Name: flat mount imaging of mouse skin and its application to the analysis of hair follicle patterning and sensory axon morphology
Uploaded: 2014-06-25T15:00:00.000+00:00
Duration: 13 min 58 s
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，用碱混合固化剂，并搅拌用塑料棒。 </li><li>移液器〜35毫升药液的Sylgard每10厘米直径的组织培养皿中。组织培养皿比标准菌菜更深;这提供了昆虫针垂直间隙。 </li><li>将二上在室温下水平表面畲族过夜。在混合过程中形成的气泡会消失。 </li><li>以下聚合中，存储的Sylgard板在室温下进行。 注意:的Sylgard板可在室温下保存多年，并重复使用多次，直至的Sylgard从板分开。 </li></ol></li><li>解决方案<ol><li>苯甲酸苄酯和苯甲醇（BBBA）。混合2卷苯甲酸苄酯和1体积的苯甲醇。存储在一个玻璃瓶中，用玻璃塞或特氟隆顶暗。 注意:使用BBBA应有机溶剂废物进行适当处理。不要让BBBA接触塑料。 </li><li>磷酸盐缓冲盐水（PBS）中。添加4克PFA至80毫升水中，加入0.1的1N氢氧化钠，搅拌，在加热板上在约70℃〜5分钟，直到煤灰完全溶解，然后加入10 ml 10X PBS中，使体积达到100毫升水。 注意:甲醛/ PFA和BBBA蒸气吸入应在有盖的容器保持这些解决方案最小化。甲醛/ PFA是一种致癌物质。 </li><li>油红O。准备一个0.5％的股票在异丙醇。在即将使用前用水稀释至0.3％的油红O的终浓度，然后通过0.2微米的过滤器进行过滤。 </li></ol></li></ol> 2，皮肤解剖，枷，及结算所<ol><li>背部皮肤的免疫染色和黑色素成像<ol><li>对于非常年幼的小鼠（ 如胎儿和出生后一天（P）0-P3的幼仔），异氟醚吸入安乐死的小鼠。异氟醚吸入或氯胺酮/赛拉嗪注射液颈椎脱位安乐死年纪较大的老鼠。 </li><li>用电动剃须刀和脱毛露脱毛的。对于小鼠年龄小于P8，这一步可以跳过。 </li><li>用剃刀刀片从沿每个侧翼的尾根部进行水平切割，通过对各肢的基部的背侧，则进行横向于耳朵和端ing在鼻子。 </li><li>轻轻剥离从下层组织程序从后方的皮肤保持戴手套的手指之间的皮肤，使其不被钳子夹住至前。当剥离皮肤的耳朵水平，先切断的耳朵用剪刀和进行特别慢可使皮肤撕裂在这个位置。 </li><li>从这点起，定向朝上内侧的皮肤上。脚皮肤的Sylgard与20〜昆虫针均匀地分布在周边;不要过度牵拉皮肤（ 图2B）。盖在10-20毫升的PBS皮肤。 </li><li>解剖相差使用成角度的或弯曲的钳朝向水平方向，以最小化该镊子将渗入皮肤的危险钳子的尖端与皮肤相关的脂肪和结缔组织。挤出已经被困在皮肤下轻轻滚动棉签在皮肤表面的气泡。 注:如果皮肤是怎么回事用于平面安装或免疫组织化学，除去尽可能多的结缔组织越好;如果皮肤将要使用基于黑色素形象化毛囊，少完全去除结缔组织是令人满意的。产前皮肤需要贴壁结缔组织极少"清洗"。 </li><li>通过加入20ml新鲜制备的4％PFA / PBS中，或10％福尔马林缓冲液中每10cm的Sylgard板固定钉扎皮肤（如皮肤，将用于黑色素成像或碱性磷酸酶（AP）组织化学）或20毫升新制备的2％PFA / PBS（如果皮肤将用于染色或可视化的转基因角蛋白（KRT）17-GFP 11），轻轻在冷室旋转过夜。第二天，在PBS洗为&gt; 10分钟。 注:标准福尔马林是37％的甲醛;因此，"10％的福尔马林"，是3.7％的甲醛。 </li><li>对于AP染色和免疫染色，继续在第3节，对于黑色素IMAGING，脱水，通过梯度乙醇系列皮肤（70％，95％，100％），一天的每个步骤中，在室温下温和水平旋转。去除残留的结缔组织。 </li><li>用在100％乙醇的皮肤，取出昆虫针，如果需要，用剪刀修剪的皮肤，除去最外围1-2毫米的皮肤与销孔。 </li><li>皮肤转移到一个直径10厘米的玻璃皿中，将两片玻璃显微镜载片上的皮肤，以防止翘曲，并添加20毫升BBBA的。 BBBA迅速变硬组织所以重要的是对皮肤的载玻片的重量下加入BBBA之前被平坦化。 </li><li>在接下来的几个小时，抬起滑离皮肤几秒钟，让BBBA洗在皮肤上。 注:与BBBA工作时戴上手套。取决于皮肤的年龄和位置可以有BBBA高达30％的收缩率。 </li></ol></li><li>足部皮肤的毛囊Patte的分析rning 注:年龄一般检查的范围为P1-P8。 <ol><li>安乐死后，切断英尺以上的踝关节，并通过脚底使沿腹面一个直切。 </li><li>轻轻地从下面的组织剥离皮肤，则进行在近侧至远侧方向。 </li><li>切位以释放在皮肤和针它SYLGARD与内表面朝上（ 图2C）。有在脚部皮肤最小的结缔组织。 </li><li>转基因KRT17-GFP的皮肤现在就可以成像。对于黑色素的成像进行固定，脱水和BBBA结算如2.1节所述。 </li></ol></li><li>足部皮肤的可视化皮脂腺<ol><li>安乐死后，通过摩擦毛发去除凝胶在皮肤表面用戴手套的手指和拇指除去毛发;等待10分钟。 注意:不要使用电动剃须刀，这将破坏脆弱的足部皮肤。 </li><li> W灰皮肤表面用自来水，解剖针和皮肤的Sylgard如2.2节所述。 </li><li>固定用1％PFA / PBS中30分钟，在室温下，然后在PBS洗。 注:有关脚，产后第一周之后，开发可视化的皮脂腺，进行分析与油红O的最佳年龄是P21，因为这标志着头发色素沉着的最低点在第一毛发周期12，因此，皮脂腺可以更容易地看出。用小白鼠这种分析可以在任何年龄进行。为了获得白化后代跨色素突变体小鼠酪氨酸酶的突变品系，如C57BL/6J-Tyr C-2J。 </li></ol></li><li>尾部皮肤准备的分析黑色素分布，毛囊方向，和皮脂腺可视化<ol><li>安乐死后，使周围的尾巴基部圆形切口，然后在运行沿其腹侧面尾部的长度的纵向狭缝。 </li> &lt;立&gt;揭去尾部皮肤牢牢抓住尾部骨和/或与一对钳子的，并与第二对钳子的皮肤结缔组织的前端开始在尖端。针尾皮肤的Sylgard。 </li><li>对于黑色素的分布和毛囊取向的分析，解决和处理如2.1节所述的皮肤。对皮脂腺的分析，切尾皮肤成一系列段〜0.5至1厘米的长度固定之前和孵化的皮肤片在PBS / 5mM EDTA的过夜，在37℃以削弱真皮-表皮粘附13。 </li><li>轻轻地从真皮层剥离表皮，脚表皮到的Sylgard（内面朝上），并固定在4％PFA / PBS中1小时，在室温下。用PBS冲洗，如下所述与油红O染色。 注意:一个电动剃须刀和/或脱毛凝胶可用于解剖较旧的尾部皮肤（ 如 P21）之前，但这种治疗方法是可选的，因为尾巴上的毛是不是作为致密作为对身体别处。 </li></ol></li></ol> 3，染色反应<ol><li>人胎盘碱性磷酸酶（AP）组织化学可视化基因标记的轴突乔木10,14,15（ 图3A，B） <ol><li>上的Sylgard PFA固定后，将皮肤固定在10厘米的玻璃盘，并在PBS / 1毫米MgCl 2淹没。轻轻旋转水浴中烹调，在70℃下进行90分钟至破坏内源磷酸酶活性。 </li><li>可视化的AP报告基因活性的组织化学与在室温下4-48小时培养中的0.1M Tris，pH值9.5，0.1M氯化钠，50毫摩尔MgCl 2，0.34μg/ ml的NBT和0.175微克/毫升BCIP，通常使用10-15毫升每P21背部皮肤染色溶液。监控解剖显微镜下的反应，注意不要让它继续下一步的最佳时间，作为判断的强度相对轴突染色的目视检查，以非特异性NBT沉积。 </li&gt; <li>停止洗涤用PBS/0.1％Tween-20中的在1小时的过程中3的变化，重新针的外观来的Sylgard，通过乙醇系列脱水的皮肤，然后将皮肤与转移到10cm的培养皿在反应BBBA。 注:BBBA慢慢溶解的NBT沉淀。在最初的几个小时中BBBA，沉淀NBT的罚款背景会溶解;作为一个结果，BBBA解决方案将变暗，并且皮肤会减轻。几个小时后变成新鲜BBBA。 </li><li>成像在AP染色的皮肤后，转移它在室温下用乙醇为&gt; 1小时，然后将其在-20℃储存在新鲜乙醇℃。 AP染色的外观可以被存储在这样的许多个月，无信号损失。 </li></ol></li><li>油红O染色，以可视化皮脂腺4（图3C-F） <ol><li>除去昆虫标签，并从板的Sylgard转移轻轻固定脚或尾部的皮肤到6孔组织培养板的孔中，与高达每孔2的皮肤样品。 5分钟，在室温下的水:用60:40异丙醇清洗。 </li><li>染色在2毫升0.3％油红O在60:40异丙醇:水（见1.2节）在室温下2小时。 </li><li>为5分钟，在室温下，接着用水洗涤水:用60:40的异丙醇洗涤。小心地修剪掉皮肤的边缘，其中的销孔，用细剪刀使皮肤的表面可制成尽可能平坦。 </li><li>放置在与外表面朝上滑动皮肤，添加几滴的水或含水介质的安装，盖上盖玻片，在皮肤上，然后轻轻地用胶带将盖玻片与滑动到进一步平坦化的皮肤。 </li></ol></li><li>平山4,16,17免疫染色（ 图3G，H） <ol><li>切PFA固定的皮肤（ 如 1厘米××0.5cm）上的一个矩形，标志着其方向与不对称剪裁（ 如夹右前角）。进行孵化和洗轻柔旋转在一个旋转的水平平台的步骤。在6井 - 或12 - 孔组织培养板，洗几次，用PBS以除去残留的PFA，洗净，用PBS超过5-8小时，在10〜变化用0.3％的Triton X-100（0.3％PBST）室温通透的皮肤。 </li><li>与第一抗体在0.3％PBST清洗，用5％山羊血清和20％DMSO的五天在室温下孵育。覆盖板与透明胶带或粘合剂的塑料，以消除蒸发损失的水井。 </li><li>用0.3％PBST中的10〜15改变洗涤过5-8小时，并用第二抗体孵育在0.3％PBST清洗，用5％山羊血清和20％DMSO的3天，在室温下进行。 </li><li>用0.3％PBST超过5-8小时10-15洗涤。 </li><li>脱水在25％，50％和75％甲醇的每个，然后3次在100％甲醇中的每个20分钟5分钟。 </li><li>明确在BBBA在室温下过夜。 注:平板安装肌肤年轻的染色比〜P1可如上所述进行，但以较短的温育和洗涤时间。例如，P1的皮肤可以用过夜培养初级抗体和几个小时的潜伏期在二级抗体进行免疫染色，并用介入的只有2-3小时洗0.3％PBST。如上面所指出的，更年轻的外观也足够薄，它们可以在不BBBA结算进行成像。 </li></ol></li><li>可视化默克尔细胞团荧光神经末梢染料吸收（ 图3I-L） AMI-43（绿色）和AM4-65（红色）是通过非选择性阳离子通道18进入细胞可固定的荧光染料。在生物皮肤，这些染料被选择性地吸收和浓缩默克尔细胞。 <ol><li>溶解1毫克2毫升的PBS中等分AM1-43或AM4-65，并储存于-80°C。 </li><li>皮下注入新生的幼崽与使用29号针头每克体重2-10微克染料。 </li><li>一天后，安乐死的小鼠和狄氏CT背侧皮肤。 </li><li>修正了在4％PFA / PBS的皮肤在4℃过夜，洗净，用PBS，并安装在3.2节中描述的皮肤。 </li></ol></li></ol> 4，成像和图像分析<ol><li>成像毛囊方向（ 图4） <ol><li>使用解剖显微镜图像背，脚，尾巴或皮肤，在玻璃皿淹没在BBBA，扁平载玻片下，仍然 - 这种方法最大限度地减少了显微镜BBBA污染。 </li><li>从下面照亮菜。为了尽量减少空间变化在整个视场中的光强度，提高了玻璃盘到一个高度〜10厘米解剖显微镜的标准工作表面上方，并放置一个漫射塑料或玻璃板在光源。 </li><li>返回到皮肤100​​％乙醇为长期贮存于-20℃。 注:将在BBBA皮肤更比一天造成的黑色素颗粒，打破了的一部分。一旦皮肤被BBBA对待它会变硬并从该点保持平稳前进。不同的外观可以在其前部和/或后部端部被编码的一系列凹口，从而使它们可以被存储在一起，以节省空间，在冰箱中。 </li></ol></li><li>成像和跟踪轴突乔木（ 图3A，B） <ol><li>将AP染色皮（节3.1.3）用于小蛋白凝胶类型的两块玻璃板之间。避免引入在板与皮肤之间的空气泡，然后小心地吸走多余的BBBA用纸巾。放置板皮板的上的显微镜载物台（ 图2D）的三明治。 </li><li>使用亮场或DIC照明用10X物镜和2微米或5微米的Z-步骤。使用机械化XY平台捕捉的XY图像的蒙太奇被缝合在一起创建一个单一的三维灰度数据集。 注:对于一个大的轴突乔木，该数据集可以是一个的大型为5 GB。 </li><li>进行手动，自动，或轴突乔木与任何各种软件包的半自动跟踪。 注:软件，如Neuromantic（http://www.reading.ac.uk/neuromantic/）是运行在PC环境中，是比较容易掌握10一个免费的神经元重建工具。 </li></ol></li></ol>

