笔者想承认儿童研究所成像核心使用共聚焦显微镜和张健博士的建议，安装使用琼脂糖珠的部分。这项工作是由美国国立卫生研究院授予DC010387。

1。内耳的分离和固定<ol><li>在Corti器的蜂窝图案分析，安乐死适当上演胚胎或成人。 </li><li>解剖奥迪胶囊，含有膜的内耳。鼠标，可以轻松完成对胚胎的胚胎（五）14.5天以上，其中E0.5检测阴道塞的一天。解剖奥迪胶囊是第一个完成斩首和开放的头骨中线。移除大脑揭露每个耳朵（图1A）。内耳可掏出完整的使用镊子（如杜蒙＃5，图1B）。如果成像Corti器，小心去除与奥迪胶囊相关的所有神经细胞和结缔组织是没有必要的。 </li><li>修复整个内耳由4％多聚甲醛浸泡在温和的摇摆（PFA）4 ° C 1.8毫升螺丝帽NUNC小瓶冷冻管，或其他适当大小的容器中过夜。煤灰的解决方案应该是在2 0 DDH取得了新的，然后储存在-20 ° C，直到需要。 </li><li>第二天，洗出三个1X磷酸盐的变化固定液缓冲液（PBS）。组织可存储天至数月，直到准备进行处理，在无菌1X PBS。成人组织，除垢在10％EDTA（0.27 M）在4 ° C为5天，其次是在1X PBS冲洗和存储。 </li></ol> 2。切割vibratome节<ol><li>准备在1X PBS 4％低熔点琼脂糖。添加低熔点琼脂糖1X PBS和微波，直到琼脂糖溶液中。该解决方案不泡过的护理 - 利用微波定期和漩涡混合。保持在55℃水浴，直到准备使用的解决方案。 </li><li>使用镊子，取出内耳1X PBS液和一个剥离一个双向嵌入模具的底部的位置。吸取了任何多余的1X PBS从模具。填充模具用大量液体4％低熔点琼脂糖（图2A），以支付组织。 </li><li>内耳切片在适当的平面位置。我已获得逼近奥迪胶囊的位置，如果它一直在原地留在头部，并在横向平面切片Corti器的横截面。 E14.5胚胎到成人，位置，以便它视为横向与外侧的半圆形运河可见（图2B），内耳。角的内耳使奥迪胶囊后形成的线大约是在一个45度角，从切片（图2A，B）的拟平面。当奥迪胶囊被认为奥迪胶囊背部分是在顶部，耳蜗底回应位于底部（箭头，图2b）和前半规管（ASC）在顶部（图2B）。 </li><li>琼脂糖将设置在室温下30分钟内。转移剥走模具含有包埋组织，以4℃，直到准备第。如果切片将被推迟到第二天，加入1X PBS覆盖在模具和存储在一个潮湿的纸毛巾浸泡在1X PBS框琼脂糖。 </li><li>剥离的塑料模具。使用刀片，切出琼脂糖框，其中包含的内耳组织。照顾，以确保截面直和平行块的对面，这将是表面上粘琼脂糖块。由于琼脂糖不渗透的组织，不从周围的组织修剪过多琼脂糖，这样的组织将是很好的支持。 </li><li>使用强力胶块切割面。 </li><li>在第40至100微米的厚度vibratome。与1X PBS，0.1％TritonX - 100填补了组织培养皿中保存的部分。一个有代表性的部分是通过耳蜗螺旋图所示。 2C。 </li><li>使用镊子，解剖远离组织琼脂糖。琼脂糖不会穿透组织，但也只是坚持到内耳的外表面。 </li><li>如果需要，池琼脂糖，切片组织一个大洞的开放使用吸管，以防止组织过度剪切。如果执行多个不同组织的多种抗体的污渍，部分可以在16 - 或24孔组织培养皿举办。 </li></ol> 3。抗体染色vibratome部分继续与抗体染色。在整个安装过程部分，在溶液中自由浮动。下面是一个例子Corti器的支柱细胞S100的标记抗体染色协议，但该协议可以用于任何抗体。除非另有说明，所有的清洗是在室温下进行。 <ol><li>在一个多井的组织培养皿中，PBT温和的摇摆（1X PBS，1％的牛血清白蛋白，0.1％Triton X一100）孵育30分钟的自由浮动的部分。 </li><li>吸取关闭旧的解决方案和块自由FLoating节30分钟的温和的摇摆PBT +正常山羊血清（50μL农工商超市，PBT / 1毫升）。 </li><li>吸取关闭旧的解决方案，并添加在PBT +农工商超市（PBT +农工商S100的1:200稀释）稀释抗体。在4℃孵育过夜彗星与温和的摇摆。 </li><li>吸取过初级抗体溶液。洗3倍，5分钟。与PBT，然后4X，30分钟。