Name: flat mount imaging of mouse skin and its application to the analysis of hair follicle patterning and sensory axon morphology
Uploaded: 2014-06-25T15:00:00
Description: Watch this Scientific Journal Video about Flat Mount Imaging of Mouse Skin and Its Application to the Analysis of Hair Follicle Patterning and Sensory Axon Morphology at JoVE.com

The authors thank Dr. Amir Rattner for helpful comments on the manuscript. Supported by the Howard Hughes Medical Institute.

严格按照本指南中的美国国立卫生研究院实验动物的护理和使用的建议进行研究。所有的动物都按照批准的机构动物护理和使用委员会的约翰霍普金斯医疗机构（IACUC）协议MO11M29处理。咨询安乐死的方法核定的当地机构动物护理和使用委员会的指导方针。处理醛固色剂或有机溶剂时，应戴上手套，实验室外套，和护目镜。 材料1。准备<ol><li>浇SYLGARD板<ol><li>按照制造商的指示...

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上述的解剖方法的掌握，只需要耐心，一个稳定的手，和几个不错的清扫工具。背部皮肤剥离是相对容易的，但尾巴和脚的皮肤解剖 - 尤其是在出生后早期的年龄 - 是更具挑战性。在早期产前年龄（ 例如 ，E15之前），皮肤，很难不将其撕破删除。方便的是，对于生长和皮肤结构小鼠构图的很多研究中，感兴趣的事件发生时出生后，如在立毛肌19的生长的研究可见，例如。 <p class...

皮肤是身体中最大的器官，在躯体感觉，绝缘/体温调节和免疫防御1重要的功能。了解皮肤的发育和功能的细胞和分子基础已经因为皮肤作为一个生物系统及其与皮肤科相关的基本重要性的长期利益。哺乳动物的皮肤中含有多种多细胞结构，包括角质形成细胞分层层，真皮结缔组织，几种类型的毛囊，皮脂腺，立毛肌，血管，至少有十几个不同的类传入的（感觉）和传出神经的纤维（ 图1）。身体的不同区域与典型不同类型的皮肤有关。在大多数哺乳动物中，几乎整个身体表面覆盖有皮肤被密集地填充有毛囊。 [人类和无毛鼹鼠构成例外TH是的图案。头发是从手和脚，这也与专门的表皮图案（皮纹），外分泌腺和感觉神经末梢相关的掌面失踪。控制生长，分化和毛囊内的细胞空间排列的细胞和分子事件是特殊利益，因为每个毛囊展品，缩影，很多器官2的主要特征。这些功能包括干细胞的存在和干细胞小生境，正是编排细胞迁移，并从胚胎学上不同的部分多细胞结构的组装。 本文介绍了解剖方法，固定，标记和成像小鼠皮肤作为一个完整的二维表，被称为“整装”或“平山”的准备。自小鼠的皮肤较薄，也能够通过图像扁平的滑雪板的整个厚度N使用常规的共聚焦显微镜。平板安装的方法来成像哺乳动物皮肤在技术上是有利的，因为它绕过了需要物理切片，从而使结构将完全由光学切片重建。因为几乎整个皮肤被处理为一个单一的对象，平面安装方法也有利于身体表面的多个区域的成像，同时保持对位置和相对方位信息到主体轴。最后，对皮肤内的结构是典型地存在于被定期重复的是，从而便于从一个给定结构的多个代表图像的集合图案。这些特征是熟悉的在视网膜上，中枢神经系统，享有类似的优势，为神经元形态3的研究了二维部分谁工作神经生物学家。 这里所描述的平面安装的方法是特殊的utility表示学习表现出空间组织上的相对大的规模对皮肤的二维平面内的结构。大型空间组织的一个例子是毛囊和毛囊相关结构的协调极性- Merkel细胞簇，立毛肌，皮脂腺和神经末梢4。毛囊被定向在相对于皮肤的平面，并且位于皮肤的2维平面内的毛囊矢量的分量的角度通常表现出的取向相对于所述本体的轴是为每个精确地确定在身体上的位置。例如，从延髓背点毛囊尾鳍和头发上的脚的背侧面从近端指向远端。毛囊的方向是由平面细胞极性的信号控制（PCP，也称为组织极性5）。这个信号系统被发现在果蝇在一个小组核心PCP基因被发现，控制表皮的毛和硬毛的方向。的核心PCP基因三个哺乳动物直向同源物-型卷曲同源物6（FZD6，也被称为FZ6），钙粘蛋白EGF LAG 7通G-型受体1（CELSR1）和旺状2（VANGL2） -玩在哺乳动物类似的角色皮肤，协调毛囊的取向与主体轴。表明，在不存在PCP信令的主要缺陷是毛囊取向的初始随机化或解体，对毛囊的内在结构没有影响FZ6基因敲除小鼠（ - / - FZD6 tm1Nat，以下简称为FZ6）的研究6-8。第二个非PCP系统后的行为，促进毛囊附近，这导致生产大规模发模式，如旋涡及束的局部比对。 大规模的第二个例子皮肤内的空间组织被认为是在感觉轴突乔木的形态。感觉神经元支配的皮肤有其胞体在背根和三叉神经节。这些神经元的检测温度，疼痛，发痒，以及各种类型的机械变形的照射在皮肤和头发9。它们可以分为基于轴突直径和传导速度，终端神经末梢结构，受体，通道和其他分子的表达模式亚型。因为皮肤内神经支配的高密度，分析了包 ​​括可视化所有轴突（ 例如 ，抗神经丝免疫染色）或一个类的甚至全部的轴突（当单个细胞类型的特点是荧光记者表达看）通常揭示了一个致密的叠加轴突，使得它不可能确定单个轴的形​​态。为了解决这个问题，我们使用了极其稀疏基因定向升abeling生产背侧皮肤样本中，个人以及隔离轴突乔木是由一个组织化学记者，人胎盘碱性磷酸酶10表达的可视化。该方法允许个别轴突乔木形态的明确的视觉效果，并根据形态学标准躯体感觉神经元的类型的定义。

