作者承认了赛默飞世尔科技的赞助，并感谢伦敦国王学院的尼康影像中心在共焦图像采集期间的支持。

所有的动物实验均受到当地的道德审查，并按照英国政府内政部执照执行。 皮肤收获和冷冻保存<ol><li> 准备工作。 <ol><li>准备一个100毫升培养皿与25毫升4％PFA和两个100毫米培养皿与25毫升1x磷酸盐缓冲盐水（PBS）。 </li><li>使用最佳切割温度的化合物（OCT），将长方形剥离的冷冻模型填充三分之二。 </li><li>放置一个金属板，将冷冻块放在其后的步骤中，放入-80°C冰箱。 </li></ol></li><li> 皮肤收获，固定和低温保存。 <ol><li>用干电动剃须刀夹住动物尸体的背部区域。 注意:在本例中，使用产后21天野生型小鼠。 </li><li>在皮肤上收获感兴趣的区域。 注意:鼠皮肤的背内侧区域<li>将收获的皮肤修剪成适当尺寸的矩形片，以适应低温塑料的底部，考虑到毛囊的定向生长。 注意:较小的皮肤切片可能会更容易处理新手，因为它们在孵化和安装过程中不太可能纠缠。这里所示的例子是约1cm 2的背部皮肤区域，其适合于22×30×20mm的低温瓶（ 图2a ）。 </li><li>根据皮肤样品的厚度，将皮肤在室温下固定在25 mL 4％PFA中10-30分钟（ 图1b ）。 </li><li>在25 mL PBS中将皮肤样品洗两次每次至少5分钟（ 图1b ）。 </li><li>用纸巾擦拭皮肤样本，小心地排出多余的PBS的组织，这可能导致冷冻过程中的结晶，并可能影响冷冻切片的结果。 注意:不需要标准的蔗糖梯度。然而，这也可以由用户自行决定并入协议。 </li><li>注意每个皮肤样品的毛囊方向。使用解剖显微镜进行视觉辅助（特别是第一次执行协议的人）。将皮肤样品插入OCT填充的低温容器中，并使用镊子除去附着在夹头毛发表面的任何气泡，均匀化OCT的所有区域（ 图2a和2b ）。 </li><li>将皮肤推入OCT填充块的底部，使其与底部齐平。 注意:皮肤可以在任何方向定向，a只要注意到适当的切割程序，就要注意毛囊的颗粒。当块被连接到低温恒温器以进行冷冻切割时，低温牛奶将被重新定向。标记低温瓶上的毛囊取向，因为该步骤确定了低温恒温器切割的后续方向。 </li><li>将冷冻块转移到-80°C冰箱的金属板上，以避免组织漂浮和脱位。 </li><li>监测冷冻过程，以保持低温塑料底部皮肤的方向，因为看不见的气泡可能导致皮肤上升到低温塑料的表面。 注意:冷冻组织的冷冻保存可以在-80°C下储存超过一年，并可以重新用于其他部分。 </li></ol></li></ol><img alt="图1" class="xfigimg" src="/files/ftp_upload/56106/56106fig1.jpg" /> 图1.小鼠皮肤的收获和固定。 </stroNG&gt; （ a ）从动物尸体的背内侧收获皮肤组织。该区域中的毛囊均匀间隔并对齐，因此允许在切片期间获得最佳取向，如箭头所示。 （ b ）切割适合大小的正方形后，将皮肤组织固定在4％PFA中15分钟，并在PBS中洗涤两次，每次5分钟。 <a href="http://ecsource.jove.com/files/ftp_upload/56106/56106fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> 厚组织横截面<ol><li> 准备和组织取向安装在低温恒温器上。 <ol><li>准备一个100毫升培养皿与15毫升的PBS。将其放置在低温恒温器上易于访问的区域。另外，制备一个12孔板，每孔2.5 mL PBS;标签根据样品长期储存在4°C。使用镊子来处理这些部分。 </li><li>将低温恒温器的温度调节至-20°C。 注意:温度可能影响切片，但是一个很好的指导是在-20°C开始。 </li><li>为了获得大约两个毛囊厚度的部分，调整低温恒温器以切割150μm厚的部分。 注意:根据用户的需要以及将用于分析的显微镜的限制，可以改变截面的厚度。 注意:样品的取向对于以适当的方向获得具有毛囊的皮肤切片是至关重要的。这通过正确安装在低温恒温器块上来实现。确保剖面与毛囊方向平行（ 图2c ）。如前所述，皮肤样本中截面的正确方向是确定将要获取的图像质量的关键步骤稍后的一步。 </li></ol></li></ol><img alt="图2" class="xfigimg" src="/files/ftp_upload/56106/56106fig2.jpg" /> 图2.嵌入，低温保存和切片。 （ a ） 在黑色箭头所示的低温乳头上标记毛囊（HF）方向对于在冷冻切割过程中正确定向是重要的。 （ b ）剖面需要与毛囊取向一致以产生毛囊完整长度保持完整的部分;（ c ）根据毛囊方向切割切片，由黑色箭头表示， cryomold。 （ d ）用金属镊子收集厚的组织横截面，（ e ）转移到含有1×PBS的100mm培养皿中。 （ f ）在室温下，PBS溶解OC.T。围绕厚组织横截面的化合物，如白色箭头所示。然后这些部分在PBS中自由浮动。 <a href="http://ecsource.jove.com/files/ftp_upload/56106/56106fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <ol start="2"><li> 冷冻切片。 <ol><li>使用低温恒温器切割部分。