与PBT。 </li><li>座与PBT +农工商超市，30分钟。 </li><li>孵育与荧光标记的二抗稀释PBT +农工商超市（山羊提出）。孵育过夜，4 ° C，或者几个小时，放疗，温和的摇摆。 </li><li>吸取过二次抗体溶液。洗3倍，5分钟，然后4X，30分钟。与PBT。 </li><li>关闭最后PBT吸取清洗和适当的水安装包含核染液的介质更换。 </li></ol> 4。安装在幻灯片上部分和共聚焦显微镜为了防止盖玻片压碎厚，安装个别路段时，一个间隔必须在显微镜载玻片和盖玻片之间插入。下面是两个替代方法安装厚vibratome部分: <ol><li><ol type="a"><li>在广场周围的组织切片置于真空润滑脂的边缘，可以创建一个间隔。因此，当放下盖玻片，盖玻片边缘将密封的真空润滑脂。按盖玻片边缘用拇指到幻灯片上，坚定不移地坚持它。如果周围盖玻片边缘整齐，均匀，是目前真空润滑脂，幻灯片将被封闭，足够为一个倒置的共聚焦显微镜的使用。 </li><li>另外，可以创建一个间隔使用已知的网目尺寸的琼脂糖层析树脂（如阿菲 - 凝胶蓝胶，Bio - Rad公司）。吸取1μL树脂到每个将被放置盖玻片的四个角落的显微镜幻灯片。吸取之一的组织切片和树脂的四个点中安装媒体。盖玻片。如果使用的是一个倒置的共聚焦显微镜，密封的显微镜玻片上，盖玻片，使用指甲油。 </li></ol></li><li>获得使用共聚焦显微镜切片染色vibratome的光学部分。为了量化Corti器内的各种细胞类型，在定义的步长（1.5至3微米）获得一个Z - ​​Stack的。部分应该反染核染料（步骤3.8）。共聚焦图像采集后的任何电脑屏幕上的图像可以检查。细胞可以算作手动图像观察原子核的外观和失踪审议通过Z - Stack的顺序。 </li></ol> 5。代表性的成果: 一个代表共聚焦的Z - Stack通过vibratome的部分，S100蛋白，这是目前在支柱产业和Deiters细胞，核染色（图3A - F）的counterstained染色。每个小组在3微米的步骤采取的共聚焦图像，与面板的一个共聚焦图像获得最接近vibratome节和面板F的共聚焦图像vibratome节内获得最内部表面。 Corti器的形态出现最小中断。特别是，支柱细胞的细胞质扩展不破。指出，通过在Z - Stack（图3A至3F）的外观和支柱细胞核消失，无论是内部和外部的支柱细胞数量可算（见编号，图3A - F）。由于少数每人共聚焦图像的细胞类型，这种方法可以用来计数Corti器内的大多数细胞类型。 <img src="/files/ftp_upload/2793/2793fig1.jpg" alt="图1"/> 图1。完好，周15岁，成人内耳在奥迪胶囊包裹。 （一）正确的滴耳胶囊（括号内）包裹在颅骨，从内部头部的制高点看，大脑取出后。前路是正确的。 （二）完整，15周龄后，从头骨解剖内耳。左侧内耳侧面可见;右内耳的内侧表面可见。标签表明前半规管（ASC），合作（耳蜗），OW（卵圆窗）的位置。 <img src="/files/ftp_upload/2793/2793fig2.jpg" alt="图2"/> 图2:（一）所有在琼脂糖嵌入在剥离的模具中心，从15周龄成年内耳。拟截面表示。 （b）关闭图像嵌入到前15周龄，整个内耳。周围骨的一部分已被解剖了，所以膜迷路可以更清楚地查看。被跟踪的横向半圆形运河（LSC），前半规管（ASC），和耳蜗（CO）（白色曲线）。耳蜗（箭头）基转，并打算平面部分（虚线）表示。 （三）代表的CROS -通过耳蜗。在一个截面通过人工耳蜗管，尔蒂（OC）的器官是盒装，血管纹（SV）括号内，Reissner变分的膜（RM）表示。 <img src="/files/ftp_upload/2793/2793fig3.jpg" alt="图3"/> 图3。尔蒂在野生型小鼠在P21的器官。 （AF）的顺序焦系列，在3微米步长，通过一个S100抗体染色Corti器，可视化的支柱细胞和Deiters，"细胞。从开始到结束的一系列内部和外部的支柱细胞计数的编号。缩写:IHC（内毛细胞），aIHC（相邻的内毛细胞），OHC（外毛细胞），PC（支柱细胞），DC（Deiters&#39;细胞）。

