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>

<ol>
	<li>Plikus, M. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+dermal+BMP+signalling+regulates+stem+cell+activation+during+hair+regeneration.">Cyclic dermal BMP signalling regulates stem cell activation during hair regeneration.</a> Nature. 451 (7176), 340-344 (2008).</li><li>Zhang, Y. V., Cheong, J., Ciapurin, N., McDermitt, D. J., Tumbar, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+self-renewal+and+differentiation+phases+in+the+niche+of+infrequently+dividing+hair+follicle+stem+cells.">Distinct self-renewal and differentiation phases in the niche of infrequently dividing hair follicle stem cells.</a> Cell Stem Cell. 5 (3), 267-278 (2009).</li><li>Jensen, U. B., Lowell, S., Watt, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+spatial+relationship+between+stem+cells+and+their+progeny+in+the+basal+layer+of+human+epidermis:+a+new+view+based+on+whole-mount+labelling+and+lineage+analysis.">The spatial relationship between stem cells and their progeny in the basal layer of human epidermis: a new view based on whole-mount labelling and lineage analysis.</a> Development. 126 (11), 2409-2418 (1999).</li><li>Braun, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manipulation+of+stem+cell+proliferation+and+lineage+commitment:+visualisation+of+label-retaining+cells+in+wholemounts+of+mouse+epidermis.">Manipulation of stem cell proliferation and lineage commitment: visualisation of label-retaining cells in wholemounts of mouse epidermis.</a> Development. 130 (21), 5241-5255 (2003).</li><li>Driskell, R. R., Giangreco, A., Jensen, K. B., Mulder, K. W., Watt, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sox2-positive+dermal+papilla+cells+specify+hair+follicle+type+in+mammalian+epidermis.">Sox2-positive dermal papilla cells specify hair follicle type in mammalian epidermis.</a> Development. 136 (16), 2815-2823 (2009).</li><li>Niculescu, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+ablation+of+integrin+alpha-6+in+mouse+epidermis+leads+to+skin+fragility+and+inflammation.">Conditional ablation of integrin alpha-6 in mouse epidermis leads to skin fragility and inflammation.</a> Eur J Cell Biol. 90 (2-3), 270-277 (2011).</li><li>Fujiwara, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+basement+membrane+of+hair+follicle+stem+cells+is+a+muscle+cell+niche.">The basement membrane of hair follicle stem cells is a muscle cell niche.</a> Cell. 144 (4), 577-589 (2011).</li><li>Desciak, E. B., Maloney, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artifacts+in+frozen+section+preparation.">Artifacts in frozen section preparation.</a> Dermatol Surg. 26 (5), 500-504 (2000).</li><li>Driskell, R., Jahoda, C. A., Chuong, C. M., Watt, F., Horsley, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+dermal+adipose+tissue.">Defining dermal adipose tissue.</a> Exp Dermatol. 23 (9), 629-631 (2014).</li><li>Driskell, R. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+fibroblast+lineages+determine+dermal+architecture+in+skin+development+and+repair.">Distinct fibroblast lineages determine dermal architecture in skin development and repair.</a> Nature. 504 (7479), 277-281 (2013).</li><li>Snippert, H. J., Schepers, A. G., Delconte, G., Siersema, P. D., Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Slide+preparation+for+single-cell-resolution+imaging+of+fluorescent+proteins+in+their+three-dimensional+near-native+environment.">Slide preparation for single-cell-resolution imaging of fluorescent proteins in their three-dimensional near-native environment.</a> Nat Protoc. 6 (8), 1221-1228 (2011).</li><li>Sada, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+the+cellular+lineage+hierarchy+in+the+interfollicular+epidermis+of+adult+skin.">Defining the cellular lineage hierarchy in the interfollicular epidermis of adult skin.</a> Nat Cell Biol. 18 (6), 619-631 (2016).</li><li>Chung, K., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CLARITY+for+mapping+the+nervous+system.">CLARITY for mapping the nervous system.</a> Nat Methods. 10 (6), 508-513 (2013).</li><li>Tetteh, P. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replacement+of+Lost+Lgr5-Positive+Stem+Cells+through+Plasticity+of+Their+Enterocyte-Lineage+Daughters.">Replacement of Lost Lgr5-Positive Stem Cells through Plasticity of Their Enterocyte-Lineage Daughters.</a> Cell Stem Cell. 18 (2), 203-213 (2016).</li></ol>

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。

<table border="0" cellpadding="0" cellspacing="0" width="658">
	<tbody>
		<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

Universidad de Cartagena UDEC

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

High-resolution Episcopic Microscopy (HREM) - Simple and Robust Protocols for Processing and Visualizing Organic Materials