使用镊子收集含有嵌入的皮肤的OCT（ 图2d ）。 注意:使用低温恒温器，可以沿X，Y和Z轴进行独立的移动和调整，以获得最佳的样品定位。这允许产生理想排列的组织横截面。 </li><li>将切片从低温恒温器中转移到装有PBS的100毫米培养皿中，并继续下一个切片。不要将样品收集在幻灯片上（ 图2e ）。 注意:在室温下，PBS将溶解掉OCT，留下易于使用镊子处理的皮肤切片（ 图2f ）。 注意:溶解许多100μm厚的组织切片后，100 mm培养皿中可能需要新鲜的PBS，可以相应地更改。 </li><li>使用镊子将漂浮的皮肤部分转移到装有2.5mL PBS的12孔板中的正确标记的孔（ 图3a ，左）。 注意:在4°C时，样品可以储存至少两天。为了在切片后长期储存含皮肤的OCT块，用新鲜的OCT液滴密封切割表面。冷冻OCT液滴后，将所用的OCT块包裹在石蜡膜中，并将其放回-80°C冷冻箱。 </li></ol></li></ol> 3.免疫荧光标记。 <ol><li>准备PB缓冲液（补充有0.5％脱脂奶粉，0.25％鱼皮明胶和0.5％Triton X-100的PBS）t 2 h提前，如前所述5 。 注意:叠氮化钠可以添加到PB缓冲液中进行抗体保存，重复使用染色缓冲液。 </li><li>标记1.5 mL微量离心管，每管加入500μLPB缓冲液。仔细使用镊子将皮肤切片从PBS转移到含有PB缓冲液的分离管中（ 图3a ，右图）。确保所有皮肤切片完全淹没。将微量离心管放置在跷跷板摇臂上，速度不得高于每分钟10次，这不应破坏组织完整性，在室温下1小时。 注意:至关重要的是，跷跷板摇杆上的速度每分钟不超过10次，因为它会引起缠结。 <ol><li>虽然可以每个微量离心管添加多个切片，以节省抗体，但每个管只放置一个切片。要减少抗体使用量，请使用体积250μL。 注意:如果需要，用例如96孔板的微量离心管替代。然而，由于增强的液体扰动，将厚的组织横截面放置在1.5-mL微量离心管中允许最有效的抗体渗透到组织中。 </li></ol></li><li>标记每个皮肤切片分开的1.5 mL微量离心管，并加入500μLPB缓冲液和适量的一级抗体。在1小时的阻断后，将皮肤切片转移到含有抗体的新鲜制备的管中。将切片在4℃孵育过夜。 注意:在该实施例中，使用以下一抗体:浓度为1:50的FITC大鼠抗人CD49f和浓度为1:100的山羊抗小鼠/大鼠整合素α8。 </li><li>第二天，准备两个单独的1.5 mL微量离心管，每个样品含有500μLPBS。在室温下洗两次皮肤切片1小时即</li><li>准备单独的1.5 mL含有适当浓度的适用二级抗体和4&#39;，6-二脒基-2-苯基吲哚（DAPI）的PB缓冲液的微量离心管。 注:本例中使用的二抗是Alexa Fluor 488驴抗大鼠IgG和浓度为1:500的Alexa Fluor 555驴抗山羊IgG。 DAPI的浓度为1:100。 </li><li>小心地将皮肤样品转移到包含二抗和DAPI的PB缓冲液中，并在旋转器或摇床上以低速将室温下皮肤切片孵育1小时。 </li><li>将切片在4℃下储存在含有二抗和DAPI的PB缓冲液中长达四天，并且如果向PBS中加入叠氮化钠，则可能更长。 </li></ol><img alt="图3" class="xfigimg" src="/files/ftp_upload/56106/56106fig3.jpg" /> 图3.免疫荧光标签和安装。 （ a ）浮体组织横截面可以在4℃下储存在12孔板中至少两天。在免疫荧光（IF）标记之前，将感兴趣的组织切片转移到含有PB缓冲液的1.5mL微量离心管中，如箭头1所示。对于IF标记，遵循步骤3中详述的多步骤程序。步骤3的一部分需要将组织横截面小心地转移到新制备的含有一次抗体溶液，二抗溶液或洗涤缓冲液的微量离心管中，其由箭头2所示。（ b ）在IF标记之后，使用解剖显微镜的帮助，切片在甘油液滴中松开并变平。 （ c ）一旦组织横截面完全平坦化到盖板的底部，则使用常规显微镜载玻片来安装该部分。 <ahref ="http://ecsource.jove.com/files/ftp_upload/56106/56106fig3large.jpg"target ="_ blank"&gt;请点击此处查看此图的较大版本。 4.用于显微镜可视化的安装<ol><li>在成像之前，将皮肤切片转移到含有500μLPBS的分离的微量离心管中以洗涤二次抗体和DAPI。 </li><li>使用1000μL移液管，但切断第一个0.5厘米的移液器吸头，以便适当吸取高粘度甘油。在解剖显微镜下将22 x 50毫米的盖玻片放在黑暗的背景下（ 图3b ）。 </li><li>在盖板上加入一滴100％甘油（ 图3b ）。将皮肤切片从微量离心管转移到甘油液滴上。使用解剖显微镜和尖锐的镊子仔细展开卷曲的皮肤切片。 注意:当切片漂浮在gly中时通过咳嗽组织的自然倾向恢复其正常形状，可以解开它。不要强迫切片的非自然矫直，因为这可能会对组织造成伤害。 </li><li>一旦皮肤部分的整个长度正确定向并平坦化在盖板上，就安装组织;使用常规显微镜幻灯片。这一步将进一步矫正皮肤切片。 注意:避免空气滞留（ 图3c ）。 </li><li>在未来两天内在甘油安装的皮肤部分中进行成像。 注意:长期储存会对组织和成像质量产生负面影响。在这个例子中，所有的图像都是用直立的共焦显微镜采用20倍的物镜。 </li></ol>