<ol>
	<li>Lim, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+structure+of+the+organ+of+Corti:+a+review.">Functional structure of the organ of Corti: a review.</a> Hear Res. 22, 117-146 (1986).</li><li>Raphael, Y., Altschuler, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+innervation+of+the+cochlea.">Structure and innervation of the cochlea.</a> Brain Res Bull. 60, 397-422 (2003).</li><li>Chung, W. S., Shin, C. H., Stainier, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bmp2+signaling+regulates+the+hepatic+versus+pancreatic+fate+decision.">Bmp2 signaling regulates the hepatic versus pancreatic fate decision.</a> Dev Cell. 15, 738-748 (2008).</li><li>Sun, X., Mariani, F. V., Martin, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functions+of+FGF+signalling+from+the+apical+ectodermal+ridge+in+limb+development.">Functions of FGF signalling from the apical ectodermal ridge in limb development.</a> Nature. 418, 501-508 (2002).</li><li>Trinh, L. A., Stainier, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibronectin+regulates+epithelial+organization+during+myocardial+migration+in+zebrafish.">Fibronectin regulates epithelial organization during myocardial migration in zebrafish.</a> Dev Cell. 6, 371-382 (2004).</li><li>Yin, C., Kikuchi, K., Hochgreb, T., Poss, K. D., Stainier, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hand2+regulates+extracellular+matrix+remodeling+essential+for+gut-looping+morphogenesis+in+zebrafish.">Hand2 regulates extracellular matrix remodeling essential for gut-looping morphogenesis in zebrafish.</a> Dev Cell. 18, 973-984 (2010).</li><li>Shim, K., Minowada, G., Coling, D. E., Martin, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sprouty2,+a+mouse+deafness+gene,+regulates+cell+fate+decisions+in+the+auditory+sensory+epithelium+by+antagonizing+FGF+signaling.">Sprouty2, a mouse deafness gene, regulates cell fate decisions in the auditory sensory epithelium by antagonizing FGF signaling.</a> Dev Cell. 8, 553-564 (2005).</li></ol>

