我们感谢澳大利亚的生物资源（加文学院，澳大利亚），生物资源中心（新南威尔士大学澳大利亚）和动物护理和伦理委员会与动物实验的支持。这项工作是由澳大利亚国家卫生和医学研究理事会（计划资助APP1062720）的支持。塞萨尔P.卡纳莱斯博士是CONICYT-Becas智利奖学金（＃72101076）的接受者。 Akladios的Bassem先生是澳大利亚新南威尔士大学国际研究生奖的获得者。

伦理学声明:所有涉及动物主题程序遵循的动物护理和伦理委员会（ACEC）在新南威尔士大学澳大利亚的批准下ACEC协议13 / 64B的准则。 1.透明小鼠皮肤组织的制备注意:在本研究中使用的所有小鼠在C57BL / 6遗传背景<ol><li>小鼠皮肤组织的集合。 <ol><li>入道安乐死颈椎脱位小鼠。 </li><li>小心地从与微调，注意不要伤到皮肤相关的皮肤区域中删除毛发。 </li><li>清洗皮肤与磷酸盐缓冲液（PBS）70％乙醇消毒。 </li><li>提起背颈部皮肤钳，使切口，用剪刀。 </li><li>背解剖老鼠的皮肤（约1.5×4厘米）的大面积。 </li><li>展平皮肤真皮面朝下放在滤纸上，并记下样品的前后方向。 </li><li>三周围的解剖皮肤μm过滤纸，并将其放置在15ml试管填充有在PBS新鲜制备的4％低聚甲醛（PFA）的解决方案。 </li><li>在4℃的冰箱中固定1小时，在室温下，或过夜。 </li><li>洗涤皮肤2×5分钟的PBS在15毫升管。 注:以下（1.1.10 - 1.1.12）是组织的长期存储可选步骤。 </li><li>脱水在PBS解剖皮肤中在室温下1小时的洗涤步骤在15毫升管增加的乙醇（25％，50％70％）的浓度。 </li><li>在4℃储存于PBS 70％乙醇15毫升管缺水的肌肤，直到进一步使用。 </li><li>结算前4小时，再水合的皮肤组织在PBS中于室温下1小时的洗涤步骤减小的在一个15毫升管乙醇（70％，50％，25％，0％）的浓度。 </li></ol></li><li>清除小鼠皮肤活检<ol><li> 3.85克尿素3.85克的N-，N，N-溶解准备CUBIC1结算解决方案&#39;，N&#39;N&#39;-四（2-羟丙基）乙二胺在5.38毫升上设置为60的加热器蒸馏水 - 70℃。用热搅拌器。 </li><li>添加2.31克聚乙二醇单对异辛基苯基醚/的Triton X-100的溶液，一旦它是明确的，冷却到室温。 </li><li>切开小鼠皮肤用锋利的剃刀刀片成尺寸约为0.2×0.5厘米活检，并在5毫升在一个15毫升管CUBIC1结算溶液淹没。为了优化毛囊的可视化，确保了活检的较长侧沿试样的前 - 后方向切割。 </li><li>放置在37℃下在杂交烘箱的旋转平台上。 </li><li> 7天之后更改结算解决方案。准备使用前新鲜CUBIC1解决方案。 </li><li>入住后7日内组织的透明度。如果有必要，离开活检CUBIC1结算解决方案，直到组织是完全透明的。 </li><li>一旦皮肤活检是透明的，除去CUBIC1溶液，并加入4毫升1-×PBS中洗涤组织4次6小时于37℃。 </li><li>在37℃下洗皮肤组织在20％重量/在PBS V蔗糖在一个15毫升管4小时。 </li><li>冻结在-80℃冷冻过夜在15ml管安装平台最佳切削温度（OCT）化合物的组织。 注意:此步骤会增加活检的渗透率为在后续步骤中抗体的渗透。 </li></ol></li></ol> 2.免疫荧光染色<ol><li>解冻从1.2.9组织），以室温下搅拌2 - 3小时。 </li><li>在15ml试管用5毫升的PBS 8小时，在室温下洗涤​​组织以除去OCT化合物。 </li><li>转印活检到2ml的管中，并孵育在1ml兔抗Keratin14或兔抗Ki67抗体组织，无论稀释1:在PBST（PBS + 0.1％的Triton-X100）在37 100 3天在振荡器上°℃烘箱。 </li><li>转印活检到15毫升管中，一个第二洗组织4次，6小时在5毫升的PBST振荡器上在37℃的烘箱。 </li><li>在37℃的烘箱在PBST 100和孵育3天振荡器上:转印活检到2ml的管中，加入1 ml的抗 - 兔Alexa594稀释的二级抗体。 </li><li>转印活检到15毫升管中，并洗净组织4次，6小时在PBST 5毫升振荡器上在37℃的烘箱。 </li><li>转印活检到2ml的管中，加入1 ml 4&#39;，6-二脒基-2-苯基吲哚（DAPI）核染剂溶液（1:1,000）在PBST和在37℃的烘箱振荡器上孵育过夜。 </li><li>除去DAPI复染溶液，和1ml的PBST加至2毫升管洗组织4次6小时在摇床上在37℃的烘箱。 注意:可选步骤:活检可以存储在黑暗中在1×PBS中的0.02％叠氮化钠，至少3周。 </li></ol> 3.