We provide simple protocols for generating digital volume data with the high-resolution episcopic microscopy (HREM) method. HREM is capable of imaging organic materials with volumes up to 5 x 5 x 7 mm3 in typical numeric resolutions between 1 x 1 x 1 and 5 x 5 x 5 &#181;m3. Specimens are embedded in methacrylate resin and sectioned on a microtome. After each section an image of the block surface is captured with a digital video camera that sits on the phototube connected to the compound microscope head. The optical axis passes through a green fluorescent protein (GFP) filter cube and is aligned with a position, at which the bock holder arm comes to rest after each section. In this way, a series of inherently aligned digital images, displaying subsequent block surfaces are produced. Loading such an image series in three-dimensional (3D) visualization software facilitates the immediate conversion to digital volume data, which permit virtual sectioning in various orthogonal and oblique planes and the creation of volume and surface rendered computer models. We present three simple, tissue specific protocols for processing various groups of organic specimens, including mouse, chick, quail, frog and zebra fish embryos, human biopsy material, uncoated paper and skin replacement material.

High-resolution Episcopic Microscopy HREM - Simple and Robust Protocols for Processing and Visualizing Organic Materials

We provide simple and robust protocols for processing biopsy material of various species, embryos of biomedical model organisms and samples of other organic tissues in order to permit digital volume data generation with the high-resolution episcopic microscopy method.

高分辨率显微镜（HREM） - 用于处理和可视化有机材料的简单和可靠的协议

We provide simple protocols for generating digital volume data with the high-resolution episcopic microscopy (HREM) method. HREM is capable of imaging ...

科研

发育生物学

Multimodal Hierarchical Imaging of Serial Sections for Finding Specific Cellular Targets within Large Volumes

Targeting specific cells at ultrastructural resolution within a mixed cell population or a tissue can be achieved by hierarchical imaging using a combination of light and electron microscopy. Samples embedded in resin are sectioned into arrays consisting of ribbons of hundreds of ultrathin sections and deposited on pieces of silicon wafer or conductively coated coverslips. Arrays are imaged at low resolution using a digital consumer like smartphone camera or light microscope (LM) for a rapid large area overview, or a wide field fluorescence microscope (fluorescence light microscopy (FLM)) after labeling with fluorophores. After post-staining with heavy metals, arrays are imaged in a scanning electron microscope (SEM). Selection of targets is possible from 3D reconstructions generated by FLM or from 3D reconstructions made from the SEM image stacks at intermediate resolution if no fluorescent markers are available. For ultrastructural analysis, selected targets are finally recorded in the SEM at high-resolution (a few nanometer image pixels). A ribbon-handling tool that can be retrofitted to any ultramicrotome is demonstrated. It helps with array production and substrate removal from the sectioning knife boat. A software platform that allows automated imaging of arrays in the SEM is discussed. Compared to other methods generating large volume EM data, such as serial block-face SEM (SBF-SEM) or focused ion beam SEM (FIB-SEM), this approach has two major advantages: (1) The resin-embedded sample is conserved, albeit in a sliced-up version. It can be stained in different ways and imaged with different resolutions. (2) As the sections can be post-stained, it is not necessary to use samples strongly block-stained with heavy metals to introduce contrast for SEM imaging or render the tissue blocks conductive. This makes the method applicable to a wide variety of materials and biological questions. Particularly prefixed materials e.g., from biopsy banks and pathology labs, can directly be embedded and reconstructed in 3D.

This protocol targets specific cells in tissue for imaging at nanoscale resolution using a scanning electron microscope (SEM). Large numbers of serial sections from resin-embedded biological material are first imaged in a light microscope to identify the target and then in a hierarchical manner in the SEM.

用于在大容量中查找特定细胞目标的串行剖面的多模式分层成像

Targeting specific cells at ultrastructural resolution within a mixed cell population or a tissue can be achieved by hierarchical imaging using a ...

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 ...

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

Skin is a highly heterogeneous tissue. Intra-dermal structures include hair follicles, arrector pili muscles, epidermal specializations (such as Merkel cell clusters), sebaceous glands, nerves and nerve endings, and capillaries. The spatial arrangement of these structures is tightly controlled on a microscopic scale - as seen, for example, in the orderly arrangement of cell types within a single hair follicle - and on a macroscopic scale - as seen by the nearly identical orientations of thousands of hair follicles within a local region of skin. Visualizing these structures without physically sectioning the skin is possible because of the 2-dimensional geometry of this organ. In this protocol, we show that mouse skin can be dissected, fixed, permeabilized, stained, and clarified as an intact two dimensional object, a flat mount. The protocol allows for easy visualization of skin structures in their entirety through the full thickness of large areas of skin by optical sectioning and reconstruction. Images of these structures can also be integrated with information about position and orientation relative to the body axes.