IACUC威斯康星医学院规定的准则和法规的规定进行了动物实验。

vibratome切片，免疫组织化学和激光共聚焦成像的过程，允许尔蒂与器官组织的损伤最小的可视化。显然，从内部在vibratome部分地区采取的共聚焦图像显示对细胞的形态，只有通过固定工件有限的优秀保存。接近两个削减了vibratome部分表面的共聚焦图像往往无法区分来自内部区域的图像，虽然偶尔蜂窝中断，如在休息的支柱细胞的细胞质扩展，观察（未显示）。这种方法很简单，主要设备的物品 - ...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td>徕卡VT1000S振动刀片切片机</td><td>徕卡</td><td></td><td></td></tr><tr><td>蔡司LSM510激光扫描共聚焦显微镜</td><td>蔡司</td><td></td><td></td></tr><tr><td>学生杜蒙＃5镊子</td><td>精细科学的工具</td><td> 91150-20 </td><td></td></tr><tr><td>超纯低熔点琼脂糖</td><td> Invitrogen公司</td><td> 16520-050 </td><td></td></tr><tr><td>剥离一个双向嵌入模具</td><td> Polysciences </td><td> 18986或18646A </td><td></td></tr><tr><td>牛血清白蛋白</td><td>杰克逊ImmunoResearch </td><td> 001-000-162 </td><td></td></tr><tr><td>山羊血清</td><td> Invitrogen公司</td><td> 16210-064 </td><td></td></tr><tr><td> S100 </td><td> Dako公司</td><td> Z0311 </td><td></td></tr><tr><td>的Alexa Fluor 568山羊抗兔IgG </td><td> Invitrogen公司</td><td> A - 11036 </td><td> 1:1000稀释</td></tr><tr><td> Vectashield </td><td>媒介实验室</td><td> H - 1000 </td><td></td></tr><tr><td> YO - PRO - 1 </td><td> Invitrogen公司</td><td> Y3603 </td><td></td></tr><tr><td>真空润滑脂</td><td>费舍尔</td><td> S41718 </td><td></td></tr><tr><td>阿菲 - 凝胶蓝胶</td><td> Bio - Rad公司</td><td> 153-7301 </td><td></td></tr></tbody></table>

vibratome sectioning for enhanced preservation of the cytoarchitecture of the mammalian organ of corti

尔蒂哺乳动物器官是一个高度有序的mechanosensory头发和nonsensory支持细胞的细胞镶嵌 （在1,2审查）。可视化的这种细胞的马赛克往往需要Corti器的横截面。特别是nonsensory支柱和Deiters细胞，其细胞核位于毛细胞基部，不能无交叉切片Co​​rti器的可视化。然而，细腻尔蒂哺乳动物器官的细胞结构，包括罚款的支柱和Deiters细胞的胞质突起，是难以保持常规病理过程，如石蜡及冷冻切片，这是与标准的免疫组织化学染色技术兼容。 在这里，我描述了一个简单而强大的程序，包括耳蜗，在整个装载这些vibratome部分的免疫组织化学染色，共聚焦显微镜，切片vibratome。这个程序已经广泛用于多个器官，包括鼠标肢芽，斑马鱼肠道，肝，胰，心（见3-6选定的例子）的immunhistochemical分析。此外，此过程成像和quantitificaton支柱细胞在胚胎和成年小鼠7尔蒂突变和控制机关数sucessful。然而，这种方法是目前没有被广泛用来研究哺乳动物器官的尔蒂。在此过程中的潜在的罚款尔蒂成人器官的细胞结构都提供了增强的保存和允许各种细胞类型的量化描述。

Watch this Scientific Journal Video about Vibratome Sectioning for Enhanced Preservation of the Cytoarchitecture of the Mammalian Organ of Corti at JoVE.com

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

一个简单的程序，切片，免疫组织化学和激光共聚焦显微镜Corti器的vibratome描述。此过程允许的罚款尔蒂哺乳动物器官的细胞结构的改善保存，因此可以准确定量的细胞类型。

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 ...

vibratome-sectioning-for-enhanced-preservation-cytoarchitecture

Research

JoVE Journal

Neuroscience

24.2K 次观看.  Medical College of Wisconsin. 一个简单的程序，切片，免疫组织化学和激光共聚焦显微镜Corti器的vibratome描述。此过程允许的罚款尔蒂哺乳动物器官的细胞结构的改善保存，因此可以准确定量的细胞类型。

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

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical and molecular methods provides information of potential target genes and signaling pathways. To further elucidate specific regulatory mechanisms requires a system allowing the manipulation of only a small number of cells of a specific tissue by either overexpression, ablation or re-introduction of specific genes and follow their fate during development. To achieve this ex utero electroporation of hippocampal structures, especially the dentate gyrus, followed by organotypic slice culture provides such a tool. Using this system to generate mosaic deletions allows determining whether the gene of interest regulates cell-autonomously developmental processes like progenitor cell proliferation or neuronal differentiation. Furthermore it facilitates the rescue of phenotypes by re-introducing the deleted gene or its target genes. In contrast to in utero electroporation the ex utero approach improves the rate of successfully targeting deeper layers of the brain like the dentate gyrus. Overall ex utero electroporation and organotypic slice culture provide a potent tool to study regulatory mechanisms in a semi-native environment mirroring endogenous conditions.