尼罗红染色<ol><li>溶解在二甲基亚砜（DMSO）尼罗红粉末1μm的最终浓度克/毫升</li><li>加入1μl尼罗红溶液到1ml PBS中至1微克/毫升的最终浓度。 </li><li>从1.2.9解冻组织），以室温下搅拌2 - 3小时，并用5ml PBS洗涤，在室温下8小时。 </li><li>转移活检到2ml的管中，并在1ml尼罗红染色溶液淹没组织在室温下2.5小时。 </li><li>洗2毫升管的皮肤活检用1ml PBST 4次在室温下30分钟。 </li><li>除去从2毫升管的PBST溶液，加入1毫升的DAPI核复染溶液（1:1,000）在PBST，在室温下孵育过夜。 </li><li>除去DAPI复染溶液，并在2毫升管中添加的PBST 1毫升洗皮肤组织4次在室温下6小时。 注意:可选步骤:活检可以存储在黑暗中在1×PBS中的0.02％叠氮化钠，至少3周。 </li></ol> 4.成像<ol><li>准备CUBIC2结算解决方案，其中包含50个％（重量/体积）蔗糖，25％（重量/体积）脲，10％（重量/体积）2,2&#39;，2&#39;&#39; - nitrilotriethanol和0.1％（体积/体积）的Triton X-100。 </li><li>孵育在37℃的烘箱中进行24小时的摇床上，在2毫升管1毫升CUBIC2溶液的皮肤组织。此步骤将连组织的折射率。 </li><li>检查组织的清晰度。一旦它是清楚的，在整个皮肤活检（0.2×0.5厘米）在其长边放置到玻璃盖玻片，使得毛囊的长度方向平行于盖玻片表面（＃1 24×60毫米） 注:可选步骤:抗体染色切片可以存储在CUBIC2溶液中约7天。尼罗红染色的活检只能存储在1天CUBIC2溶液，并应尽快成像。 </li><li>制备成像室（ 图1） 注:成像室的准备所需耗材主要有:蓝色粘性，玩面团或相似，且2盖玻片（24×50毫米）（Figure 1A）。 <ol><li>制备蓝色粘性的两个薄带（直径约1mm×2厘米），和两个盖玻片（ 图1B）。 </li><li>放置在一个盖玻片蓝色钉条，允许皮肤活检（ 图1C）足够的空间。 4.4.3） </li><li>放置皮肤活检中条之间关于在CUBIC2溶液滴（ 图1C）的盖玻片。 </li><li>覆盖皮肤活检与第二盖玻片（ 图1D）。 </li></ol></li><li>与安装的皮肤活检成像室放置到共聚焦显微镜的阶段，移动组织进入光通道。 </li><li>扫描用水银或卤素光源和标准表面荧光过滤器（ 例如，DAPI / GFP / CY3 / CY5）识别感兴趣荧光染色区样品。 </li><li>使用10X和20X物镜（NA 0.75）和标准共聚焦荧光成像技术（ 例如 ，与DA感兴趣图像区域PI染色样品照亮有405nm激光器和收集425之间的荧光信号 - 475纳米，并用的Alexa Fluor 594或尼罗红染色的样品照射与561纳米的激光，并收集570之间的荧光信号 - 620纳米）。 </li><li>生成的使用显微镜Z堆叠图像采集软件每个关心区域的图像的Z堆叠（ 例如 ，使用NIS元件成像软件4.13如下所述）。 <ol><li>确保最佳的激光功率和PMT HV /偏移设置已经选定，收集样品荧光。 </li><li>从"采集控制"打开"ND采集"工具栏。 </li><li>选择"Z系列设置"选项卡（确保所有其他选项卡选择）。 </li><li>虽然现场扫描，聚焦到样品的顶部，然后按"顶部"按钮。 </li><li>聚焦到样品的底部，然后按"底部"按钮。 </li><li>输入所需的步长（或按优化步长按钮）。 </li><li>预SS的"立即运行"按钮。 </li><li>保存生成的Z-堆栈作为单独的TIFF图像堆栈（每图像Z堆栈1荧光染料）。 </li></ol></li><li>使用图像的Z堆栈重建的使用三维分析软件感兴趣的3D体积的区域（ 例如 ，使用如下所述了Imaris 7.2.3 64位）。 <ol><li>启动分析软件。 </li><li>在工具栏的三维体积代选择"超越"按钮。 </li><li>敞开的第一彩色图象Z堆叠（ 例如 ，DAPI）。 </li><li>使用"添加频道"工具来添加额外的颜色通道，每个荧光染料一个通道。因而含有DAPI和Alexa Fluor 594的荧光的样本将需要两个信道。 </li><li>使用"图片属性"工具来设置正确的像素（像素），尺寸（XYZ），在4.7以上决定。 </li><li>根据需要使用"显示调整"工具来改变颜色通道（如 DAPI通道设置为蓝色，的Alexa Fluor 594通道，红色）。 </li><li>宝习得在图像窗口中的3D体积，使用计算机鼠标根据需要点击并拖动音量。 </li><li>在工具栏中使用"快照"工具来生成3D图像的屏幕截图。 </li><li>在工具栏中使用"动画"工具来生成3D样本轮换的电影。 </li></ol></li></ol>