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作者想透露，该手稿的出版由Thermo-Fisher Scientific Inc.出资

Here, we present the "horizontal whole mount" technique, which has several advantages over classical frozen sections. One advantage of our technique is that it can easily be performed with standard histology equipment and a confocal microscope. Second, the tissue integrity is preserved in a superior manner when compared to standard cryosections. For example, adipocytes within the hypodermal layer (i.e., dermal white adipocytes (DWAT))9 are severely disrupted in standard cryosections, which makes studying tissues that are adipose-laden impossible. Our technique allows for the full analysis of the adipocytes in this layer and may be adapted to other tissues with high adipocyte contents. Furthermore, this allows for the improved detection of mesenchymal cells in lineage-tracing studies 2,10. 
Standard cryosections, by their very nature, can never reveal the true 3D aspects of a tissue through confocal microscopy. This means that thicker sections are advantageous in the analysis of skin, since hair follicles, as well as their substructures, can traverse a depth that exceeds the standard thickness of 10 &#181;m used in classical crysosections. We are intrigued that other tissues, such as the intestine and brain, for example, possess similar challenges with regard to viewing the tissue as a 3D structure2,7,11,12,13. Intriguingly, our technique has already been shown to be beneficial for use in certain applications in the intestines14. We believe that our horizontal whole-mount protocol has the potential be applied to any other tissue requiring 3D analysis.

