentifying novel genetic factors involving lipid droplet metabolism in oenocytes and other tissues.

Dissection and Lipid Droplet Staining of Oenocytes in Drosophila Larvae

Presented here are detailed methods for the dissection and lipid droplet staining of oenocytes in Drosophila larvae using BODIPY 493/503, a lipid droplet-specific fluorescent dye.

果蝇幼虫的欧诺细胞的解剖和脂滴染色

Lipids are essential for animal development and physiological homeostasis. Dysregulation of lipid metabolism results in various developmental defects and ...

Isolation and Differentiation of Primary White and Brown Preadipocytes from Newborn Mice

The understanding of the mechanisms underlying adipocyte differentiation and function has greatly benefited from the use of immortalized white preadipocyte cell lines. These cultured cell lines, however, have limitations. They do not fully capture the diverse functional spectrum of the heterogenous adipocyte populations that are now known to exist within white adipose depots. To provide a more physiologically relevant model to study the complexity of white adipose tissue, a protocol has been developed and optimized to enable simultaneous isolation of primary white and brown adipocyte progenitors from newborn mice, their rapid expansion in culture, and their differentiation in vitro into mature, fully functional adipocytes. The primary advantage of isolating primary cells from newborn, rather than adult mice, is that the adipose depots are actively developing and are, therefore, a rich source of proliferating preadipocytes. Primary preadipocytes isolated using this protocol differentiate rapidly upon reaching confluence and become fully mature in 4-5 days, a temporal window that accurately reflects the appearance of developed fat pads in newborn mice. Primary cultures prepared using this strategy can be expanded and studied with high reproducibility, making them suitable for genetic and phenotypic screens and enabling the study of the cell-autonomous adipocyte phenotypes of genetic mouse models. This protocol offers a simple, rapid, and inexpensive approach to study the complexity of adipose tissue in vitro.

This report describes a protocol for the simultaneous isolation of primary brown and white preadipocytes from newborn mice. Isolated cells can be grown in culture and induced to differentiate into fully mature white and brown adipocytes. The method enables genetic, molecular, and functional characterization of primary fat cells in culture.

初白和棕色预生细胞与新生小鼠的隔离和分化

The understanding of the mechanisms underlying adipocyte differentiation and function has greatly benefited from the use of immortalized white ...

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to understand the pathophysiology of ocular diseases because of their similarities with human anatomy and physiology. Alterations in the retinal pigment epithelium (RPE), including changes in morphology and function, are common features shared by many ocular disorders. However, successful isolation and culture of primary mouse RPE cells is very challenging. This paper is an updated audiovisual version of the protocol previously published by Fernandez-Godino et al. in 2016 to efficiently isolate and culture primary mouse RPE cells. This method is highly reproducible and results in robust cultures of highly polarized and pigmented RPE monolayers that can be maintained for several weeks on Transwells. This model opens new avenues for the study of the molecular and cellular mechanisms underlying eye diseases. Moreover, it provides a platform to test therapeutic approaches that can be used to treat important eye diseases with unmet medical needs, including inherited retinal disorders and macular degenerations.

This protocol, which was originally reported by Fernandez-Godino et al. in 20161, describes a method to efficiently isolate and culture mouse RPE cells, which form a functional and polarized RPE monolayer within one week on Transwell plates. The procedure takes approximately 3 hours.

原发性小鼠视网膜色素上皮细胞的有效解剖与培养

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to ...

Dissection of the Endolymphatic Sac from Mice

The study of mutant mouse models of human hearing and balance disorders has unraveled many structural and functional changes which may contribute to the human phenotypes. Although important progress has been done in the understanding of the development and function of the neurosensory epithelia of the cochlea and vestibula, limited knowledge is available regarding the development, cellular composition, molecular pathways and functional characteristics of the endolymphatic sac. This is, in large part, due to the difficulty of visualizing and microdissecting this tissue, which is an epithelium comprised of only one cell layer. The study presented here describes an approach to access and microdissect the endolymphatic sac from the wild-type mouse inner ear at different ages. The result of a similar dissection is shown in a pendrin-deficient mouse model of enlargement of the vestibular aqueduct. A transgenic mouse with a fluorescent endolymphatic sac is presented. This reporter mouse can be used to readily visualize the endolymphatic sac with limited dissection and determine its size. It can also be used as an educational tool to teach how to dissect the endolymphatic sac. These dissection procedures should facilitate further characterization of this understudied part of the inner ear.

<meta charset="utf8"></head><body>小鼠内淋巴囊解剖

This study describes how to dissect the endolymphatic sac from the inner ear of the mouse at different ages. The result of a similar dissection is shown in an Slc26a4-null mouse model of enlargement of the endolymphatic sac. A transgenic mouse with a fluorescent reporter expressed in the endolymphatic sac is presented as a model to readily visualize the endolymphatic sac, and educational tool.

小鼠内淋巴囊解剖

The study of mutant mouse models of human hearing and balance disorders has unraveled many structural and functional changes which may contribute to the ...