<table border="0" cellpadding="0" cellspacing="0" width="824">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Name of Material/ Equipment</td>
			<td style="width:256px;">Company</td>
			<td style="width:145px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5-bromo-4-chloro-indolyl phosphate (BCIP)</td>
			<td>Roche</td>
			<td>11383221001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AM1-43</td>
			<td>Biotium</td>
			<td>70024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AM4-65</td>
			<td>Biotium</td>
			<td>70039</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Benzyl alcohol</td>
			<td>Sigma</td>
			<td>402834</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Benzyl benzoate</td>
			<td>Sigma&#160;</td>
			<td>B-6630</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Confocal microscope</td>
			<td style="width:256px;">Zeiss</td>
			<td style="width:145px;">LSM700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cy3-alpha smooth muscle actin antibody</td>
			<td>Sigma&#160;</td>
			<td>C6198</td>
			<td>1:400</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cytokeratin-8&#160;</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>TROMA-I-c</td>
			<td>1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissecting microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissection tools&#160;</td>
			<td>Fine Science Tools</td>
			<td></td>
			<td>scissors and forceps</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Electric razor</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluoromount G</td>
			<td>EM Sciences</td>
			<td>17984-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Formalin</td>
			<td>Sigma</td>
			<td>HT501320</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass dishes</td>
			<td>Pyrex&#160;</td>
			<td></td>
			<td>6 cm and 10 cm diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass plates</td>
			<td>Amersham Biosciences</td>
			<td>SE202P-10</td>
			<td>10 cm x 8 cm x 1 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hair remover&#160;</td>
			<td>Nair</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Horizontal rotating platform&#160;</td>
			<td style="width:256px;">Hoefer</td>
			<td style="width:145px;">PR250 Orbital shaker</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insect pins</td>
			<td>Fine Science Tools&#160;</td>
			<td>26002-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ketamine/xylazine</td>
			<td>Sigma</td>
			<td>K113</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nitroblue tetrazolium (NBT)</td>
			<td>Roche&#160;</td>
			<td>11383213001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Oil Red O</td>
			<td>Sigma</td>
			<td>O0625</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde</td>
			<td>Sigma&#160;</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Razor Blades</td>
			<td style="width:256px;">VWR</td>
			<td style="width:145px;">55411-055</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Secondary antibodies&#160;</td>
			<td>Invitrogen</td>
			<td></td>
			<td>Alexa-dye conjugated&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sylgard-184</td>
			<td>Fisher Scientific</td>
			<td>NC9020938</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue culture plastic dishes</td>
			<td></td>
			<td></td>
			<td>10 cm diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue culture plates</td>
			<td></td>
			<td></td>
			<td>6- and 12-well&#160;</td>
		</tr>
	</tbody>
</table>

flat mount imaging of mouse skin and its application to the analysis of hair follicle patterning and sensory axon morphology