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The authors have nothing to disclose.

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。 成像深入到一台安装配置的皮肤是具有挑战性的，因为皮肤是一个相对折光组织。这一挑战变得更大作为皮肤的成熟由于分化和其构成层的厚度增加。对这个问题的一个部分解决方案是分离的真皮和表皮，并分析表皮是作为一个单独的结构（"表皮全坐骑"）。这种方法是用于可视化的毛囊及其相关结构，特别是对于小鼠尾部皮肤有益的。然而，获得高品质的表皮整体安装件从小鼠背部皮肤是困难的，这可能是由于:（1）高密度毛囊延伸深入到真皮和滤泡间表皮13的（2）的薄。 在我们的经验，BBBA治疗是一种高效的方法来呈现皮肤光学透明，同时保留免疫荧光信号，而无需分离表皮和真皮层的需要。苄基苯甲酸酯已用于清除组织几十年20，它被广泛地用来清除青蛙和鱼胚胎。然而，BBBA具有其变性的GFP等荧光蛋白的缺点，它溶于油红O，并且它可以污染设备（ 例如显微镜）。这将是INTERES的吨，有系统地比较BBBA治疗皮肤和其它澄清方法如SeeDB 21，ClearT 22，刻度23，3DISCO 24和 ​​净度25，其中有一些是用荧光蛋白质相容的。如果BBBA处理的样品是利用解剖显微镜进行成像，常规的光/荧光显微镜或共焦显微镜中，样品应置于玻璃皿或两个大的玻璃板之间，以减少BBBA污染。 我们还没有讨论数据分析的任何细节，因为这是更多的特质被调查的生物学问题。然而，如在引言中所述，许多基本相同的结构，如毛囊，Merkel细胞群集，和神经末梢和完整性与这些结构可以在平片进行成像的存在提供了一个机会来测量的结构或形态参数在统计学上严格的马新新人类创新，如最近皮肤感觉轴突形态10和毛囊和毛囊相关的结构在野生型和FZ6突变小鼠4,8研究见过的例子。与更复杂的图像分析工具的发展，有可能实现自动化或半自动化一些，目前，正在手动地执行，从而有利于更广泛使用的皮肤的分析平板装载成像作为调查的基本原则的方法多细胞组织。