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Here we present a protocol providing a tool to examine regulatory mechanisms of specific genes during hippocampal development. Employing ex utero electroporation and organotypic slice culture allows the up- and down-regulation of the expression of genes of interest in single cells and follow their fate during development.

小鼠海马组织的前子宫内电和器官切片文化

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical ...

科研

发育生物学

An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as well as differentiation and integration of new neurons into existing neural circuits. For postnatal neurogenesis in the olfactory bulbs, these events are segregated within three anatomically distinct domains: proliferation largely occurs in the subependymal zone (SEZ) of the lateral ventricles, migrating neuroblasts traverse through the rostral migratory stream (RMS), and new neurons differentiate and integrate within the olfactory bulbs (OB). The three domains serve as ideal platforms to study the cellular, molecular, and physiological mechanisms that regulate each of the biological events distinctly. This paper describes an organotypic slice assay optimized for postnatal brain tissue, in which the extracellular conditions closely mimic the in vivo environment for migrating neuroblasts. We show that our assay provides for uniform, oriented, and speedy movement of neuroblasts within the RMS. This assay will be highly suitable for the study of cell autonomous and non-autonomous regulation of neuronal migration by utilizing cross-transplantation approaches from mice on different genetic backgrounds.

This protocol describes an organotypic slice assay optimized for the postnatal brain and high-resolution time-lapse imaging of neuroblast migration in the rostral migratory stream.

对于高分辨率延时成像神经细胞迁移的一个器官型产后脑切片检测

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as ...

神经科学

Preparation of Parasagittal Slices for the Investigation of Dorsal-ventral Organization of the Rodent Medial Entorhinal Cortex

Computation in the brain relies on neurons responding appropriately to their synaptic inputs. Neurons differ in their complement and distribution of membrane ion channels that determine how they respond to synaptic inputs. However, the relationship between these cellular properties and neuronal function in behaving animals is not well understood. One approach to this problem is to investigate topographically organized neural circuits in which the position of individual neurons maps onto information they encode or computations they carry out1. Experiments using this approach suggest principles for tuning of synaptic responses underlying information encoding in sensory and cognitive circuits2,3.
The topographical organization of spatial representations along the dorsal-ventral axis of the medial entorhinal cortex (MEC) provides an opportunity to establish relationships between cellular mechanisms and computations important for spatial cognition. Neurons in layer II of the rodent MEC encode location using grid-like firing fields4-6. For neurons found at dorsal positions in the MEC the distance between the individual firing fields that form a grid is on the order of 30 cm, whereas for neurons at progressively more ventral positions this distance increases to greater than 1 m. Several studies have revealed cellular properties of neurons in layer II of the MEC that, like the spacing between grid firing fields, also differ according to their dorsal-ventral position, suggesting that these cellular properties are important for spatial computation2,7-10.
Here we describe procedures for preparation and electrophysiological recording from brain slices that maintain the dorsal-ventral extent of the MEC enabling investigation of the topographical organization of biophysical and anatomical properties of MEC neurons. The dorsal-ventral position of identified neurons relative to anatomical landmarks is difficult to establish accurately with protocols that use horizontal slices of MEC7,8,11,12, as it is difficult to establish reference points for the exact dorsal-ventral location of the slice. The procedures we describe enable accurate and consistent measurement of location of recorded cells along the dorsal-ventral axis of the MEC as well as visualization of molecular gradients2,10. The procedures have been developed for use with adult mice (&gt; 28 days) and have been successfully employed with mice up to 1.5 years old. With adjustments they could be used with younger mice or other rodent species. A standardized system of preparation and measurement will aid systematic investigation of the cellular and microcircuit properties of this area.