显微成像设备的革命提供了复杂的高分辨率成像仪器。然而，当获得完整的三维（3D）组织横截面的显微图像时，样品制备提出了相当大的挑战，并且可能是定义图像质量的限制因素。为了保持组织形态和靶蛋白的抗原性，为了最小化加工诱发的人工制品，并且最大化最终的图像质量，每个单独的步骤都值得仔细考虑。例如，皮肤的传统分析需要与被适当地定向的表皮和真皮，与毛囊的视图的图像，从而允许的干细胞区室的贡献皮肤稳态1，2中的解剖分析。这需要对皮肤如何嵌入和切片进行全面的分析。重要的是，毛囊可以更厚大于100μm，这大大超过了标准石蜡或冰冻切片厚度，从而导致相比于整体安装件或厚的横截面3，4，5分析的较低的标准。 总之，用于显微镜分析的样品制备的每个步骤是影响图像分析的关键决定因素。在这里，提出了一种用于粗，3D组织横截面分析的新颖处理方案，我们称之为"水平整体安装"。该协议高度保留抗原性，并通过使用标准共聚焦成像设备实现对皮肤厚部的全面开发。这是使用皮肤进行厚组织横截面加工和成像的完整指南，包括组织收获和多聚甲醛（PFA）辅助冷冻保存（步骤1），用低温恒温器生成100μm厚的组织横截面（步骤2）和免疫荧光标记和安装（步骤3和4）。代表性的结果比较了两种不同组织学制备技术的共焦图像 - 经典的冷冻切片和厚的3D组织横截面 - 突出了该方案的潜在用户的"水平整体安装"的优点。

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		<tr height="21">
			<td height="21" style="height:21px;width:273px;">PBS</td>
			<td style="width:99px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">homemade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Gelatin, from cold water fish skin</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:119px;">G7765</td>
			<td>250ML</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:273px;">Glycerol</td>
			<td style="width:99px;">BDH Laboratory Supplies</td>
			<td style="width:119px;">444482V</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:273px;">O.C.T. compound</td>
			<td style="width:99px;">VWR chemicals</td>
			<td style="width:119px;">361603E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Peel-A-Way embedding molds</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:119px;">E6032-1CS</td>
			<td>Square S-22</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Non-Fat Powdered Milk</td>
			<td style="width:99px;">Bio Basic Inc.</td>
			<td style="width:119px;">NB0669</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Triton X-100</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:119px;">T9284</td>
			<td>500ML</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:273px;">4&#8242;,6-Diamidino-2-phenylindole, dilactate (DAPI)</td>
			<td style="width:99px;">Invitrogen</td>
			<td style="width:119px;">D3571</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:273px;">FITC Rat Anti-Human CD49f</td>
			<td style="width:99px;">BD Pharmingen</td>
			<td style="width:119px;">555735</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Mouse/Rat Integrin alpha 8 Antibody</td>
			<td style="width:99px;">R&#38;D Systems</td>
			<td style="width:119px;">AF4076</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:273px;">Alexa Fluor 488 donkey anti-rat IgG</td>
			<td style="width:99px;">Life Technologies</td>
			<td style="width:119px;">A21208</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:273px;">Alexa Fluor 555 donkey anti-goat IgG</td>
			<td style="width:99px;">Life Technologies</td>
			<td style="width:119px;">A21432</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:273px;">CryoStar NX70</td>
			<td style="width:99px;">Thermo Fisher Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">100mm Culture dishes</td>
			<td style="width:99px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:273px;">Disposable scalpels</td>
			<td style="width:99px;">Swann-Morton</td>
			<td style="width:119px;">Ref 0501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">pointed metal forceps</td>
			<td style="width:99px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">1.5 ml microcentrifuge tubes</td>
			<td style="width:99px;">VWR</td>
			<td dir="LTR">211-2130</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">12 Well plates</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:119px;">CLS3513</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Dissecting microscope</td>
			<td style="width:99px;">Nikon</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">20 ul and 1000 ul Pipette</td>
			<td style="width:99px;">Gilson</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;">1000&#181;l XL Graduated TipOne Filter Tip (Sterile)</td>
			<td style="width:99px;">Star Lab</td>
			<td dir="LTR">S1122-1830</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;">20&#181;l Bevelled TipOne Filter Tip (Sterile)</td>
			<td style="width:99px;">Star Lab</td>
			<td>S1120-1810</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Rocking shaker plate</td>
			<td style="width:99px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:273px;">Microscope slides, menzel Glaeser</td>
			<td style="width:99px;">Thermos Scientific</td>
			<td style="width:119px;">631-9483</td>
			<td>Superfrost Plus</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:273px;">Cover Glasses, Menzel Glaeser</td>
			<td style="width:99px;">Thermos Scientific</td>
			<td style="width:119px;">MENZBB024060AB</td>
			<td>24 x 60 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Confocal microscope</td>
			<td style="width:99px;">Nikon</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

horizontal whole mount: a novel processing and imaging protocol for thick, three-dimensional tissue cross-sections of skin