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><tbody><tr><td>5-bromo-4-chloro-indolyl phosphate (BCIP)</td><td>Roche</td><td>11383221001</td><td></td></tr><tr><td>AM1-43</td><td>Biotium</td><td>70024</td><td></td></tr><tr><td>AM4-65</td><td>Biotium</td><td>70039</td><td></td></tr><tr><td>Benzyl alcohol</td><td>Sigma</td><td>402834</td><td></td></tr><tr><td>Benzyl benzoate</td><td>Sigma&amp;nbsp;</td><td>B-6630</td><td></td></tr><tr><td>Confocal microscope</td><td>Zeiss</td><td>LSM700</td><td></td></tr><tr><td>Cy3-alpha smooth muscle actin antibody</td><td>Sigma&amp;nbsp;</td><td>C6198</td><td>1:400</td></tr><tr><td>Cytokeratin-8&amp;nbsp;</td><td>Developmental Studies Hybridoma Bank</td><td>TROMA-I-c</td><td>1:500</td></tr><tr><td>Dissecting microscope</td><td></td><td></td><td></td></tr><tr><td>Dissection tools&amp;nbsp;</td><td>Fine Science Tools</td><td></td><td>scissors and forceps</td></tr><tr><td>Electric razor</td><td></td><td></td><td></td></tr><tr><td>Fluoromount G</td><td>EM Sciences</td><td>17984-25</td><td></td></tr><tr><td>Formalin</td><td>Sigma</td><td>HT501320</td><td></td></tr><tr><td>Glass dishes</td><td>Pyrex&amp;nbsp;</td><td></td><td>6 cm and 10 cm diameter</td></tr><tr><td>Glass plates</td><td>Amersham Biosciences</td><td>SE202P-10</td><td>10 cm x 8 cm x 1 mm</td></tr><tr><td>Hair remover&amp;nbsp;</td><td>Nair</td><td></td><td></td></tr><tr><td>Horizontal rotating platform&amp;nbsp;</td><td>Hoefer</td><td>PR250 Orbital shaker</td><td></td></tr><tr><td>Insect pins</td><td>Fine Science Tools&amp;nbsp;</td><td>26002-20</td><td></td></tr><tr><td>Ketamine/xylazine</td><td>Sigma</td><td>K113</td><td></td></tr><tr><td>Nitroblue tetrazolium (NBT)</td><td>Roche&amp;nbsp;</td><td>11383213001</td><td></td></tr><tr><td>Oil Red O</td><td>Sigma</td><td>O0625</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma&amp;nbsp;</td><td>P6148</td><td></td></tr><tr><td>Razor Blades</td><td>VWR</td><td>55411-055</td><td></td></tr><tr><td>Secondary antibodies&amp;nbsp;</td><td>Invitrogen</td><td></td><td>Alexa-dye conjugated&amp;nbsp;</td></tr><tr><td>Sylgard-184</td><td>Fisher Scientific</td><td>NC9020938</td><td></td></tr><tr><td>Tissue culture plastic dishes</td><td></td><td></td><td>10 cm diameter</td></tr><tr><td>Tissue culture plates</td><td></td><td></td><td>6- and 12-well&amp;nbsp;</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）的毛囊，可视化与KRT17-GFP转基因或用抗KRT17抗体染色4（ 图3E-G，K，L）。它是直接通过将已知的方向矢量的结构使用Adobe Photoshop或Illustrator，然后quantifyin手动比分结构的此类图像的方向和空间安排克所得载体种群。虽然这种方法就足够了规模为几百向量，这将是令人望而却步乏味的10 - 或100倍更大的规模。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51749/51749fig1highres.jpg" width="500" /> 图1原理图的哺乳动物毛茸茸的皮肤呈现的主要结构。（版权所有，Terese温斯洛。） <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51749/51749fig2highres.jpg" width="500" /> 图2。皮肤解剖和样品处理。一）解剖工具。B）P5背侧皮肤固定到一个的Sylgard板，C）P5背后脚皮钉在的Sylgard板，D）背侧皮肤安装2个玻璃凝胶板成像之间瓦特第i个10倍的目标与DIC或明照明。 <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51749/51749fig3highres.jpg" width="500" /> 图3。皮肤结构扁平安装的看法。 A，B）单个皮肤感觉乔木（左）和其跟踪的图像（右）从一个BRN3A CKOAP / + A P21背部皮肤; NFL-CREER / +小鼠暴露于低剂量的他莫昔芬在妊娠第17天这个协议结果在记者在背根神经节神经元的一个非常小的部分，因此对皮肤感觉乔木一小部分标以人胎盘碱性磷酸酶（AP）报告的Cre重组酶介导的重组。标记的轴突可视化与NBT / BCIP组织化学。BRN3A是等价称为POU4F1 C，D）的尾部皮肤沾上油红O从可视化皮脂腺WT（左）和FZ6 - / - （右）小鼠P21。该FZ6 - / -结构杂乱无章。 P，近端;从WT（左）和FZ6 D，远端E，F）足背皮肤- / - （右）携带KRT17-GFP的小鼠。皮肤收获在P21和沾有油红O的FZ6 - / -皮肤有毛发螺纹在它的中心。 P，近端; 。面板G）和立毛肌[可视化与抗平滑肌肌动蛋白（SMA; D，前端G，H）背从WT小鼠在P21表示毛囊（可视化与KRT17-GFP转基因和抗GFP免疫染色皮肤）免疫组化;面板H]。共焦Z堆栈图像中深度为彩色编码。 A，前; P，后，IL）一个Merkel细胞簇（可视化与cytokeratin8免疫染色或AM11-43上染率），其核心毛囊（可视化面板K和L与KRT17-GFP）。图像被从标明的基因型P1背部皮肤获得。该FZ6 - / - Merkel细胞群形成了一个封闭的圈子，而WT Merkel细胞簇是向着前牙开。 A，前; P，后路。 比例尺:A和B，300微米; C和D，500微米; E和F，500微米; G和H，200微米;白细胞介素，50微米。 （图A和B的e生活和PROC转载。国家科学院科学。美国 ，经许可4,10） <a href="https://www.jove.com/files/ftp_upload/51749/51749fig3highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" fo:content-width="5in" src="/files/ftp_upload/51749/51749fig4highres.jpg" width="500" /> 图4。头发和背部皮肤在P1和P7毛囊可视化与黑色素沉着。从WT和FZ6 A，B）P1皮肤</eM&gt; - / -小鼠C，D）P7皮肤FZ6 - / -小鼠。 比例尺:A和B，500微米; C和D，1毫米。