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控制皮肤发育和稳态的调节机制是最常见的2D采用组织切片和组织染色标记的抗体，它能使皮肤只形态，细胞群或蛋白表达的限制升值的影响。许多方法已被开发，以改善细胞和蛋白质中的单细胞分辨率的空间组织的可视化在表皮整体坐骑三月10日至13日的尺寸。其中的一些但涉及从真皮，这在技术上是挑战性的表皮层的分离特别是在使用有毛皮肤，并经常导致毛囊的组织损伤和破裂。而且，表皮和真皮也妨碍学习胚胎发育和组织稳态期间它们之间发生的细胞和分子的相互作用的分离。 在&#39;平片的做法"是另一种方法，其中全厚鼠标皮肤解剖和免疫染色，然后用苯甲酸苄酯和苯甲醇（BBBA），同时保持免疫荧光信号14澄清。此方法不需要从真皮表皮的技术上具有挑战性的分离。然而，BBBA是剧毒，它变性一些荧光蛋白，它污染显微镜物镜，不允许进行调查的组织细胞和分子的相互作用所需要的样品的高分辨率成像。 这份报告描述了三次协议5-7澄清任何所需解剖区域和年龄使用相对安全和廉价的试剂全层小鼠皮肤活检。该技术简单协议允许的蛋白表达和增殖模式的可视化在全层皮肤活检在单细胞分辨率在三个维度与DAPI复染，使免疫前所未有的和高度精确的资格赛权威的评估和蛋白表达，增殖，组织形态和毛囊尺寸的定量。这个方法也适合于使用尼罗红染色尽管澄清过程中除去组织脂类的皮脂细胞的可视化。这种方法的一个重要的限制是抗体有效地结合和使用这种皮肤澄清过程允许各自靶蛋白的可视化的可用性。此外，所生成的图像和电影文件是大的，并如与强大的处理器和充足的数据存储空间的计算机良好IT资源是必要的。 在过程中的一个关键步骤是皮肤活检的制备。为了显现完好毛囊解剖学，皮肤活检的取向，应考虑到，并且活检的长度应平行切成毛囊的长度的方向。此外，小和非常薄的滑雪ñ活检很难正确安装显微镜。较大的活检将需要更多的时间来清除，并可能产生导致抗原检测抗体的文物渗透问题。因此，每个实验建议削减，清晰，色斑多点活检。 紧紧地密封该试管，同时准备立方溶液，以防止在加热过程中，这导致在试剂浓度的变化的内容蒸发，并可能导致受损组织清除也很重要。下一个重要步骤是该组织将在37℃下加以澄清，以加速清理过程。经过7天，CUBIC1解决方案将需要准备新的来代替旧的。 在未来，多种抗体将确定了与该立方皮肤澄清过程兼容，并分析可以扩展到可视化和表皮和黑素细胞（干）细胞标记的定量S和真皮的蛋白质。这种技术也将在在突变小鼠和转基因报告小鼠模型，包括真皮和之间的信令交互皮肤异常和正常表皮生物学基础的细胞和分子机制的阐明起到皮肤病理学和解剖学，并有助于三维研究的基础性作用表皮。 立方协议5-7可能是适用于其他脊椎动物的动物模型的皮肤，并且对人体皮肤钻取活组织检查。光片镜将使表皮解剖，蛋白表达模式，和毛囊周期阶段的进展的可视化较大皮肤样品英寸

皮肤是我们的生存至关重要。它由三个主要层的外表皮，真皮​​和皮下组织。表皮是一个高度可再生的组织。这是一个鳞状复层上皮，主要由角质形成细胞。角质形成细胞诞生于基底层，并通过基底层向上移动，而差异化，最终他们在他们出生后一个月外角化层脱落。表皮开发了一些附加物，包括毛囊和皮脂腺。毛囊也再生以循环的方式在整个生命1。表皮的再生能力是由位于滤泡表皮和毛囊2基底层干细胞和祖细胞的存在使能。 许多信号传导途径已牵涉在表皮发育和再生。其中的一些发生内仅表皮，如Hedgehog通路。其他信号事件采取真皮和表皮3之间。例如，从真皮的Wnt信号被认为是对毛囊发育重要的，它们是由毛乳头在生长期激活的发作分泌毛囊膨出干/祖细胞的增殖和毛囊生长4。要明白，控制表皮发育和再生，以便更好地理解它们是如何可能在再生皮肤疾病被扰动如皮肤癌的细胞和分子机制是很重要的。 本文介绍了一个C李尔，U nobstructed 乙雨我 maging鸡尾酒和C omputational分析（立方）协议5-7澄清整装皮肤的准备，并在激光共聚焦显微镜单细胞分辨率的3维可视化表达模式。三次方法涉及皮肤浸泡组织中的两个基于氨基醇化学鸡尾酒。这些解决方案调整皮肤样品中的折射率，而使组织透明和蛋白质的完整，从而允许在单细胞分辨率的免疫检测。 在滤泡表皮和毛囊使用该立方协议，基底和增殖角化细胞种群在使用抗Keratin14（K14）和抗Ki67抗体野生型小鼠的全层皮肤活检成像。在野生型皮肤活检皮脂腺用尼罗红染色也观察。最后，在野生型和增生YAP2-5SA-ΔC皮肤活检的基部角质细胞群体进行比较8。 这一立方协议允许在单细胞分辨率的全层皮肤活检蛋白表达的视觉评估，并以欣赏的转基因皮肤表皮的解剖和形态缺陷的重要工具小鼠，并调查表皮发育和再生基础的细胞和分子机制。