皮肤是一种高度非均质的组织。真皮内的结构包括毛囊，立毛肌，表皮特殊化（如Merkel细胞簇），皮脂腺，神经和神经末梢和毛细血管。这些结构的空间布置受到严格控制在微观尺度上 - 如看到的那样，例如，在在一个单一的毛囊细胞类型的有序排列 - 和在宏观尺度上 - 所看到的数以千计的头发在几乎相同的方向皮肤的局部区域​​内的毛囊。可视化这些结构而不物理切片的皮肤是因为这个器官的2维几何的可能。在这个协议中，我们证明了小鼠皮肤可解剖，固定，透，染色，明确作为一个完整的二维物体，一台安装。该协议允许对皮肤结构的可视化容易以其全部通过大面积的皮肤被选择的整个厚度iCal的切片和重建。这些结构中的图像也可以与左右位置和相对方位信息集成到主体轴。

皮肤铺片的明场成像可用于基于黑色素色素沉着（ 图4）的图像的皮肤感觉传入（ 图3A 10）和毛囊的图案。皮肤铺片的共焦成像可以被用来定义（1）Merkel细胞簇，可视化与抗细胞角蛋白6或带AM染料的吸收（ 图3I-L），（2）立毛肌，可视化与抗几何平滑肌肌动蛋白（ 图3G，3H），（3）皮脂腺，可视化与油红O（ 图3C-F）和（4）的...

Watch this Scientific Journal Video about Flat Mount Imaging of Mouse Skin and Its Application to the Analysis of Hair Follicle Patterning and Sensory Axon Morphology at JoVE.com

Flat Mount Imaging of Mouse Skin and Its Application to the Analysis of Hair Follicle Patterning and Sensory Axon Morphology

哺乳动物的皮肤中含有结构的多样化 - 如毛囊和神经末梢 - 表现出独特的空间组织模式。作为平坦安装分析皮肤借此组织的2维几何的优点，以产生皮肤结构的全厚的高清晰度图像。

小鼠皮肤及其毛囊图案和感觉轴突形态分析中的应用平山成像

Skin is a highly heterogeneous tissue. Intra-dermal structures include hair follicles, arrector pili muscles, epidermal specializations (such as Merkel ...

Research

JoVE Journal

Neuroscience

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

多鼠神经解剖学磁共振成像

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

科研

神经科学

Tissue Preparation and Immunostaining of Mouse Sensory Nerve Fibers Innervating Skin and Limb Bones

鼠标感觉神经支配的皮肤和四肢骨骼纤维组织的制备及免疫染色

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

嗅觉受体神经元在全山免疫标记<em&gt;果蝇</em&gt;天线

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

Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

小鼠胚胎感觉轴突预测整个安装成像

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

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

成像钙<sup&gt; 2+</sup&gt;在小鼠视网膜锥光感受器轴突终末动态

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

Whole Mount Dissection and Immunofluorescence of the Adult Mouse 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 ...

Combined Recording of Mechanically Stimulated Afferent Output and Nerve Terminal Labelling in Mouse Hair Follicle Lanceolate Endings

机械刺激传入输出和神经末梢标签联合录制在小鼠毛囊披针形结局

A novel dissection and recording technique is described for monitoring afferent firing evoked by mechanical displacement of hairs in the mouse pinna. The ...

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

在毛囊披针形结局生活神经末端标记的光学监测<em&gt;离体</em&gt;鼠耳皮肤

A novel dissection and recording technique is described for optical monitoring staining and de-staining of lanceolate terminals surrounding hair follicles ...

Patch Clamp Recording of Starburst Amacrine Cells in a Flat-mount Preparation of Deafferentated Mouse Retina

星爆无长突细胞的膜片钳在Deafferentated小鼠视网膜的平板安装准备

The mammalian retina is a layered tissue composed of multiple neuronal types. To understand how visual signals are processed within its intricate synaptic ...