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 ...

flat-mount-imaging-mouse-skin-its-application-to-analysis-hair

Nanyang Technological University

Research

JoVE Journal

Neuroscience

21.4K 次观看.  Howard Hughes Medical Institute, Johns Hopkins University School of Medicine. 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。 

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

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

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in animals and humans. These peripheral sensory nerve fibers express a plethora of receptors and ion channel proteins which detect and initiate specific sensory stimuli. Methods are available to characterize the electrical properties of peripheral sensory nerve fibers innervating the skin, which can also be utilized to identify the functional expression of specific ion channel proteins in these fibers. However, similar electrophysiological methods are not available (and are also difficult to develop) for the detection of the functional expression of receptors and ion channel proteins in peripheral sensory nerve fibers innervating other visceral organs, including the most challenging tissues such as bone. Moreover, such electrophysiological methods cannot be utilized to determine the expression of non-excitable proteins in peripheral sensory nerve fibers. Therefore, immunostaining of peripheral/visceral tissue samples for sensory nerve fivers provides the best possible way to determine the expression of specific proteins of interest in these nerve fibers. So far, most of the protein expression studies in sensory neurons have utilized immunostaining procedures in sensory ganglia, where the information is limited to the expression of specific proteins in the cell body of specific types or subsets of sensory neurons. Here we report detailed methods/protocols for the preparation of peripheral/visceral tissue samples for immunostaining of peripheral sensory nerve fibers. We specifically detail methods for the preparation of skin or plantar punch biopsy and bone (femur) sections from mice for immunostaining of peripheral sensory nerve fibers. These methods are not only key to the qualitative determination of protein expression in peripheral sensory neurons, but also provide a quantitative assay method for determining changes in protein expression levels in specific types or subsets of sensory fibers, as well as for determining the morphological and/or anatomical changes in the number and density of sensory fibers during various pathological states. Further, these methods are not confined to the staining of only sensory nerve fibers, but can also be used for staining any types of nerve fibers in the skin, bones and other visceral tissue.