We describe procedures for preparation and electrophysiological recording from brain slices that maintain the dorsal-ventral axis of the medial entorhinal cortex (MEC). Because neural encoding of location follows a dorsal-ventral organization within the MEC, these procedures facilitate investigation of cellular mechanisms important for navigation and memory.

背腹鼠​​类内侧嗅皮层组织的调查矢状窦片的制备

Computation in the brain relies on neurons responding appropriately to their synaptic inputs. Neurons differ in their complement and distribution of ...

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.

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 ...

生物学

Vibratome Sectioning Mouse Retina to Prepare Photoreceptor Cultures

The retina is a part of the central nervous system that has organized architecture, with neurons in layers from the photoreceptors, both rods and cones in contact with the retinal pigmented epithelium in the most distant part on the retina considering the direction of light, and the ganglion cells in the most proximal distance. This architecture allows the isolation of the photoreceptor layer by vibratome sectioning. The dissected neural retina of a mouse aged 8 days is flat-embedded in 4% gelatin on top of a slice of 20% gelatin photoreceptor layer facing down. Using a vibratome and a double edged razor blade, the 100 &#181;m thick inner retina is sectioned. This section contains the ganglion cells and the inner layer with notably the bipolar cells. An intermediary section of 15 &#181;m is discarded before 200 &#181;m of the outer retina containing the photoreceptors is recovered. The gelatin is removed by heating at 37 &#176;C. Pieces of outer layer are incubated in 500 &#181;l of Ringer&#39;s solution with 2 units of activated papain for 20 min at 37 &#176;C. The reaction is stopped by adding 500 &#181;l 10% fetal calf serum (FCS) in Dulbecco&#39;s Modified Eagle Medium (DMEM), then 25 units of DNAse I is added before centrifugation at RT, washed several times to remove serum and the cells are resuspended in 500 &#181;l of DMEM and seeded at 1 x 105 cells/cm2. The cells are grown to 5 days in vitro and their viability scored using live/dead assay. The purity of the culture is first determined by microscopic observation during the experiment. The purity is then validated by seeding and fixing cells on a histological slide and analyzing using a rabbit polyclonal anti-SAG, a photoreceptor marker and mouse monoclonal anti-RHO, a rod photoreceptor specific marker. Alternatively, the photoreceptor layer (97% rods) can be used for gene or protein expression analysis and for transplantation.

Neural retina of a mouse aged 8 days is on top of a 4% gelatin block. After isolation of the photoreceptor layer (200 &#181;m) by vibratome, the photoreceptors are seeded after mechanical and enzymatic dissociation for culture. The photoreceptor layer can be used for molecular, biochemical analyses or transplantation.

的vibratome切片小鼠视网膜准备感光文化

The retina is a part of the central nervous system that has organized architecture, with neurons in layers from the photoreceptors, both rods and cones in ...

Fixation of Embryonic Mouse Tissue for Cytoneme Analysis

Developmental tissue patterning and postdevelopmental tissue homeostasis depend upon controlled delivery of cellular signals called morphogens. Morphogens act in a concentration- and time-dependent manner to specify distinct transcriptional programs that instruct and reinforce cell fate. One mechanism by which appropriate morphogen signaling thresholds are ensured is through delivery of the signaling proteins by specialized filopodia called cytonemes. Cytonemes are very thin (&#8804;200 nm in diameter) and can grow to lengths of several hundred microns, which makes their preservation for fixed-image analysis challenging. This paper describes a refined method for delicate handling of mouse embryos for fixation, immunostaining, and thick sectioning to allow for visualization of cytonemes using standard confocal microscopy. This protocol has been successfully used to visualize cytonemes that connect distinct cellular signaling compartments during mouse neural tube development. The technique can also be adapted to detect cytonemes across tissue types to facilitate the interrogation of developmental signaling at unprecedented resolution.

Here, an optimized step-by-step protocol is provided for fixation, immunostaining, and sectioning of embryos to detect specialized signaling filopodia called cytonemes in developing mouse tissues.