Mammalian skin contains a diverse array of structures - such as hair follicles and nerve endings - that exhibit distinctive patterns of spatial organization. Analyzing skin as a flat mount takes advantage of the 2-dimensional geometry of this tissue to produce full-thickness high-resolution images of skin structures.

小鼠皮肤及其毛囊图案和感觉轴突形态分析中的应用平山成像

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

神经科学

Three-Dimensional Imaging of Tumor-Bearing Tissue Using the Iterative Bleaching Extends Multiplexity Approach

The spatial heterogeneity of the tumor microenvironment (TME) is a critical determinant of therapeutic response, particularly for immune-oncology agents, where success depends on the distribution of specific immune cell subpopulations. Over the past decade, multiple sophisticated technologies have been introduced to achieve detailed resolution of the TME using two-dimensional sections from either formalin-fixed, paraffin-embedded (FFPE) or fixed-frozen tissues. While these thin sections are easier to procure and analyze, they lack the three-dimensional architecture needed to reliably and comprehensively characterize a tumor. To address this limitation, a tissue mounting and imaging technique was developed to enable the three-dimensional analysis of tumor lesions in their native in vivo state. This protocol outlines the procurement of human tumor tissue, the mounting of samples on custom-printed platforms, and staining procedures for post-fixation samples. The multiplexed immunofluorescence technique, IBEX (Iterative Bleaching Extends Multiplexity), was adapted to characterize the three-dimensional TME with up to 15 markers for tumor, immune, and stromal cells using commercially available antibodies. Imaging depths of up to 100 &#181;m were achieved using an inverted white-light laser confocal microscope with a custom-printed imaging adaptor and commercial glass-bottom dishes to ensure optimal tissue orientation. This protocol highlights the potential of the IBEX method to expand multiplexed immunofluorescence studies, providing a more comprehensive understanding of TME composition.

This article describes a protocol for multiplex immunofluorescence optimized to characterize the three-dimensional architecture of peritoneal metastases.

The spatial heterogeneity of the tumor microenvironment (TME) is a critical determinant of therapeutic response, particularly for immune-oncology agents, ...

癌症研究

Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in every tissue to be identified and characterized. Our laboratory is actively working to make technological advances in X-ray micro-computed tomography (micro-CT) that will bring the diagnostic power of histology to the study of full tissue volumes at cellular resolution (i.e., an X-ray Histo-tomography modality). Toward this end, we have made targeted improvements to the sample preparation pipeline. One key optimization, and the focus of the present work, is a straightforward method for rigid embedding of fixed and stained millimeter-scale samples. Many of the published methods for sample immobilization and correlative micro-CT imaging rely on placing the samples in paraffin wax, agarose, or liquids such as alcohol. Our approach extends this work with custom procedures and the design of a 3-dimensional printable apparatus to embed the samples in an acrylic resin directly into polyimide tubing, which is relatively transparent to X-rays. Herein, sample preparation procedures are described for the samples from 0.5 to 10 mm in diameter, which would be suitable for whole zebrafish larvae and juveniles, or other animals and tissue samples of similar dimensions. As proof of concept, we have embedded the specimens from Danio, Drosophila, Daphnia, and a mouse embryo; representative images from 3-dimensional scans for three of these samples are shown. Importantly, our methodology leads to multiple benefits including rigid immobilization, long-term preservation of laboriously-created resources, and the ability to re-interrogate samples.

We developed protocols and designed a custom apparatus to enable embedding of millimeter-scale specimens. We present sample preparation procedures with an emphasis on embedding in acrylic resin and polyimide tubing to achieve rigid immobilization and long-term storage of specimens for the interrogation of tissue architecture and cell morphology by micro-CT.

用微计算机断层扫描法对无节段3D 组织学进行固定染色、全尺寸、毫米度试样的刚性嵌入

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in ...

生物工程

Generation of Self-assembled Vascularized Human Skin Equivalents

Human skin equivalents (HSEs) are tissue engineered constructs that model epidermal and dermal components of human skin. These models have been used to study skin development, wound healing, and grafting techniques. Many HSEs continue to lack vasculature and are additionally analyzed through post-culture histological sectioning which limits volumetric assessment of the structure. Presented here is a straightforward protocol utilizing accessible materials to generate vascularized human skin equivalents (VHSE); further described are volumetric imaging and quantification techniques of these constructs. Briefly, VHSEs are constructed in 12 well culture inserts in which dermal and epidermal cells are seeded into rat tail collagen type I gel. The dermal compartment is made up of fibroblast and endothelial cells dispersed throughout collagen gel. The epidermal compartment is made up of keratinocytes (skin epithelial cells) that differentiate at the air-liquid interface. Importantly, these methods are customizable based on needs of the researcher, with results demonstrating VHSE generation with two different fibroblast cell types: human dermal fibroblasts (hDF) and human lung fibroblasts (IMR90s). VHSEs were developed, imaged through confocal microscopy, and volumetrically analyzed using computational software at 4- and 8-week timepoints. An optimized process to fix, stain, image, and clear VHSEs for volumetric examination is described. This comprehensive model, imaging, and analysis techniques are readily customizable to the specific research needs of individual labs with or without prior HSE experience.