<table><tbody><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich&amp;nbsp;</td><td>P6418</td><td></td></tr><tr><td>Ethanol 96% (undenaturated)</td><td>Chem-supply</td><td>UN1170</td><td></td></tr><tr><td>Nile Red</td><td>Sigma-Aldrich&amp;nbsp;</td><td>72485-100MG</td><td></td></tr><tr><td>4&amp;rsquo;,6-diamidino-2-phenylindole (DAPI)</td><td>Roche</td><td>10236276001</td><td></td></tr><tr><td>&lt;em&gt;N,N,N&amp;rsquo;,N&amp;rsquo;&lt;/em&gt;-tetrakis (2-hydroxypropyl) ethylenediamine</td><td>Merck Millipore</td><td>821940</td><td></td></tr><tr><td>Polyethylene glycol mono-p-isooctylphenyl ether</td><td>Merck Millipore</td><td>648462</td><td></td></tr><tr><td>Triton X-100</td><td>Merck Millipore</td><td>648462</td><td></td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich&amp;nbsp;</td><td>S0389</td><td></td></tr><tr><td>Optimal Cutting Temperature (OCT) Compound</td><td>Tissue-Tek</td><td>4583</td><td></td></tr><tr><td>anti-Keratin14 antibody</td><td>Covance</td><td>PRB-155P</td><td></td></tr><tr><td>anti-Ki67 antibody&amp;nbsp;</td><td>Abcam</td><td>ab16667</td><td></td></tr><tr><td>Donkey anti-rabbit Alexa 594</td><td>Life Technologies</td><td>A21207</td><td></td></tr><tr><td>Dimethylsulfoxide</td><td>Sigma-Aldrich&amp;nbsp;</td><td>D2650</td><td></td></tr><tr><td>Urea</td><td>Merck Millipore</td><td>66612</td><td></td></tr><tr><td>2,2&amp;prime;,2&amp;prime;&amp;rsquo;-nitrilotriethanol</td><td>Merck Millipore</td><td>137002</td><td></td></tr><tr><td>Confocal Microscope</td><td>Nikon Instruments Inc</td><td>Nikon A1 - Confocal Microscope</td><td></td></tr><tr><td>cruZer6 Face Trimmer</td><td>Braun</td><td>Braun cruZer6 Face</td><td></td></tr><tr><td>Sodium azide</td><td>Sigma-Aldrich&amp;nbsp;</td><td>438456</td><td></td></tr></tbody></table>

cubic protocol visualizes protein expression at single cell resolution in whole mount skin preparations

皮肤是我们的生存至关重要。外表皮层由滤泡表皮，这是覆盖最我们的身体，和表皮附属物的复层鳞状上皮，例如毛囊和汗腺。表皮经历整个生命，并且响应于损伤的再生。这是通过K14表达被紧紧由表皮内和表皮和真皮之间活动多调节机制调节基底表皮干细胞/祖细胞群启用。本文介绍了一种简单的方法来阐明全层小鼠皮肤活检，和可视化K14蛋白的表达模​​式，Ki67的标记的增殖细胞，尼罗红标记的皮脂腺细胞，并在3D单细胞分辨率DAPI细胞核标记。这种方法可以准确的评估和皮肤的解剖学和病理学的量化，并在转基因小鼠品系的表皮异常表型。立方协议是日E，可到目前为止，调查在单细胞分辨率的全层皮肤活检的分子和细胞相互作用最好的方法。