Immunocytochemical identification of peripheral sensory nerve fiber subtypes (and detection of protein expression therein) are key to the understanding of molecular mechanisms underlying peripheral sensation. Here we describe methods for preparation of peripheral/visceral tissue samples, such as skin and limb bones, for specific immunostaining of peripheral sensory nerve fibers.

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 ...

科研

神经科学

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

水平整体安装:厚三维组织横截面皮肤的新型加工和成像方案

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

发育生物学

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 ...

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their close-to-surface positioning facilitates microscopic imaging of skin nerve endings in living intact animal. Using multiphoton microscopy, it is possible to obtain fine images overcoming the problem of strong light scattering of the skin tissue. Reporter transgenic mice that express EYFP under the control of Thy-1 promoter in neurons (including periphery sensory neurons) are well suited for the longitudinal studies of individual nerve endings over extended periods of time up to several months or even life-long. Furthermore, using the same femtosecond laser as for the imaging, it is possible to produce highly selective lesions of nerve fibers for the studies of the nerve fiber restructuring. Here, we present a simple and reliable protocol for longitudinal multiphoton in vivo imaging and laser-based microsurgery on mouse skin nerve endings.

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

The protocol for dynamic longitudinal imaging and selective laser lesion of nerve endings in reporter transgenic mice is presented.&#160;

在皮肤中的单神经末梢体内双光子显微镜

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their ...

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity. As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses. Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain. Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo. This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex. Therefore, images can be acquired immediately after surgery is performed. The experimental procedure can be performed repetitively over various time intervals ranging from hours to years. The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions.

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Time-lapse imaging in the living animal provides valuable information on structural reorganization in the intact brain. Here, we introduce a thinned-skull preparation that allows transcranial imaging of fluorescently labeled synaptic structures in the living mouse cortex by two-photon microscopy.

双光子<em&gt;在体内</em&gt;树突棘中的鼠标皮质用稀疏的头颅成像准备

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

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

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

小鼠胚胎感觉轴突预测整个安装成像

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

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

A novel dissection and recording technique is described for monitoring afferent firing evoked by mechanical displacement of hairs in the mouse pinna. The technique is very cost-effective and easily undertaken with materials commonly found in most electrophysiology laboratories, or easily purchased. The dissection is simple and fast, with the mechanical displacement provided by a generic electroceramic wafer controlled by proprietary software. The same software also records and analyses the electroneurogram output. The recording of the evoked nerve activity is through a commercial differential amplifier connected to fire-polished standard glass microelectrodes. Helpful tips are given for improving the quality of the preparation, the stimulation and the recording conditions to optimize recording quality. The system is suitable for assaying the electrophysiological and optical properties of lanceolate terminals of palisade endings of hair follicles, as well as the outcomes from their pharmacological and/or genetic manipulation. An example of combining electrical recording with mechanical stimulation and labeling with a styryl pyridinium vital dye is given.

A simple and novel technique for recording afferent discharge due to mechanical stimulation of lanceolate terminals of palisade endings innervating mouse ear skin hair follicles is presented.

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

A novel dissection and recording technique is described for optical monitoring staining and de-staining of lanceolate terminals surrounding hair follicles in the skin of the mouse pinna. The preparation is simple and relatively fast, reliably yielding extensive regions of multiple labeled units of living nerve terminals to study uptake and release of styryl pyridinium dyes extensively used in studies of vesicle recycling. Subdividing the preparations before labeling allows test vs. control comparisons in the same ear from a single individual. Helpful tips are given for improving the quality of the preparation, the labeling and the imaging parameters. This new system is suitable for assaying pharmacologically and mechanically-induced uptake and release of these vital dyes in lanceolate terminals in both wild-type and genetically modified animals. Examples of modulatory influences on labeling intensity are given.