The goal of this protocol is to describe the generation and volumetric analysis of vascularized human skin equivalents using accessible and simple techniques for long term culture. To the extent possible, the rationale for steps is described to allow researchers the ability to customize based on their research needs.

自组装血管化人体皮肤等价物的生成

Human skin equivalents (HSEs) are tissue engineered constructs that model epidermal and dermal components of human skin. These models have been used to ...

Reconstruction of 3-Dimensional Histology Volume and its Application to Study Mouse Mammary Glands

Histology volume reconstruction facilitates the study of 3D shape and volume change of an organ at the level of macrostructures made up of cells. It can also be used to investigate and validate novel techniques and algorithms in volumetric medical imaging and therapies. Creating 3D high-resolution atlases of different organs1,2,3 is another application of histology volume reconstruction. This provides a resource for investigating tissue structures and the spatial relationship between various cellular features. We present an image registration approach for histology volume reconstruction, which uses a set of optical blockface images. The reconstructed histology volume represents a reliable shape of the processed specimen with no propagated post-processing registration error. The Hematoxylin and Eosin (H&#38;E) stained sections of two mouse mammary glands were registered to their corresponding blockface images using boundary points extracted from the edges of the specimen in histology and blockface images. The accuracy of the registration was visually evaluated. The alignment of the macrostructures of the mammary glands was also visually assessed at high resolution.
This study delineates the different steps of this image registration pipeline, ranging from excision of the mammary gland through to 3D histology volume reconstruction. While 2D histology images reveal the structural differences between pairs of sections, 3D histology volume provides the ability to visualize the differences in shape and volume of the mammary glands.

We present an image registration approach for 3-dimensional (3D) histology volume reconstruction, which facilitates the study of the changes of an organ at the level of macrostructures made up of cells . Using this approach, we studied the 3D changes between wild-type and Igfbp7-null mammary glands.

3维组织学音量和其研究小鼠乳腺腺体的应用改造

Histology volume reconstruction facilitates the study of 3D shape and volume change of an organ at the level of macrostructures made up of cells. It can ...

生物学

通过荧光报告基因定量进行全器官病原体追踪

High&#45;Resolution Three&#45;Dimensional Whole&#45;Organ Tomography of Microbial Infections

Most infections take place within three-dimensional host tissues with intricate anatomy and locally varying host physiology. The positioning of pathogen cells within this diverse environment significantly affects their stress levels, responses, fate, and contribution to the overall progression of the disease and treatment failure. However, due to the technical difficulties in locating &#181;m-sized pathogen cells within cm-sized host organs, this area of research has been relatively unexplored. Here, we present a method for addressing this challenge. We employ serial two-photon tomography and AI-enhanced image analysis to locate individual Salmonella cells throughout the entire spleen, liver lobes, and whole lymph nodes of infected mice. Using fluorescent reporters and in vivo antibody administration, the replication rate of single Salmonella cells, their local interaction with specific immune cells, and bacterial responses to antibiotics can be determined. These methodologies open avenues for a comprehensive examination of infections, their prevention, and treatment within the three-dimensional tissue context.

作者聚焦:揭示细菌对组织中抗生素和免疫系统的反应

Here, we describe a procedure that enables organ-wide detection of pathogenic bacteria during infection and quantification of fluorescent reporter activities.

微生物感染的高分辨率三维全器官断层扫描

Most infections take place within three-dimensional host tissues with intricate anatomy and locally varying host physiology. The positioning of pathogen ...

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

摘要

探索更多视频

此视频中的章节

高分辨率显微镜（HREM） - 用于处理和可视化有机材料的简单和可靠的协议

用于在大容量中查找特定细胞目标的串行剖面的多模式分层成像

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

小鼠皮肤及其毛囊图案和感觉轴突形态分析中的应用平山成像

Three-Dimensional Imaging of Tumor-Bearing Tissue Using the Iterative Bleaching Extends Multiplexity Approach

用微计算机断层扫描法对无节段3D 组织学进行固定染色、全尺寸、毫米度试样的刚性嵌入

自组装血管化人体皮肤等价物的生成

3维组织学音量和其研究小鼠乳腺腺体的应用改造

微生物感染的高分辨率三维全器官断层扫描

微生物感染的高分辨率三维全器官断层扫描