处理感兴趣的组织以产生支持科学论证的微观图像可能是具有挑战性的。获取高质量的显微图像并不完全取决于显微镜的质量，而且还取决于组织处理的方法，其通常涉及多个关键动作或步骤。此外，皮肤和其他组织中的间充质细胞类型代表组织制备和成像的新挑战。在这里，我们提出了一个完整的过程，从组织收获到显微镜。我们的技术，称为"水平整体安装"，是新手可以快速熟练的技术，并允许在用低温恒温器切割的60-300微米厚的部分进行抗原保存和检测。这种厚度的部分提供了在三维环境中组织微架构的增强的可视化。此外，协议保留间叶细胞，以增强图像质量与标准低温恒温器或石蜡切片相比，从而提高免疫染色的功效和可靠性。我们认为，这个协议将有益于所有实验室，可视化的皮肤，可能的其他组织和器官。

为了强调我们技术的优势，我们将我们厚实的3D组织横切技术"水平整体安装"与经典冷冻切片进行了比较。如前所述切割经典冷冻切片5 。为了在显微镜图像中提供表皮的视觉结构，我们对整合素α-6（Itga6）进行免疫染色，整合素α-6（Itga6）是将表皮细胞锚定在下面的基底膜6上的组分。我们还用整联蛋白α-8 7标记了负责皮肤刺激（也称为"鹅肝"）的牵引力皮肌（APM）。在经典冷冻切片中，与Itga6一起显现的大多数毛囊在整个长度上没有切片，与每个部分相比主要产生不完全的毛囊，与水平整体安装（ 图4a -4d </strong&gt;）。与传统的10-μm部分相比，厚的组织横截面使得可以获得更多的Z堆叠层，从而允许更完整的3D图像。当研究与毛囊和上覆的基底膜相关的APM的完整性时，这变得更加明显。在经典的冷冻切片中，大部分的APM被分级（ 图4a- 4d ）。此外，与冷冻切片连接到温暖的玻璃载玻片上的脂肪细胞的破坏相比，低层皮肤隔室的组织完整性保持在水平整体安装，这是众所周知的冻融假象（ 图4a- b ，比较皮下地区） 8 。 <img alt="图4" class="xfigimg" src="/files/ftp_upload/56106/56106fig4.jpg" /> 图4.水平整体相比传统的冷冻切片。 （ a ）使用整合素α-6（Itga6）和整联蛋白α-8（Itga8）标记10μm厚的常规获得的皮肤冷冻切片和（ b ）100μm厚的3D组织横截面以显现表皮隔室和牵引器皮肌， 分别。厚组织横截面的图像被表示为大Z堆叠的最大投影。白色框架表示放大的区域，显示在（ c ）古典和（ d ）水平整体安装部分中。 （ e ）完整的毛囊和（ f ）完整的放射性皮肌在经典和水平整体安装部分进行定量。比例尺表示100μm。数据表示为平均值±标准误差（SEM）。对每个生物复制物（ n = 3）进行一个部分的定量。未配对t检验* P &lt;0.05，*** P &lt;0.0005。 <a href="http://ecsource.jove.com/files/ftp_upload/56106/56106fig4large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin at JoVE.com

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

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

这项工作提出了一种新颖的加工和成像协议，用于厚的三维组织横截面分析，可以充分利用共聚焦成像模式。该协议保留抗原性，并代表一个强大的系统来分析皮肤组织学和潜在的其他组织类型。

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

horizontal-whole-mount-novel-processing-imaging-protocol-for-thick

Research

JoVE Journal

Developmental Biology

15.0K 次观看.  King's College London. 这项工作提出了一种新颖的加工和成像协议，用于厚的三维组织横截面分析，可以充分利用共聚焦成像模式。该协议保留抗原性，并代表一个强大的系统来分析皮肤组织学和潜在的其他组织类型。 