成年野生型小鼠的全层背部皮肤活检进行了澄清，与抗体结合基础角质标记Keratin14（K14）染色，细胞核复染用DAPI染色溶液（ 图2和电影1）。 DAPI阳性细胞核整个样品（ 图2A，C）是可见的，和K14染色只在滤泡表皮的单细胞厚基底层是可见的，并概述皮脂腺（黑星号），外根鞘毛囊，和在二次发病菌（ 图2B，C）中 ，如先前所发表的9。 K14染色强度是在滤泡表皮，远侧毛囊和次级头发病菌（白星号）高，并且它是在毛囊（卜在图2C）的凸出区域相对较低。真皮乳头也明显visiblE到DAPI标记（ 如图2C箭头）。 为了显现增殖细胞，全厚度背部皮肤活检成年野生型小鼠的休止期得到澄清并用抗Ki67抗体染色，并且细胞核复染用DAPI溶液（ 图3，和电影2）。 在基底滤泡表皮（ 图3C IFE）检测增殖的角质形成细胞，并且在峡部（在图3C是）毛囊的，但不是在隆起区域（卜在图3C）。 为了显现皮脂腺，全厚度背部皮肤活检成年野生型小鼠的生长期进行澄清和尼罗红该标记皮脂细胞的脂肪含量染色，细胞核复染用DAPI染色溶液（ 图4和电影3）。尼罗红阳性sebaceou在毛囊（SB在图4C）的峡部区域观察到腺，显示出三次协议不显著影响这些含脂肪结构的形态。出乎意料的是，毛发干还标记尼罗红（HS在图4C）。 为了显现在YAP2-5SA-ΔC转基因小鼠表皮中表达基底角质形成细胞8过动YAP突变蛋白的形态变化，成年野生型和YAP2-5SA-ΔC转基因同窝小鼠的全层背部皮肤活检澄清并与染色抗K14抗体和细胞核用DAPI溶液（ 图5，和电影4（野生型）和电影5（YAP2-5SA-ΔC））的复染。 在YAP2-5SA-ΔC转基因皮肤，使表皮滤泡的增生（双顶图4F箭头</strong&gt;）是清晰可见，和毛囊更长并且在异常毛囊（ 图5F低级双箭头）向近侧显示K14标记细胞块，占扩大的干细胞群，如前所述8。 <img alt="图1" src="/files/ftp_upload/54401/54401fig1.jpg" /> 图1:成像室的制备。答 :成像室的准备所需耗材是蓝色的粘性，玩面团或相似，且2盖玻片（24×50毫米）B:准备蓝钉（直径约2毫米×2厘米）的两根细细的条，和2盖玻片C:在一个盖玻片放置蓝色钉条，允许在皮肤活检足够的空间。放置在皮肤活检在CUBIC2解决方案在两带之间降D:蓝色钉条将第二盖玻片覆盖皮肤活检。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54401/54401fig1large.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/54401/54401fig2.jpg" /> 图 2: 在 成年 野生型小鼠 的 休止期 背部 皮肤 的 K14 / DAPI标记 ING背侧皮肤活检的3D体积重建在标有K14抗体（红在B，C）的成年野生型小鼠的休止期，和DAPI核复染剂。 （在A，C蓝色）。 K14染色强于滤泡表皮（IFE），峡部和皮脂腺（黑星号），并且次级头发病菌（白星号），以及相对低的隆起<html>区（BU）。比例尺50微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54401/54401fig2large.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/54401/54401fig3.jpg" /> 图 3: 背皮肤 的Ki67标记 成年野生型小鼠标记有Ki67的抗体的成年野生型小鼠的背部皮肤活检3D体积重建的休止期 （红色中B，C），和DAPI核复染剂（在一个蓝色， C）。增殖的Ki67阳性的角质形成细胞是在基底滤泡表皮（IFE）明显的，并且在峡部区域（是），但不是在休止期毛囊的隆起（BU）。比例尺为50μm。rget ="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图4" src="/files/ftp_upload/54401/54401fig4.jpg" /> 图 4:在成年野生型小鼠的生长期背部皮肤的尼罗红染色背侧皮肤活检的3D体积重建标记的尼罗红（红色中B，C），和DAPI核复染剂（蓝在成人野生型小鼠的生长期。 A，C）。尼罗红染色在皮脂细胞（SB）和在毛干（HS）可见。比例尺50微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54401/54401fig4large.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/54401/54401fig5.jpg" /> 数字5:形态异常的YAP2-5SA-ΔC表皮成年野生型（A - C）的背部皮肤活检的3D体积重建和YAP2-5SA-ΔC转基因鼠同窝（D - F）标有K14抗体（红英寸 B，C，E，F）和DAPI核染剂（蓝色A，C，D，F）。所述YAP2-5SA-ΔC表皮的表皮增生是在滤泡表皮和毛囊（F中顶部的双箭头），该近侧显示特性干/祖细胞块（F中较低的双箭头）8显而易见。比例尺50微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54401/54401fig5large.jpg" target="_blank">请点击此处查看大图</a><html>这一数字。 <img alt="电影1" src="/files/ftp_upload/54401/54401movie1.jpg" /> 电影1 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54401/Movie_1.mov" target="_blank">，请点击此处下载这部电影。</a> <img alt="电影2" src="/files/ftp_upload/54401/54401movie2.jpg" /> 电影2 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54401/Movie_2.avi" target="_blank">，请点击此处下载这部电影。</a> <img alt="电影3" src="/files/ftp_upload/54401/54401movie3.jpg" /> 电影3. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54401/Movie_3.mov" target="_blank">请点击此处下载这部电影。</a> 01movie4.jpg"/&gt; 电影4. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54401/Movie_4.mov" target="_blank">请点击此处下载这部电影。</a> <img alt="电影5" src="/files/ftp_upload/54401/54401movie5.jpg" /> 电影5. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54401/Movie_5.mov" target="_blank">请点击此处下载这部电影。</a>

Watch this Scientific Journal Video about CUBIC Protocol Visualizes Protein Expression at Single Cell Resolution in Whole Mount Skin Preparations at JoVE.com