固定胚胎小鼠组织以进行细胞内膜分析

Developmental tissue patterning and postdevelopmental tissue homeostasis depend upon controlled delivery of cellular signals called morphogens. Morphogens ...

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.

We present a method to isolate the adult organ of Corti as three intact cochlear turns (apex, middle, and base). We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies. Together these techniques allow the study of hair cells, supporting cells, and other cell types found in the cochlea.

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 ...

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 ...

Stereocilia Bundle Imaging with Nanoscale Resolution in Live Mammalian Auditory Hair Cells

Inner ear hair cells detect sound-induced displacements and transduce these stimuli into electrical signals in a hair bundle that consists of stereocilia that are arranged in rows of increasing height. When stereocilia are deflected, they tug on tiny (~5 nm in diameter) extracellular tip links interconnecting stereocilia, which convey forces to the mechanosensitive transduction channels. Although mechanotransduction has been studied in live hair cells for decades, the functionally important ultrastructural details of the mechanotransduction machinery at the tips of stereocilia (such as tip link dynamics or transduction-dependent stereocilia remodeling) can still be studied only in dead cells with electron microscopy. Theoretically, scanning probe techniques, such as atomic force microscopy, have enough resolution to visualize the surface of stereocilia. However, independent of imaging mode, even the slightest contact of the atomic force microscopy probe with the stereocilia bundle usually damages the bundle. Here we present a detailed protocol for the hopping probe ion conductance microscopy (HPICM) imaging of live rodent auditory hair cells. This non-contact scanning probe technique allows time lapse imaging of the surface of live cells with a complex topography, like hair cells, with single nanometers resolution and without making physical contact with the sample. The HPICM uses an electrical current passing through the glass nanopipette to detect the cell surface in close vicinity to the pipette, while a 3D-positioning piezoelectric system scans the surface and generates its image. With HPICM, we were able to image stereocilia bundles and the links interconnecting stereocilia in live auditory hair cells for several hours without noticeable damage. We anticipate that the use of HPICM will allow direct exploration of ultrastructural changes in the stereocilia of live hair cells for better understanding of their function.

Here we present a protocol for the Hopping Probe Ion Conductance Microscopy (HPICM), a non-contact scanning probe technique that allows nanoscale imaging of stereocilia bundles in live auditory hair cells.

立体纤毛束成像在活哺乳动物听觉毛细胞中的纳米级分辨率

Inner ear hair cells detect sound-induced displacements and transduce these stimuli into electrical signals in a hair bundle that consists of stereocilia ...

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 ...

Vibratome切片增强保尔蒂哺乳动物器官的细胞构筑

摘要

探索更多视频

此视频中的章节

小鼠海马组织的前子宫内电和器官切片文化

对于高分辨率延时成像神经细胞迁移的一个器官型产后脑切片检测

背腹鼠类内侧嗅皮层组织的调查矢状窦片的制备

原代培养及质粒电穿孔尔蒂小鼠器官。

的vibratome切片小鼠视网膜准备感光文化

固定胚胎小鼠组织以进行细胞内膜分析

全山解剖和成年小鼠耳蜗的免疫

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

立体纤毛束成像在活哺乳动物听觉毛细胞中的纳米级分辨率

体外延时活细胞成像探索细胞向科尔蒂器官迁移

Vibratome切片增强保尔蒂哺乳动物器官的细胞构筑

摘要

探索更多视频

此视频中的章节

小鼠海马组织的前子宫内电和器官切片文化

对于高分辨率延时成像神经细胞迁移的一个器官型产后脑切片检测

背腹鼠​​类内侧嗅皮层组织的调查矢状窦片的制备

原代培养及质粒电穿孔尔蒂小鼠器官。

的vibratome切片小鼠视网膜准备感光文化

固定胚胎小鼠组织以进行细胞内膜分析

全山解剖和成年小鼠耳蜗的免疫

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

立体纤毛束成像在活哺乳动物听觉毛细胞中的纳米级分辨率

体外延时活细胞成像探索细胞向科尔蒂器官迁移

背腹鼠类内侧嗅皮层组织的调查矢状窦片的制备