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

A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is routinely used as an in vivo model to study embryo implantation and pregnancy progression. Unfortunately, such murine studies require pregnancy interruption to enable follow-up phenotypic analysis. To address this issue, we used three-dimensional (3-D) reconstruction of HFUS imaging data for early detection and characterization of murine embryo implantation sites and their individual developmental progression in utero. Combining HFUS imaging with 3-D reconstruction and modeling, we were able to accurately quantify embryo implantation site number as well as monitor developmental progression in pregnant C57BL6J/129S mice from 5.5 days post coitus (d.p.c.) through to 9.5 d.p.c. with the use of a transducer. Measurements included: number, location, and volume of implantation sites as well as inter-implantation site spacing; embryo viability was assessed by cardiac activity monitoring. In the immediate post-implantation period (5.5 to 8.5 d.p.c.), 3-D reconstruction of the gravid uterus in both mesh and solid overlay format enabled visual representation of the developing pregnancies within each uterine horn. As genetically engineered mice continue to be used to characterize female reproductive phenotypes derived from uterine dysfunction, this method offers a new approach to detect, quantify, and characterize early implantation events in vivo. This novel use of 3-D HFUS imaging demonstrates the ability to successfully detect, visualize, and characterize embryo-implantation sites during early murine pregnancy in a non-invasive manner. The technology offers a significant improvement over current methods, which rely on the interruption of pregnancies for gross tissue and histopathologic characterization. Here we use a video and text format to describe how to successfully perform ultrasounds of early murine pregnancy to generate reliable and reproducible data with reconstruction of the uterine form in mesh and solid 3-D images.

Mice are widely used to study gestational biology. However, pregnancy termination is required for such studies which precludes longitudinal investigations and necessitates the use of large numbers of animals. Therefore, we describe a non-invasive technique of high-frequency ultrasonography for early detection and monitoring of post-implantation events in the pregnant mouse.

三维高频超声对小鼠早期妊娠特征的新应用

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is ...

科研

发育生物学

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and optical transparency. In particular, the zebrafish embryo has become an important organism to study vertebrate kidney organogenesis as well as multiciliated cell (MCC) development. To visualize MCCs in the embryonic zebrafish kidney, we have developed a combined protocol of whole-mount fluorescent in situ hybridization (FISH) and whole mount immunofluorescence (IF) that enables high resolution imaging. This manuscript describes our technique for co-localizing RNA transcripts and protein as a tool to better understand the regulation of developmental programs through the expression of various lineage factors.

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

Cilia development is vital to proper organogenesis. This protocol describes an optimized method to label and visualize ciliated cells of the zebrafish.

全贴装荧光原位杂交和免疫荧光法在斑马鱼中 Multiciliated 细胞的可视化

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and ...

Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques

Due to its formidable tools for molecular genetic studies, Arabidopsis thaliana is one of the most prominent model species in plant biology and, especially, in plant reproductive biology. However, plant morphological, anatomical, and ultrastructural analyses traditionally involve time-consuming embedding and sectioning procedures for bright field, scanning, and electron microscopy. Recent progress in confocal fluorescence microscopy, state-of-the-art 3-D computer-aided microscopic analyses, and the continuous refinement of molecular techniques to be used on minimally processed whole-mount specimens, has led to an increased demand for developing efficient and minimal sample processing techniques. In this protocol, we describe techniques for properly dissecting Arabidopsis flowers and siliques, basic clearing techniques, and some staining procedures for whole-mount observations of reproductive structures.

Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques

In this protocol, we describe techniques for the proper dissection of Arabidopsis flowers and siliques, some basic clearing techniques, and selected staining procedures for whole-mount observations of reproductive structures.

拟南芥花器官和角果的全装清和染色

Due to its formidable tools for molecular genetic studies, Arabidopsis thaliana is one of the most prominent model species in plant biology and, ...

Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development and organogenesis are two areas of research that have benefitted greatly from these advances, due to the high quality of data that can be obtained directly from imaging pre-implantation embryos or ex vivo organs. While pre-implantation embryos have yielded data with especially high spatial resolution, later stages have been less amenable to three-dimensional reconstruction. Obtaining high-quality 3D or volumetric data for known embryonic structures in combination with fate mapping or genetic lineage tracing will allow for a more comprehensive analysis of the morphogenetic events taking place during embryogenesis.
This protocol describes a whole-mount immunofluorescence approach that allows for the labeling, visualization, and quantification of progenitor cell populations within the developing cardiac crescent, a key structure formed during heart development. The approach is designed in such a way that both cell- and tissue-level information can be obtained. Using confocal microscopy and image processing, this protocol allows for three-dimensional spatial reconstruction of the cardiac crescent, thereby providing the ability to analyze the localization and organization of specific progenitor populations during this critical phase of heart development. Importantly, the use of reference antibodies allows for successive masking of the cardiac crescent and subsequent quantitative measurements of areas within the crescent. This protocol will not only enable a detailed examination of early heart development, but with adaptations should be applicable to most organ systems in the gastrula to early somite stage mouse embryo.

Here, we describe a protocol for whole mount immunofluorescence and image-based quantitative volumetric analysis of early stage mouse embryos. We present this technique as a powerful approach to qualitatively and quantitatively assess cardiac structures during development, and propose that it may be widely adaptable to other organ systems.

小鼠胚胎心肌内祖群的定量全贴免疫荧光分析

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development ...

Three-dimensional Rendering and Analysis of Immunolabeled, Clarified Human Placental Villous Vascular Networks

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that makes up much of the parenchyma of the human placenta. The distal villous capillary network is the terminus of the fetal blood supply after several generations of branching of vessels extending out from the umbilical cord. This network has a contiguous cellular sheath, the syncytial trophoblast barrier layer, which prevents mixing of fetal blood and the maternal blood in which it is continuously bathed. Insults to the integrity of the placental capillary network, occurring in disorders such as maternal diabetes, hypertension and obesity, have consequences that present serious health risks for the fetus, infant, and adult. To better define the structural effects of these insults, a protocol was developed for this study that captures capillary network structure on the order of 1 - 2 mm3 wherein one can investigate its topological features in its full complexity. To accomplish this, clusters of terminal villi from placenta are dissected, and the trophoblast layer and the capillary endothelia are immunolabeled. These samples are then clarified with a new tissue clearing process which makes it possible to acquire confocal image stacks to z- depths of ~1 mm. The three-dimensional renderings of these stacks are then processed and analyzed to generate basic capillary network measures such as volume, number of capillary branches, and capillary branch end points, as validation of the suitability of this approach for capillary network characterization.

This study presents a protocol for the reversible tissue clearing, immunostaining, 3D-rendering and analysis of vascular networks in human placenta villi samples on the order of 1 - 2 mm3.

Immunolabeled, 澄清人胎盘长柔毛血管网络的三维渲染与分析

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that ...

Three-dimensional Inflammatory Human Tissue Equivalents of Gingiva

Periodontal diseases (such as gingivitis and periodontitis) are the leading causes of tooth loss in adults. Inflammation in gingiva is the fundamental physiopathology of periodontal diseases. Current experimental models of periodontal diseases have been established in various types of animals. However, the physiopathology of animal models is different from that of humans, making it difficult to analyze cellular and molecular mechanisms and evaluate new medicines for periodontal diseases. Here, we present a detailed protocol for reconstructing human inflammatory tissue equivalents of gingiva (iGTE) in vitro. We first build human tissue equivalents of gingiva (GTE) by utilizing two types of human cells, including human gingival fibroblasts (HGF) and human skin epidermal keratinocytes (HaCaT), under three-dimensional conditions. We create a wound model by using a tissue puncher to punch a hole in the GTE. Next, human THP-1 monocytes mixed with collagen gel are injected into the hole in the GTE. By adimistration of 10 ng/mL phorbol 12-myristate 13-acetate (PMA) for 72 h, THP-1 cells differentiated into macrophages to form inflammatory foci in GTE (iGTE) (IGTE also can be stumilated with 2 &#181;g/mL of lipopolysaccharides (LPS) for 48 h to initiate inflammation). IGTE is the first in vitro model of inflammatory gingiva using human cells with a three-dimensional architecture. IGTE reflects major pathological changes (immunocytes activition, intracellular interactions among fibryoblasts, epithelial cells, monocytes and macrophages) in periodontal diseases. GTE, wounded GTE, and iGTE can be used as versatile tools to study wound healing, tissue regeneration, inflammation, cell-cell interaction, and screen potential medicines for periodontal diseases.