CUBIC Protocol Visualizes Protein Expression at Single Cell Resolution in Whole Mount Skin Preparations

这份报告描述了三次协议，以澄清全层小鼠皮肤活检，并在3D单细胞分辨率的可视化表达方式，增殖细胞，皮脂腺细胞和。这种方法使皮肤的解剖学和病理学的准确评估，并在转基因小鼠品系的表皮异常表型。

CUBIC协议可视化在全山皮肤配方单细胞分辨率蛋白表达

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium ...

cubic-protocol-visualizes-protein-expression-at-single-cell

Nanyang Technological University

Research

JoVE Journal

Biology

11.5K 次观看.  University of New South Wales Australia. 这份报告描述了三次协议，以澄清全层小鼠皮肤活检，并在3D单细胞分辨率的可视化表达方式，增殖细胞，皮脂腺细胞和。这种方法使皮肤的解剖学和病理学的准确评估，并在转基因小鼠品系的表皮异常表型。 

视频: CUBIC Protocol Visualizes Protein Expression at Single Cell Resolution in Whole Mount Skin Preparations

Localizing Protein in 3D Neural Stem Cell Culture: a Hybrid Visualization Methodology

The importance of 3-dimensional (3D) topography in influencing neural stem and progenitor cell (NPC) phenotype is widely acknowledged yet challenging to study. When dissociated from embryonic or post-natal brain, single NPCs will proliferate in suspension to form neurospheres. Daughter cells within these cultures spontaneously adopt distinct developmental lineages (neurons, oligodendrocytes, and astrocytes) over the course of expansion despite being exposed to the same extracellular milieu. This progression recapitulates many of the stages observed over the course of neurogenesis and gliogenesis in post-natal brain and is often used to study basic NPC biology within a controlled environment. Assessing the full impact of 3D topography and cellular positioning within these cultures on NPC fate is, however, difficult. To localize target proteins and identify NPC lineages by immunocytochemistry, free-floating neurospheres must be plated on a substrate or serially sectioned. This processing is required to ensure equivalent cell permeabilization and antibody access throughout the sphere. As a result, 2D epifluorescent images of cryosections or confocal reconstructions of 3D Z-stacks can only provide spatial information about cell position within discrete physical or digital 3D slices and do not visualize cellular position in the intact sphere. Here, to reiterate the topography of the neurosphere culture and permit spatial analysis of protein expression throughout the entire culture, we present a protocol for isolation, expansion, and serial sectioning of post-natal hippocampal neurospheres suitable for epifluorescent or confocal immunodetection of target proteins. Connexin29 (Cx29) is analyzed as an example. Next, using a hybrid of graphic editing and 3D modelling softwares rigorously applied to maintain biological detail, we describe how to re-assemble the 3D structural positioning of these images and digitally map labelled cells within the complete neurosphere. This methodology enables visualization and analysis of the cellular position of target proteins and cells throughout the entire 3D culture topography and will facilitate a more detailed analysis of the spatial relationships between cells over the course of neurogenesis and gliogenesis in vitro.
Both Imbeault and Valenzuela contributed equally and should be considered joint first authors.

Here, we describe how to produce, expand, and immunolabel postnatal hippocampal neural progenitor cells (NPCs) in three-dimensional (3D) culture. Next, using hybrid visualization technologies, we demonstrate how digital images of immunolabelled cryosections can be used to reconstruct and map the spatial position of immunopositive cells throughout the entire 3D neurosphere.

本地化的蛋白质三维神经干细胞培养：一个混合的可视化方法

The importance of 3-dimensional (3D) topography in influencing neural stem and progenitor cell (NPC) phenotype is widely acknowledged yet challenging to ...

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

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular level. To date the shear complexity of biological systems has caused precise single-molecule experimentation to be far too demanding, instead focusing on studies of single systems using relatively crude bulk ensemble-average measurements. However, many important processes occur in the living cell at the level of just one or a few molecules; ensemble measurements generally mask the stochastic and heterogeneous nature of these events. Here, using advanced optical microscopy and analytical image analysis tools we demonstrate how to monitor proteins within a single living bacterial cell to a precision of single molecules and how we can observe dynamics within molecular complexes in functioning biological machines. The techniques are directly relevant physiologically. They are minimally-perturbative and non-invasive to the biological sample under study and are fully attuned for investigations in living material, features not readily available to other single-molecule approaches of biophysics. In addition, the biological specimens studied all produce fluorescently-tagged protein at levels which are almost identical to the unmodified cell strains ("genomic encoding"), as opposed to the more common but less ideal approach for generating significantly more protein than would occur naturally ('plasmid expression'). Thus, the actual biological samples which will be investigated are significantly closer to the natural organisms, and therefore the observations more relevant to real physiological processes.

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Here we demonstrate the protocols for performing single-molecule fluorescence microscopy on living bacterial cells to enable functional molecular complexes to be detected, tracked and quantified.

可视化单分子络合物在体内使用高级荧光显微

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular ...