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

Here we describe a novel preparation for imaging live lanceolate sensory terminals of palisade endings that innervate mouse ear skin hair follicles during staining and destaining with styryl pyridinium dyes.

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 ...

Visualization of Motor Axon Navigation and Quantification of Axon Arborization In Mouse Embryos Using Light Sheet Fluorescence Microscopy

Spinal motor neurons (MNs) extend their axons to communicate with their innervating targets, thereby controlling movement and complex tasks in vertebrates. Thus, it is critical to uncover the molecular mechanisms of how motor axons navigate to, arborize, and innervate their peripheral muscle targets during development and degeneration. Although transgenic Hb9::GFP mouse lines have long served to visualize motor axon trajectories during embryonic development, detailed descriptions of the full spectrum of axon terminal arborization remain incomplete due to the pattern complexity and limitations of current optical microscopy. Here, we describe an improved protocol that combines light sheet fluorescence microscopy (LSFM) and robust image analysis to qualitatively and quantitatively visualize developing motor axons. This system can be easily adopted to cross genetic mutants or MN disease models with Hb9::GFP lines, revealing novel molecular mechanisms that lead to defects in motor axon navigation and arborization.

Here, we describe a protocol for visualizing motor neuron projection and axon arborization in transgenic Hb9::GFP mouse embryos. After immunostaining for motor neurons, we used light sheet fluorescence microscopy to image embryos for subsequent quantitative analysis. This protocol is applicable to other neuron navigation processes in the central nervous system.

用光片荧光显微镜观察运动轴突导航和小鼠胚轴突树枝状的定量化

Spinal motor neurons (MNs) extend their axons to communicate with their innervating targets, thereby controlling movement and complex tasks in ...

Non-Invasive Visualization of Nailbed Microvascular Morphology in Mice Using Capillaroscopy

Imaging microcapillary networks of the skin in humans using nailfold capillaroscopy (NFC) has underscored the importance of microcirculation as a target organ system in critical systemic illnesses. Nailfold capillaroscopy is applied clinically to detect peripheral microvascular dysfunction and abnormalities in a range of systemic conditions, including rheumatic, cardiac, ocular (e.g., glaucoma), and endocrine disorders (e.g., hypertension and diabetes mellitus). NFC is useful not only in detecting peripheral systemic microvasculature disruption but also in assessing drug efficacy. However, translating clinical NFC findings to animal disease models can be challenging. Detecting microvascular dysfunction or abnormalities in animals is often invasive (e.g., endoscopic), carried out ex vivo (e.g., post-mortem imaging of tissues), or expensive, requiring specialized equipment such as those used in microcomputed tomography and photoacoustic imaging techniques. Developing quick, non-invasive, and inexpensive techniques to image peripheral microvasculature in animal models of disease is warranted to decrease research expenses and increase translatability to the clinic.
Capillaroscopy has previously been used to visualize the nailfold microvasculature in animal models, including in guinea pigs and mice, thus demonstrating the capability of capillaroscopy as a non-invasive imaging tool in animal models. This study provides a protocol that applies capillaroscopy to a mouse nailbed, allowing researchers to easily and inexpensively assess the morphology of its microvasculature. Representative images of typical nailbed microvascular architecture in wild-type mice using two commonly used laboratory strains, SV129/S6 and C57/B6J, are provided. Further studies using this method are essential for applying nailbed capillaroscopy to a wide range of mouse disease models with peripheral microvascular abnormalities.

Capillaroscopy is an accessible tool for direct, inexpensive, and non-invasive visualization of microvasculature. The goal of this protocol is to enable researchers to use capillaroscopy for the visualization of peripheral microvascular morphology in the nailbeds of mice.

使用毛细血管镜检查小鼠甲床微血管形态的无创可视化

Imaging microcapillary networks of the skin in humans using nailfold capillaroscopy (NFC) has underscored the importance of microcirculation as a target ...