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?...

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 border="0" cellpadding="0" cellspacing="0" width="1082">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Paraformaldehyde</td>
			<td style="width:195px;">Sigma-Aldrich&#160;</td>
			<td style="width:229px;">P6418</td>
			<td style="width:236px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Ethanol 96% (undenaturated)</td>
			<td style="width:195px;">Chem-supply</td>
			<td style="width:229px;">UN1170</td>
			<td style="width:236px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Nile Red</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>72485-100MG</td>
			<td style="width:236px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4&#8217;,6-diamidino-2-phenylindole (DAPI)</td>
			<td>Roche</td>
			<td>10236276001</td>
			<td style="width:236px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N,N,N&#8217;,N&#8217;-tetrakis (2-hydroxypropyl) ethylenediamine</td>
			<td style="width:195px;">Merck Millipore</td>
			<td style="width:229px;">821940</td>
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			<td style="width:229px;">648462</td>
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			<td height="21" style="height:21px;width:423px;">Triton X-100</td>
			<td style="width:195px;">Merck Millipore</td>
			<td style="width:229px;">648462</td>
			<td style="width:236px;"></td>
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			<td height="21" style="height:21px;width:423px;">Sucrose</td>
			<td style="width:195px;">Sigma-Aldrich&#160;</td>
			<td style="width:229px;">S0389</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Optimal Cutting Temperature (OCT) Compound</td>
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			<td height="21" style="height:21px;width:423px;">anti-Keratin14 antibody</td>
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			<td height="21" style="height:21px;width:423px;">anti-Ki67 antibody&#160;</td>
			<td style="width:195px;">Abcam</td>
			<td style="width:229px;">ab16667</td>
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			<td height="20" style="height:20px;width:423px;">Donkey anti-rabbit Alexa 594</td>
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			<td style="width:229px;">A21207</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Dimethylsulfoxide</td>
			<td style="width:195px;">Sigma-Aldrich&#160;</td>
			<td style="width:229px;">D2650</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Urea</td>
			<td style="width:195px;">Merck Millipore</td>
			<td style="width:229px;">66612</td>
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		<tr height="21">
			<td height="21" style="height:21px;">2,2&#8242;,2&#8242;&#8217;-nitrilotriethanol</td>
			<td style="width:195px;">Merck Millipore</td>
			<td style="width:229px;">137002</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:423px;">Confocal Microscope</td>
			<td style="width:195px;">Nikon Instruments Inc</td>
			<td style="width:229px;">Nikon A1 - Confocal Microscope</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">cruZer6 Face Trimmer</td>
			<td style="width:195px;">Braun</td>
			<td style="width:229px;">Braun cruZer6 Face</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Sodium azide</td>
			<td style="width:195px;">Sigma-Aldrich&#160;</td>
			<td style="width:229px;">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...

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

Research

JoVE Journal

Biology

11.4K 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

Method for Whole Mount Antibody Staining in Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

整装在鸡的抗体染色方法

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

科研

生物学

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

斑马鱼整装高分辨率双荧光原位杂交

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching morphogenesis. We have developed the limb skin vasculature model to study vascular development in which a pre-existing primitive capillary plexus is reorganized into a hierarchically branched vascular network. Whole-mount confocal microscopy with multiple labelling allows for robust imaging of intact blood vessels as well as their cellular components including endothelial cells, pericytes and smooth muscle cells, using specific fluorescent markers. Advances in this limb skin vasculature model with genetic studies have improved understanding molecular mechanisms of vascular development and patterning. The limb skin vasculature model has been used to study how peripheral nerves provide a spatial template for the differentiation and patterning of arteries. This video article describes a simple and robust protocol to stain intact blood vessels with vascular specific antibodies and fluorescent secondary antibodies, which is applicable for vascularized embryonic organs where we are able to follow the process of vascular development.

We introduce a whole-mount immunohistochemistry and laser scanning confocal microscopy with multiple labelling for analyzing intricate vascular network formation in mouse embryonic limb skin.

全安装胚胎肢体皮肤血管的免疫组织化学分析:一个模型系统，研究在胚胎血管分支形态发生

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching ...

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures are lengthy and technically demanding with multiple important steps that collectively contribute to the quality of the final result. This protocol describes in detail several key quality control steps for optimizing probe labeling and performance. Overall, our protocol provides a detailed description of the critical steps necessary to reproducibly obtain high quality results. First, we describe the generation of digoxygenin (DIG) labeled RNA probes via in vitro transcription of DNA templates generated by PCR. We describe three critical quality control assays to determine the amount, integrity and specific activity of the DIG-labeled probes. These steps are important for generating a probe of sufficient sensitivity to detect endogenous mRNAs in a whole mouse embryo. In addition, we describe methods for the fixation and storage of E8.5-E11.5 day old mouse embryos for in situ hybridization. Then, we describe detailed methods for limited proteinase K digestion of the rehydrated embryos followed by the details of the hybridization conditions, post-hybridization washes and RNase treatment to remove non-specific probe hybridization. An AP-conjugated antibody is used to visualize the labeled probe and reveal the expression pattern of the endogenous transcript. Representative results are shown from successful experiments and typical suboptimal experiments.

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

This whole mount in situ hybridization protocol discusses critical steps that ensure reproducible high quality results for gene expression studies in E8.5-E11.5 day old mouse embryos.

全山原位杂交E8.5，E11.5小鼠胚胎

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures ...

Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies during chromosome separation are a hallmark of malignancy and associated with progressive disease 2-4. The spindle assembly checkpoint (SAC) is a mitotic surveillance mechanism that holds back cells at metaphase until every single chromosome has established a stable bipolar attachment to the mitotic spindle1. The SAC exerts its function by interference with the activating APC/C subunit Cdc20 to block proteolysis of securin and cyclin B and thus chromosome separation and mitotic exit. Improper attachment of chromosomes prevents silencing of SAC signaling and causes continued inhibition of APC/CCdc20 until the problem is solved to avoid chromosome missegregation, aneuploidy and malignant growths1.
Most studies that addressed the influence of improper chromosomal attachment on APC/C-dependent proteolysis took advantage of spindle disruption using depolymerizing or microtubule-stabilizing drugs to interfere with chromosomal attachment to microtubules. Since interference with microtubule kinetics can affect the transport and localization of critical regulators, these procedures bear a risk of inducing artificial effects 5.
To study how the SAC interferes with APC/C-dependent proteolysis of cyclin B during mitosis in unperturbed cell populations, we established a histone H2-GFP-based system which allowed the simultaneous monitoring of metaphase alignment of mitotic chromosomes and proteolysis of cyclin B 6.
To depict proteolytic profiles, we generated a chimeric cyclin B reporter molecule with a C-terminal SNAP moiety 6 (Figure 1). In a self-labeling reaction, the SNAP-moiety is able to form covalent bonds with alkylguanine-carriers (SNAP substrate) 7,8 (Figure 1). SNAP substrate molecules are readily available and carry a broad spectrum of different fluorochromes. Chimeric cyclin B-SNAP molecules become labeled upon addition of the membrane-permeable SNAP substrate to the growth medium 7 (Figure 1). Following the labeling reaction, the cyclin B-SNAP fluorescence intensity drops in a pulse-chase reaction-like manner and fluorescence intensities reflect levels of cyclin B degradation 6 (Figure 1). Our system facilitates the monitoring of mitotic APC/C-dependent proteolysis in large numbers of cells (or several cell populations) in parallel. Thereby, the system may be a valuable tool to identify agents/small molecules that are able to interfere with proteolytic activity at the metaphase to anaphase transition. Moreover, as synthesis of cyclin B during mitosis has recently been suggested as an important mechanism in fostering a mitotic block in mice and humans by keeping cyclin B expression levels stable 9,10, this system enabled us to analyze cyclin B proteolysis as one element of a balanced equilibrium 6.

Metaphase to anaphase transition is triggered through anaphase-promoting complex (APC/C)-dependent ubiquitination and subsequent destruction of cyclin B. Here, we established a system which, following pulse-chase labeling, allows monitoring cyclin B proteolysis in entire cell populations and facilitates the detection of interference by the mitotic checkpoint.

在整个细胞群在单细胞水平上研究细胞周期蛋白B的蛋白水解

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies ...

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression both in terms of tissue distribution and developmental stage. Protocols are described for the use of WISH of zebrafish embryos using antisense RNA probes labeled with digoxigenin. Probes are generated by incorporating digoxigenin-linked nucleotides through in vitro transcription of gene templates that have been cloned and linearized. The chorions of embryos harvested at defined developmental stages are removed before incubation with specific probes. Following a washing procedure to remove excess probe, embryos are incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase. By employing a chromogenic substrate for alkaline phosphatase, specific gene expression can be assessed. Depending on the level of gene expression the entire procedure can be completed within 2-3 days.

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

The zebrafish, a small tropical fish, has become a popular model for studying gene function during vertebrate development and disease. The temporal and spatial expression of target genes can be determined by in situ hybridization. Our improved protocol allows for the detection of low abundant transcripts with low non-specific background signal.

高解析度全山<em&gt;在原位</em杂交斑马鱼胚胎内，研究基因表达和功能

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

全球合成的RNA在单细胞水平以下的低氧分析

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues.
Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

We provide here an efficient and reliable protocol for immunostaining, Fluorescence in&#160;situ Hybridization, DNA staining followed by quantitative, high-resolution imaging in whole-mount Arabidopsis thaliana ovules. This method was successfully used to analyze chromatin modifications and nuclear architecture.

定量，单细胞在全安装胚珠在染色质修饰和核体系结构分析的有效方法<em&gt;拟南芥</em

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the ...

Visualizing Clathrin-mediated Endocytosis of G Protein-coupled Receptors at Single-event Resolution via TIRF Microscopy

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological assays, measuring global changes in endocytosis, have identified over 30 known components participating in CME, and biochemical studies have generated an interaction map of many of these components. It is becoming increasingly clear, however, that CME is a highly dynamic process whose regulation is complex and delicate. In this manuscript, we describe the use of Total Internal Reflection Fluorescence (TIRF) microscopy to directly visualize the dynamics of components of the clathrin-mediated endocytic machinery, in real time in living cells, at the level of individual events that mediate this process. This approach is essential to elucidate the subtle changes that can alter endocytosis without globally blocking it, as is seen with physiological regulation. We will focus on using this technique to analyze an area of emerging interest, the role of cargo composition in modulating the dynamics of distinct clathrin-coated pits (CCPs). This protocol is compatible with a variety of widely available fluorescence probes, and may be applied to visualizing the dynamics of many cargo molecules that are internalized from the cell surface.

Clathrin-mediated endocytosis, a rapid and highly dynamic process internalizes many proteins, including signaling receptors. The protocol described here directly visualizes the kinetics of individual endocytic events. This is essential for understanding how core members of the endocytic machinery coordinate with each other, and how protein cargo influence this process.

通过全内反射荧光显微镜可视化G蛋白偶联受体的单事件分辨率网格蛋白介导的内吞作用

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological ...

CUBIC协议可视化在全山皮肤配方单细胞分辨率蛋白表达

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