生物学

Isolation and Staining of Mouse Skin Keratinocytes for Cell Cycle Specific Analysis of Cellular Protein Expression by Mass Cytometry

The goal of this protocol is to detect and quantify protein expression changes in a cell cycle-dependent manner using single cells isolated from the epidermis of mouse skin. There are seven important steps: separation of the epidermis from the dermis, digestion of the epidermis, staining of the epidermal cell populations with cisplatin, sample barcoding, staining with metal tagged antibodies for cell cycle markers and proteins of interest, detection of metal-tagged antibodies by mass cytometry, and the analysis of expression in the various cell cycle phases. The advantage of this approach over histological methods is the potential to assay the expression pattern of &#62;40 different markers in a single cell at different phases of the cell cycle. This approach also allows for the multivariate correlation analysis of protein expression that is more quantifiable than histological/imaging methods. The disadvantage of this protocol is that a suspension of single cells is needed, which results in the loss of location information provided by the staining of tissue sections. This approach may also require the inclusion of additional markers to identify different cell types in crude cell suspensions. The application of this protocol is evident in the analysis of hyperplastic skin disease models. Moreover, this protocol can be adapted for the analysis of specific sub-type of cells (e.g., stem cells) by the addition of lineage-specific antibodies. This protocol can also be adapted for the analysis of skin cells in other experimental species.

This protocol describes how to isolate skin keratinocytes from mouse models, to stain with metal-tagged antibodies, and to analyze stained cells by mass cytometry in order to profile the expression pattern of proteins of interest in the different cell cycle phases.

小鼠皮肤角质细胞的分离和染色,用于细胞周期特定分析细胞蛋白表达的量度细胞测定

The goal of this protocol is to detect and quantify protein expression changes in a cell cycle-dependent manner using single cells isolated from the ...

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern tissue engineering. A technical bottleneck is the deposition of collagen in vitro, as it is notoriously slow, resulting in sub-optimal formation of connective tissue and subsequent tissue cohesion, particularly in skin models. Here, we describe a method which involves the addition of differentially-sized sucrose co-polymers to skin cultures to generate macromolecular crowding (MMC), which results in a dramatic enhancement of collagen deposition. Particularly, dermal fibroblasts deposited a significant amount of collagen I/IV/VII and fibronectin under MMC in comparison to controls.
The protocol also describes a method to decellularize crowded cell layers, exposing significant amounts of extracellular matrix (ECM) which were retained on the culture surface as evidenced by immunocytochemistry. Total matrix mass and distribution pattern was studied using interference reflection microscopy. Interestingly, fibroblasts, keratinocytes and co-cultures produced cell-derived matrices (CDM) of varying composition and morphology. CDM could be used as &#34;bio-scaffolds&#34; for secondary cell seeding, where the current use of coatings or scaffolds, typically from xenogenic animal sources, can be avoided, thus moving towards more clinically relevant applications.
In addition, this protocol describes the application of MMC during the submerged phase of a 3D-organotypic skin co-culture model which was sufficient to enhance ECM deposition in the dermo-epidermal junction (DEJ), in particular, collagen VII, the major component of anchoring fibrils. Electron microscopy confirmed the presence of anchoring fibrils in cultures developed with MMC, as compared to controls. This is significant as anchoring fibrils tether the dermis to the epidermis, hence, having a pre-formed mature DEJ may benefit skin graft recipients in terms of graft stability and overall wound healing. Furthermore, culture time was condensed from 5 weeks to 3 weeks to obtain a mature construct, when using MMC, reducing costs.

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

We present a protocol to obtain cell-derived matrices rich in extracellular matrix proteins, using macromolecular crowders (MMC). In addition, we present a protocol which incorporates MMC in 3D organotypic skin co-culture generation, which reduces culture time while maintaining maturity of construct.

提高2D和3D皮肤<em&gt;体外</em&gt;模型使用大分子拥挤

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern ...

生物工程

In Vivo Two-Color 2-Photon Imaging of Genetically-Tagged Reporter Cells in the Skin

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located in. Although traditional imaging techniques are useful in identifying protein interactions and morphology in fixed tissue, they are not able to give insight as to how quickly cells are able to bind and uptake proteins, and once activated how their morphology changes in vivo. In the present study, we ask 2 major questions: 1) what is the time-course of fibroblast activation via toll-like receptor-4 (TLR4) and lipopolysaccharide (LPS) interaction and 2) how do these cells behave once activated? Using 2-photon imaging, we have developed a novel technique to assess the ability of LPS-FITC to bind to its cognate receptor, TLR4, expressed on peripheral fibroblasts in the genetic reporter mouse line; FSP1cre; tdTomatolox-stop-lox&#160;in vivo. This unique approach allows researchers to create in-depth, time-lapse videos and/or pictures of proteins interacting with live cells that allows one to have an increased level of granularity in understanding how proteins can alter cellular behavior.

Morphological changes occur in immune responsive fibroblast cells following activation and promote alterations in cellular recruitment. Utilizing 2-photon imaging in conjunction with a genetically engineered Fibroblast-specific protein 1 (FSP1)-cre; tdTomato floxed-stop-floxed (TB/TB) mouse line and green fluorescently tagged lipopolysaccharide-FITC, we can illustrate highly specific uptake of lipopolysaccharide in dermal fibroblasts and morphological changes in vivo.

在 Vivo 双色 2 光子成像中,基因标记报告器细胞在皮肤中

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located ...

Three-dimensional Imaging of Bacterial Cells for Accurate Cellular Representations and Precise Protein Localization

The shape of a bacterium is important for its physiology. Many aspects of cell physiology such as cell motility, predation, and biofilm production can be affected by cell shape. Bacterial cells are three-dimensional (3D) objects, although they are rarely treated as such. Most microscopy techniques result in two-dimensional (2D) images leading to the loss of data pertaining to the actual 3D cell shape and localization of proteins. Certain shape parameters, such as Gaussian curvature (the product of the two principal curvatures), can only be measured in 3D because 2D images do not measure both principal curvatures. Additionally, not all cells lie flat when mounting and 2D imaging of curved cells may not accurately represent the shapes of these cells. Accurately measuring protein localization in 3D can help determine the spatial regulation and function of proteins. A forward convolution technique has been developed that uses the blurring function of the microscope to reconstruct 3D cell shapes and to accurately localize proteins. Here, a protocol for preparing and mounting samples for live cell imaging of bacteria in 3D both to reconstruct an accurate cell shape and to localize proteins is described. The method is based on simple sample preparation, fluorescent image acquisition, and MATLAB-based image processing. Many high-quality fluorescent microscopes can be simply modified to take these measurements. These cell reconstructions are computationally intensive and access to high-throughput computational resources is recommended, although not necessary. This method has been successfully applied to multiple bacterial species and mutants, fluorescent imaging modalities, and microscope manufacturers.

This protocol explains how to prepare and mount bacterial samples for live three-dimensional imaging and how to reconstruct the three-dimensional shape of E. coli from those images.

细菌细胞的三维成像，实现精确的细胞表现和精确的蛋白质定位

The shape of a bacterium is important for its physiology. Many aspects of cell physiology such as cell motility, predation, and biofilm production can be ...

免疫与感染

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

软件辅助跟踪动态细胞突变中蛋白质浓度的图形用户界面

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Staining and High-Resolution Imaging of Three-Dimensional Organoid and Spheroid Models

In vitro three-dimensional (3D) cell culture models, such as organoids and spheroids, are valuable tools for many applications including development and disease modeling, drug discovery, and regenerative medicine. To fully exploit these models, it is crucial to study them at cellular and subcellular levels. However, characterizing such in vitro 3D cell culture models can be technically challenging and requires specific expertise to perform effective analyses. Here, this paper provides detailed, robust, and complementary protocols to perform staining and subcellular resolution imaging of fixed in vitro 3D cell culture models ranging from 100 &#181;m to several millimeters. These protocols are applicable to a wide variety of organoids and spheroids that differ in their cell-of-origin, morphology, and culture conditions. From 3D structure harvesting to image analysis, these protocols can be completed within 4-5 days. Briefly, 3D structures are collected, fixed, and can then be processed either through paraffin-embedding and histological/immunohistochemical staining, or directly immunolabeled and prepared for optical clearing and 3D reconstruction (200 &#181;m depth) by confocal microscopy.

Here, we provide detailed, robust, and complementary protocols to perform staining and subcellular resolution imaging of fixed three-dimensional cell culture models ranging from 100 &#181;m to several millimeters, thus enabling the visualization of their morphology, cell-type composition, and interactions.

三维类器官和球状体模型的染色和高分辨率成像

In vitro three-dimensional (3D) cell culture models, such as organoids and spheroids, are valuable tools for many applications including development and ...

3D Visualization of Immune Cell Populations in HIV-Infected Tissues via Clearing, Immunostaining, Confocal, and Light Sheet Fluorescence Microscopy

Human Immunodeficiency Virus (HIV), the causative agent of Acquired Immune Deficiency Syndrome (AIDS), is a major global health concern with nearly 40 million individuals infected worldwide and no widely accessible cure. Despite intensive efforts, a detailed understanding of virus and host cell interactions in tissues during infection and in response to therapy remains incomplete. To address these limitations, water-based tissue clearing techniques CUBIC (Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis) and CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/Immunostaining/in situ-hybridization-compatible Tissue hYdrogel) are applied to visualize complex virus host-cell interactions in HIV-infected tissues from animal models and humans using confocal and light sheet fluorescence microscopy. Optical sectioning of intact tissues and image analysis allows rapid reconstruction of spatial information contained within whole tissues and quantification of immune cell populations during infection. These methods are applicable to most tissue sources and diverse biological questions, including infectious disease and cancer.

Tissue clearing, combined with immunofluorescence microscopy, allows spatial visualization and quantification of immune-cell populations and virus proteins within intact tissues. Optical sectioning of cleared tissues with confocal and light sheet fluorescence microscopy can generate 3D models of complex tissue environments and reveal spatial heterogeneity exhibited during HIV infection.

Human Immunodeficiency Virus (HIV), the causative agent of Acquired Immune Deficiency Syndrome (AIDS), is a major global health concern with nearly 40 ...