The goal of the protocol is to build an inflammatory human gingiva model in vitro. This tissue model co-cultivates three types of human cells, HaCaT keratinocytes, gingival fibroblasts, and THP-1 macrophages, under three-dimensional conditions. This model can be applied to investigating periodontal diseases, such as gingivitis and periodontitis.

牙龈三维炎症人体组织当量

Periodontal diseases (such as gingivitis and periodontitis) are the leading causes of tooth loss in adults. Inflammation in gingiva is the fundamental ...

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 ...

Three-dimensional Organotypic Cultures of Vestibular and Auditory Sensory Organs

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. Moreover, although existing culture techniques allow biochemical perturbations, these methods do not provide a means to study the effects of mechanical force and tissue stiffness during development of the inner ear sensory organs. Here we describe a method for three-dimensional organotypic culture of the intact murine utricle and cochlea that overcomes these limitations. The technique for adjustment of a three-dimensional matrix stiffness described here permits manipulation of the elastic force opposing tissue growth. This method can therefore be used to study the role of mechanical forces during inner ear development. Additionally, the cultures permit virus-mediated gene delivery, which can be used for gain- and loss-of-function experiments. This culture method preserves innate hair cells and supporting cells and serves as a potentially superior alternative to the traditional two-dimensional culture of vestibular and auditory sensory organs.

Three-dimensional organotypic cultures of the murine utricle and cochlea in optically clear collagen I gels preserve innate tissue morphology, allow for mechanical stimulation through adjustment of matrix stiffness, and permit virus-mediated gene delivery.

三维脊髓前庭和听觉感官器官的培养

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. ...

Whole Mount Immunofluorescence and Follicle Quantification of Cultured Mouse Ovaries

Research in the field of mammalian reproductive biology often involves evaluating the overall health of ovaries and testes. Specifically, in females, ovarian fitness is often assessed by visualizing and quantifying follicles and oocytes. Because the ovary is an opaque three-dimensional tissue, traditional approaches require laboriously slicing the tissue into numerous serial sections in order to visualize cells throughout the entire organ. Furthermore, because quantification by this method typically entails scoring only a subset of the sections separated by the approximate diameter of an oocyte, it is prone to inaccuracy. Here, a protocol is described that instead utilizes whole organ tissue clearing and immunofluorescence staining of mouse ovaries to visualize follicles and oocytes. Compared to more traditional approaches, this protocol is advantageous for visualizing cells within the ovary for numerous reasons: 1) the ovary remains intact throughout sample preparation and processing; 2) small ovaries, which are difficult to section, can be examined with ease; 3) cellular quantification is more readily and accurately achieved; and 4) the whole organ imaged.

Here, we present a protocol to quantify follicles in cultured ovaries of young mice without serial sectioning. Using whole organ immunofluorescence and tissue clearing, physical sectioning is replaced with optical sectioning. This method of sample preparation and visualization maintains organ integrity and facilitates automated quantification of specific cells.

培养小鼠卵巢的全贴装免疫荧光和卵泡定量研究

Research in the field of mammalian reproductive biology often involves evaluating the overall health of ovaries and testes. Specifically, in females, ...

A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical applications. The conventional isolation method of human keratinocytes involves a two-step sequential enzymatic digestion procedure, which has been proven to be inefficient in generating primary cells from adult tissues due to the low cell recovery rate and reduced cell viability. We recently reported an advanced method to isolate human primary epidermal progenitor cells from skin tissues that utilizes the Rho kinase inhibitor Y-27632 in the medium. Compared with the traditional protocol, this new method is simpler, easier, and less time-consuming, and increases epithelial stem cell yield and enhances their stem cell characteristics. Moreover, the new methodology does not require the separation of the epidermis from the dermis, and, therefore, is suitable for isolating cells from different types of adult tissues. This new isolation method overcomes the major shortcomings of conventional methods and is more suitable for producing large numbers of epidermal cells with high potency both for laboratory and for clinical applications. Here, we describe the new method in detail.

Here we present a protocol to efficiently isolate primary human keratinocytes from adult skin tissues. This method simplifies the conventional procedure by using the ROCK Inhibitor Y-27632 in the inoculation medium to spontaneously separate epidermal cells from dermal cells.

一种简便有效的成人皮肤组织中人角质形成细胞的分离方法

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical ...