This research was supported by the Brain Research Program through the National Research Foundation (NRF) funded by the Korean Ministry of Science, ICT, and Future Planning (NRF-2015M3C7A1028790).

动物实验必须遵守所有相关的政府和机构的规定。 1.试剂的制备<ol><li>对于水凝胶单体溶液（A4P0）:加入20克丙烯酰胺，以450毫升0.1×磷酸盐缓冲盐水的（PBS）中，并调整体积至500毫升0.1×PBS中。 注意:丙烯酰胺单体是有毒的。个人防护装备执行在通风橱中的所有程序，包括面罩，实验室外套，手套和封闭趾鞋。 <ol><li>使用，在50mL锥形管在通风橱下加入100毫克的2,2&#39;-偶氮二[2-（2-咪唑啉-2-基）丙烷]二盐酸盐，以40毫升的水凝胶单体溶液（A4P0）的前立即。 </li></ol></li><li>对于电泳组织清算（ETC）的缓冲:加40克十二烷基硫酸钠（SDS）和12.37克硼酸800毫升去离子H 2 O（卫生署2 O）的。将pH调节至8.5，用NaOH和音量调整到1升额外卫生署2 O. 注意:SDS是一种有害化学;因此，小心处理。 </li><li>为立方贴装溶液:加入250克蔗糖125克尿素和125克N，N，N&#39;，N&#39;，N&#39;-四（2-羟丙基）乙二胺至150毫升的dh 2 O和使体积500毫升卫生署2 O. </li></ol> 2.样品制备和固定<ol><li>鼠标安乐死。 <ol><li>准备在同一注射器alfaxalone（1毫克/千克）和赛拉嗪（0.5毫克/千克）。 </li><li>腹膜内注射alfaxalone和甲苯噻嗪的混合物，并允许鼠标​​3分钟以不省人事。 </li><li>等到麻醉的老鼠不再响应痛苦的刺激，如一条尾巴捏，然后再继续。 注意:按照处理动物适当的机构的指导方针。 </li></ol></li><li> Transcardially灌注用100毫升的0.9％NaCl（pH值7.4-7.5）溶液中的鼠标含有肝素（100U / mL）添加到抽血后，再用100毫升的4％多聚甲醛（PFA）在1X PBS（pH为7.4）14。 注意:PFA解决方案是有毒的。的PFA溶液和所有的后续处理的制备必须在通风橱并与个人防护设备进行。 </li><li>切断鼠标的头部，打通皮肤，并打破用小剪刀两眼之间的头骨。 </li><li>删除使用小弯钳头骨碎片。 </li><li>取出脑或期望的器官。 </li><li>孵育在修复后的4％PFA溶液大脑和器官在4℃下过夜。 注:对于特定区域组织结算，剖析特定区域或修剪组织固定后的组织中。 注意:PFA解决方案是有毒的。该穿心灌注和所有后续鼠标操作必须在通风橱和个人防护装备进行。 </li></ol> 3，水凝胶单体输液和Polymerization <ol><li>孵育A4P0水凝胶单体溶液中的固定机构，在4℃下轻轻摇动12-24小时。 </li><li>水凝胶单体，注入组织聚合。 <ol><li>样品用5ml水凝胶单体溶液转移至10毫升圆底管中。总结圆底管封口膜的顶部。 </li><li>从含有由通过液体1分钟氮气鼓泡水凝胶的注入全鼠脑样品的圆底管除去氧气。迅速盖紧含有样品的试管。 </li></ol></li><li>管转移到水浴（37℃）2小时。 </li><li>用0.1X PBS洗涤简单聚合样品以除去多余的水凝胶。 注意:水凝胶的单体是有毒的。为了避免与水凝胶单体皮肤接触，在通风橱和个人防护装备，包括面罩，实验室外套和手套完成所有手续。 注:聚合组织可以在4℃储存在包含0.1×PBS中的叠氮化钠为2周以上。 注意:叠氮化钠是高度和急性毒性。在通风橱和个人防护装备执行所有程序，包括面罩，实验室外套和手套。 </li></ol> 4.电泳组织清算（ETC） <ol><li>聚合样品转移到组织容器，并将组织容器中的ETC室。 </li><li>填充用蠕动泵ETC缓冲区ETC室。 </li><li>设置ETC条件和运行ETC.使用以下设置。 <ol><li>有关整个器官ETC设置，请使用以下设置:（1.5 AMP），温度（37℃），运行时间（6.0 h）中，泵的转速（30转）。 </li><li>为1至2毫米厚的小鼠的脑切片的ETC设置，使用以下设置:当前（1.5 AMP），温度（37℃），运行时间（2.0小时），泵的转速（每分钟30转）。 </li></ol></li><li>传输ŧ他清除样品的50mL锥形管中以45毫升0.1×PBS中。用0.1X PBS洗涤清除样本几次，直到当管短暂动摇无气泡被视为（确认彻底清除SDS的）。 </li></ol> 5.免疫标记ACT加工组织的<ol><li>小鼠全脑:在含有1x PBS中，6％的牛血清白蛋白（BSA），抗体溶液3毫升体积孵育样品0.1％的Triton X-100和0.01％叠氮化钠（抗体稀释溶液）用稀释对酪氨酸羟化酶（TH）抗体4天，在37℃轻微摇动的1/500因子。取代在第2天的末端抗体溶液。 <ol><li>用45 0.1×毫升PBS中3洗样品中的50-mL锥形管 - 5小时，并改变它的缓冲器每隔一小时。 </li><li>孵育样品中的具有1/500的稀释因子抗体稀释溶液3毫升体积为驴抗兔488第二抗体4天，在37℃轻微摇动。 REPL王牌第2天的稀释抗体溶液。 </li></ol></li><li>为1至2毫米厚的切片的脑组织:孵育样品过夜或1天中有37的1/500的稀释因子为IV型胶原抗体的0.5至1毫升体积的抗体稀释溶液°下用温和的晃动。 <ol><li>用0.1×PBS的1-2小时的12毫升体积洗样品中的15毫升锥形管中。更改缓冲区每隔一小时。 </li><li>孵育样品中的1/500的Cy3缀合的抗兔次级抗体过夜或1天，在37℃轻微摇动的稀释因子0.5至1毫升体积的抗体稀释溶液。 </li></ol></li><li>洗样品中0.1倍PBS 3 - 5小时;更改缓冲区每隔一小时。 </li><li>成像之前，孵育在立方贴装溶液适量的样品，在室温下轻轻摇动1小时。用新鲜的CUBIC安装解决方案替换和孵育一个额外的1小时。 注意: </b&gt; 76型抗体的小鼠ACT处理的组织工作良好，其中包括31单克隆抗体14。预标记的组织可以用两个以上的荧光染料中的ACT-加工的组织的免疫标记进行组合。 </li></ol> 6.密集组织的免疫标记（PRESTO） <ol><li> C-PRESTO 注:C-PRESTO适合小型的样品，例如全睾丸，整个肾脏，或更大的器官的小部分。 <ol><li>将样品转移到1.5毫升的管中，具有1/500为IV型胶原抗体的稀释因子添加的抗体稀释液500微升，并在600×g下离心反应管2小时。 </li><li>离心在600×g下洗涤用0.1×PBS中染色样品30分钟。 </li><li>同的1/500的Cy3缀合的抗兔次级抗体和离心机的稀释因子，在600×g下添加的抗体稀释液500微升2小时。 </li><li> 0洗染色样品。1×PBS中通过在600×g的30分钟离心。 </li></ol></li><li> S-PRESTO 注:S-PRESTO是适合较大的组织。 <ol><li>准备的注射器（30毫升体积）并将其连接到一个三通阀（ 图1A）。胶用胶枪的高压条件下（ 图1B）的阀。 </li><li>将样品转移到三通阀连接注射器在关闭阀位置。 </li><li>加入5 - 7毫升抗体稀释溶液的1/500为IV型胶原抗体的稀释因子。通过打开阀释放抗体溶液进入样品。 </li><li>设定注射器活塞以17毫升位置，以提供足够的空间用于输注/抽出运动。这可以在开阀位置来完成。注射器设置完成后，关闭三通阀。 </li><li>把含有在注射器泵的样品的注射器。设定注射器泵的条件为10毫升/分钟的输注/抽出容积和4-分暂停时间连续循环模式（ 图1C）。 注:泵注入，直到它到达目标体积（10毫升），然后的流量的变化的简短暂停（4分钟）后的方向。 </li><li>运行3注射泵-在室温下（ 图1F）24小时。 </li><li>打开三通阀和替换0.1倍的PBS溶液中。 </li><li>两次用0.1×PBS中，每次使用注射器泵洗涤染色样品1小时。 </li><li>用含有的Cy3缀合的抗兔二抗（1/500稀释因子）抗体稀释溶液改变和运行注射器泵3 - 24小时。 </li><li>打开三通阀和替换0.1倍的PBS溶液中。 </li></ol></li><li>成像之前，孵育在立方贴装溶液适量染色样品在室温下轻轻摇动1小时。用新鲜的CUBIC安装解决方案替换和孵育一个额外的1小时。 </li></ol> 7.我maging <ol><li>放置标记组织共焦菜，直到组织覆盖添加CUBIC安装解决方案。放置在组织上盖玻片。使用激光共聚焦显微镜图像组织14 10倍的目标之下。 </li></ol>

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作者宣称，他们没有竞争的经济利益。

差固定或水凝胶浸入到组织可导致蛋白的损失和组织中的组织结算处理的失真。整个成年小鼠脑应在4％的多聚甲醛孵育过夜，然后浸没在20毫升12水凝胶单体溶液的最小体积 - 轻轻摇动18小时。对于大的组织中，包括人体组织，如脑切片和脊髓，扩展需要在水凝胶单体溶液中浸泡的时间。因为组织固定和聚合物注入步骤分开的ACT协议是很容易适用于固定的组织的清算。组织清理后，人体组织进行精心染色与几个抗体。需要注意的是对ACT结算技术不是用酒精固定剂，诸如乙醇和甲醇兼容。 该组织的水凝胶的聚合步骤也很关键生产高质量的组织下清算过程。因为氧抑制丙烯酰胺的聚合，它应该通过脱气在氮气气体注入系统的含有组织的溶液中除去。或者，脱氧化可以利用真空室23小时的热块来执行。 信息交换速率取决于许多因素，包括器官大小，脂质或ECM纤维蛋白的含量，定影条件等多数成年小鼠组织可以由ETC过夜清零。然而，存在的风险不必要过度清算和组织肿胀;由此，在结算时的差异是重要的考虑因素。组织清除条件应根据经验确定。重要的是，ECM的内容可能会影响清除组织的外观。组织的颜色仍然是白色或不透明的，甚至在ETC缓冲区中的ACT之后，因为法案没有明确密集的蛋白质纤维。然而，当这些器官浸渍于RI匹配溶液，该Ÿ变得透明。 组织肿胀率似乎是依赖于组织的细胞外基质的内容，和柔软的器官，如脑，表现出比稠密器官更大溶胀比。因为增加的组织肿胀将有助于组织清除程序，ACT常规利用蒸馏水水性缓冲在大多数情况下。在某些情况下，当组织肿胀或瞬态畸形是不可取的，如在胚，含缓冲液0.1×PBS可以应用于高分辨率成像。还应当指出的是在缓冲器中较高的盐浓度会导致组织的收缩。 act方法可以澄清一个鼠标14的整个器官，甚至全身。然而，透明组织的深层组织成像需要特殊的显微镜和目标。因此，脑组织1到2毫米厚的是最有效的用于与常规共焦显微镜成像。在我们的手中，去除标签从ACT处理的组织编着的抗体是抗体依赖的，因此不推荐使用不同的抗体标记的回合。一些抗体，如TH和GFAP，工作特别好为全脑免疫标记14。另一方面，一些抗体，如TUJ1或地图2，通常标组织的唯一的表面上。这可以通过其他方法，如开关15得到改善。另一个潜在的局限性在于它可能很难保持精蛋白结构中的ACT-加工的组织，因为在组织清除步骤的组织肿胀和收缩。 该PRESTO技术适用于多种组织标记技术。例如，在使用预标记的组织如SeeDB 4和iDISCO 7组织透明的方法，致密组织的免疫标记需要比数天至数周更长的潜伏期。然而，PRESTO可以缩短孵化时间为几个小时，Enabling用1毫米厚的致密组织的一天内的整个过程的完成。 PRESTO可以增强深标记厚，致密组织，或甚至未清除厚组织的功效。因为PRESTO使用台式离心机或注射器泵，并且不需要任何特殊的设备，这是比较容易实现与常规实验室程序这种技术。

一个在神经科学挑战是神经元电路布线和完整的脑组织内各个单元的可视化。直到最近，这表明这样的神经元之间的连接需要1）组织连续切片; 2）具体的目标，如轴突或蛋白质分子标记;和3）通过经由计算登记或2D序列图像1的对准三维重建整个大脑的可视化。这些步骤是费力的，需要大量时间，并且容易切片和标签，使神经网络映射非常困难的过程中丢失信息。然而，许多方法，允许对完整的组织，而不切片的可视化得到了发展。生物组织可以通过组织清除技术2-10进行光学透明的。其中一个主要的方法是减少完整的组织，并且为了浸渍溶液之间的折射率差异以减少在完整的组织的光散射，从而使该组织透明和有利的深层结构的观察。某些类型的浸没溶液的具有疏水性能，其脱水过程中导致快速荧光猝灭。因此，这些方法都没有超过的时间11,12长时间荧光成像相容。代替疏水试剂，其他方法用于组织清算亲水试剂例如SeeDB 4和SCA L E 6，其保持生物组织4,6,8的结构信息和荧光。但是，大分子，包括抗体，不能由扩散单独达到完整的组织的核心。因此，在密集的包装组织深区域目标分子的预标记在技术上具有挑战性。 在最近开发的组织清除的方法，CLARITY 3，水凝胶包埋生物组织我s的丙烯酰胺形成，和类脂物通过电泳下十二烷基硫酸钠（SDS）的含液中除去。然后将样品浸入与匹配折射率的溶液，以减少光散射3。脂质清除系统先进CLARITY 13和PACT（被动清晰技术）10后来被修改。聚合后，丙烯酰胺链交联与蛋白质，形成水凝胶的组织。脂质组分不能与丙烯酰胺交联;因此，脂质可以通过电泳的含有SDS的缓冲液下消除。通过积极消除血脂，脑组织明显增加透明度9。然而，这些方法不被自由扩散处理深标签是不可能的。为了克服这一限制，需要用于试剂的主动运输成厚组织的最深部分的技术。 虽然CLARITY允许组织清算和深层组织可视化，这不是一个容易或快速的过程。它可能需要几个星期来清除整个鼠脑7,14。组织快速清除是这样的方法，为生命科学研究或体积诊断基础或临床实验室设置的应用程序是必不可少的。目前的协议通过加压辅助分娩免疫染色为生物组织间隙和随后的蛋白质检测的简化过程。它适用于通过与成像方法组合大日提交的三维数据处理系统的高分辨率体积成像。

<table><tbody><tr><td>4% Paraformaldehyde</td><td>Lugen SCI&amp;nbsp;</td><td>LGB-1175-4B</td><td>sample fixation&amp;nbsp;</td></tr><tr><td>Acrylamide</td><td>Affymetrix</td><td>75820</td><td>Hydrogel monomer solution&amp;nbsp;</td></tr><tr><td>2,2&amp;rsquo;-Azobis[2-(2-imidazolin-2-yl) propane] dihydrochloride</td><td>Wako Pure Chemical Industries&amp;nbsp;&amp;nbsp;</td><td>VA-044</td><td>Hydrogel monomer solution&amp;nbsp;</td></tr><tr><td>Boric acid</td><td>Affymetrix</td><td>76324</td><td>ETC buffer</td></tr><tr><td>Sodium Dodecyl Sulfate (SDS)</td><td>Affymetrix</td><td>18220</td><td>ETC buffer</td></tr><tr><td>Sodium hydroxide pellets</td><td>Junsei chemical</td><td>1310-73-2</td><td>ETC buffer</td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Santa Cruz Biotechnology</td><td>sc-2323A</td><td>Immunolabeling</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T8787</td><td>Immunolabeling</td></tr><tr><td>Sodium azide</td><td>Sigma-Aldrich</td><td>S2002</td><td>Immunolabeling</td></tr><tr><td>Tyrosine hydroxylase (TH)</td><td>Millipore</td><td>AB152</td><td>Antibody/immunolabeling</td></tr><tr><td>Alexa Fluor Cy3 Donkey anti-Rabbit IgG (H+L)</td><td>Jackson ImmunoResearch</td><td>711-165-152</td><td>Secondary antibody/ immunolabeling</td></tr><tr><td>Alexa Fluor 488 Donkey anti-Rabbit IgG (H+L)</td><td>Life Technologies - Molecular Probes</td><td>A21206</td><td>Secondary antibody/ immunolabeling</td></tr><tr><td>Confocal dish</td><td>SPL lifesciences</td><td>101350</td><td>Imaging</td></tr><tr><td>Confocal microscope</td><td>Leica</td><td>SP8</td><td>Imaging</td></tr><tr><td>Sucrose</td><td>Junsei chemical</td><td>31365-0301</td><td>CUBIC-mount solution&amp;nbsp;</td></tr><tr><td>Urea</td><td>Affymetrix</td><td>23036</td><td>CUBIC-mount solution&amp;nbsp;</td></tr><tr><td>&lt;em&gt;N,N,N&amp;rsquo;,N&amp;rsquo;&lt;/em&gt;-tetrakis(2-hydroxypropyl)ethylenediamine</td><td>Sigma-Aldrich</td><td>122262</td><td>CUBIC-mount solution&amp;nbsp;</td></tr><tr><td>ECT chamber</td><td>Logos Biosystems, Inc.</td><td>C10101</td><td>ETC system</td></tr><tr><td>ECT chamber controller</td><td>Logos Biosystems, Inc.</td><td>C10201</td><td>ETC system</td></tr><tr><td>Temperature probe</td><td>Logos Biosystems, Inc.</td><td>C12101</td><td>ETC system</td></tr><tr><td>Peristatic pump</td><td>Baoding longer precision pump Co., Ltd</td><td>YZ1515X</td><td>ETC system</td></tr><tr><td>Buffer reservoir</td><td>Logos Biosystems, Inc.</td><td>C10401</td><td>ETC system</td></tr><tr><td>Tissue container&amp;nbsp;</td><td>Logos Biosystems, Inc.</td><td>C12001</td><td>ETC system</td></tr><tr><td>Container holder for 1 tissue container</td><td>Logos Biosystems, Inc.</td><td>C12002</td><td>ETC system</td></tr><tr><td>Mouse brain slice holder</td><td>Logos Biosystems, Inc.</td><td>C12004</td><td>ETC system</td></tr><tr><td>Whole rat brain holder</td><td>Logos Biosystems, Inc.</td><td>C12007</td><td>ETC system</td></tr><tr><td>Peristaltic pump tubing</td><td>Logos Biosystems, Inc.</td><td>C12104</td><td>ETC system</td></tr><tr><td>Tabletop-centrifuge</td><td>Hanil science industrial Co., Ltd</td><td>MICRO 12</td><td>c-PRESTO</td></tr><tr><td>Syringe pump</td><td>Baoding longer precision pump Co., Ltd</td><td>LSP02-1B</td><td>s-PRESTO</td></tr><tr><td>Syringe (30 mL)</td><td>Korea vaccine Co., Ltd.</td><td>KV-S30</td><td>s-PRESTO</td></tr><tr><td>3-way stopcock</td><td>Hyupsung medical Co.,Ltd.</td><td>HS-T-01N</td><td>s-PRESTO</td></tr></tbody></table>

act-presto: biological tissue clearing and immunolabeling methods for volume imaging

识别和器官或细胞水平全身的具体组织的探索是生物学的基本挑战。过渡方法需要的时间和努力来获得3D图像的大量和包括切片的完整组织，免疫标记和成像串联切片组织，这在过程的每一步产生的信息的损失。在完整的组织内的高分辨率成像最近开发的方法，分子特征已经被限制蛋白质的标签。然而，对于器官结算现有协议需要相当长的处理时间，使得难以实现在实验室组织交换技术。我们最近成立了一个快速和高重复性的协议被称为ACT-PRESTO（ 一莫如çlarity 牛逼 echnique- p ressureř扬眉吐气Ëfficient和s表T转让（BOT）大分子转换为o rgans），允许在数小时内组织间隙。此外，ACT-PRESTO能够快速免疫标记与传统方法和通过施加压力或对流加速抗体渗入密集成形的，厚的样品的深层。我们描述了如何准备组织，如何通过脂质去除使用电泳清除，并通过压力辅助分娩如何免疫染色。该协议的快速性和一致性，将加快3D的组织学研究和基于卷的诊断性能。

ACT组织清算 影响组织清算的一个重要因素是组织固定。 PFA固定和丙烯酰胺注射是在这个协议单独的步骤。组织的水凝胶聚合步骤之后，自由A4P0溶液不聚合，虽然组织，注入水凝胶是交联的内源性大分子。因此，没有凝胶应该形成的组织（ 图2A）的外部。在交联少对ACT过程导致蛋白质和丙烯酰胺之间相比的净度的协议。因此，组织的水凝胶样品是更加多孔的。此功能使脂质的快速提取和大分子物质的扩散。 2小时（ 图2B），和5 - -小鼠脑1至2毫米厚1内进行充分清除的部分的ETC的6小时是足够的对于C整个老鼠大脑（ 图2C）的learance。水凝胶的介导的组织清除引起的ETC工序后的组织的膨胀，但组织返回到折射率匹配溶液原始大小。形成的ACT过程中的组织的水凝胶样品是高度多孔的，并且因此，小分子化合物和大分子能有效穿透和扩散容易（ 图3）。的1毫米的脑切片的2小时培养用酪氨酸羟化酶（TH）和IV型胶原的抗体后，用二级抗体随后2小时孵育足以标记在500微米深度多巴胺能神经元（ 图3） 。 PRESTO组织免疫标记 密器官通常具有高浓度的ECM纤维，这会影响抗体的穿透进入组织的。因此，抗体penet额定的只有20-30微米的致密组织，如肾组织的深度，孵育12小时后。为了克服这一限制，我们设计了活性大分子渗透方法，使递送试剂到深部组织，即PRESTO（压力相关的大分子的有效和稳定的转印到器官）（ 图4）。相比ACT处理肾组织用抗体溶液，离心力（600 XG）用台式离心机的应用（离心PRESTO和c-PRESTO）大大提高抗体递送到深部组织（ 图5）的静态培养。当我们应用了C-PRESTO过程致密的组织，培养3小时足以标签250到300微米的深层结构。为了改善无显著的组织损伤的组织的失真，我们还设计了一个机提供与注射器泵（注射器PRESTO，S-PRESTO）对流。这一过程同样提高抗体PEnetration相比被动扩散（ 图5）。 <img alt="图1" src="/files/ftp_upload/54904/54904fig1.jpg" /> 图1.制备S-PRESTO设备。答 :注射器（30毫升）和三通阀。 B的三路活塞相连并粘到注射器。 C.含有样品和抗体溶液的注射器，并放置在注射器泵的注射器。 D.注射泵和工作条件设置。 E的泵注入含有标记试剂到样品溶液中。当达到指定体积，该泵送方向的变化和撤回的时间的指定时间后的溶液。 <a href="http://ecsource.jove.com/files/ftp_upload/54904/54904fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 鼠脑图2. ACT组织清算。 A.聚合后，自由A4P0解决方案并不聚合;组织，注入的水凝胶是由交联与内源性大分子凝胶化。 B. 1-至2毫米厚的脑切片进行1被清除- 2小时，和扩展及大小恢复在反射率的程度（RI）-matching溶液进行记录。 C.通过全鼠脑的厚度为约0.8厘米，和5 - ETC 6小时就足够了到大脑完全清楚。 ETC的3小时后，出现了非清除组织的显著量。通过ETC 6小时，但是，已经发生的整个大脑的清除。在ETC缓冲器ACT后组织的颜色为白色或不透明的。由RI调整，组织变得透明。 <a href="http://ecsource.jove.com/files/ftp_upload/54904/54904fig2large.jpg" target="_blank">请点击她的</a>E要查看此图的放大版本。 <img alt="图3" src="/files/ftp_upload/54904/54904fig3.jpg" /> 图3.免疫标记与ACT-清除小鼠脑组织。 ACT-处理后的1毫米小鼠脑切片示出与抗体至IV和与抗体染色中脑的一部分型胶原染色小鼠大脑皮质的图像酪氨酸羟化酶（TH）。比例尺，100微米。 <a href="http://ecsource.jove.com/files/ftp_upload/54904/54904fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54904/54904fig4.jpg" /> 图4. PRESTO免疫标记方法。抗体不能渗透到致密组织中扩散。 PRESTO免疫标记的方法被设计为主动注入大分子和递送试剂到致密的组织。使用标准的台式离心机（离心魄力，C-PRESTO）离心力的应用显着地促进抗体的交付。 Appling与提高抗体的渗透注射泵（注射器魄力，S-PRESTO）对流。 <a href="http://ecsource.jove.com/files/ftp_upload/54904/54904fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54904/54904fig5.jpg" /> 图5. ACT-清除致密组织PRESTO免疫标记。对c-PRESTO，组织在600 xg离心离心以加速初级和次级抗体的穿透使用标准台式离心机3小时。对于s-PRESTO，注射器泵用于递送抗体。小鼠器官，如肾脏和肝脏，分别标有第IV型胶原。与传统方法相比，3小时C或S-PRESTO显着增强了标签的深度。在肾脏和肝脏，Z轴的深度达到300 - PRESTO样品（右）在350微米或更深，而对照样品在只有40的深度标记 - 50微米（左）。用共聚焦显微镜获得的三维重建图像。比例尺，100微米。 <a href="http://ecsource.jove.com/files/ftp_upload/54904/54904fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about ACT-PRESTO: Biological Tissue Clearing and Immunolabeling Methods for Volume Imaging at JoVE.com

ACT-PRESTO: Biological Tissue Clearing and Immunolabeling Methods for Volume Imaging

ACT-PRESTO（大分子活性清晰度技术压力相关的有效和稳定的转印到器官）实现快速，高效，但廉价的组织清算和快速抗体渗透到通过被动扩散或压力辅助输送清除组织。使用这种方法，广泛的组织中可以清除，由多个抗体免疫标记，和体积成像。

ACT-PRESTO:生物组织清算和体积成像方法免疫标记

The identification and exploration of the detailed organization of organs or of the whole body at the cellular level are fundamental challenges in ...

act-presto-biological-tissue-clearing-immunolabeling-methods-for

Research

JoVE Journal

Biology

12.2K 次观看.  Korea University College of Medicine.  ACT-PRESTO（大分子活性清晰度技术压力相关的有效和稳定的转印到器官）实现快速，高效，但廉价的组织清算和快速抗体渗透到通过被动扩散或压力辅助输送清除组织。使用这种方法，广泛的组织中可以清除，由多个抗体免疫标记，和体积成像。 

视频: ACT-PRESTO: Biological Tissue Clearing and Immunolabeling Methods for Volume Imaging

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 ...

科研

发育生物学

Imaging Cleared Intact Biological Systems at a Cellular Level by 3DISCO

Tissue clearing and subsequent imaging of transparent organs is a powerful method to analyze fluorescently labeled cells and molecules in 3D, in intact organs. Unlike traditional histological methods, where the tissue of interest is sectioned for fluorescent imaging, 3D imaging of cleared tissue allows examination of labeled cells and molecules in the entire specimen. To this end, optically opaque tissues should be rendered transparent by matching the refractory indices throughout the tissue. Subsequently, the tissue can be imaged at once using laser-scanning microscopes to obtain a complete high-resolution 3D image of the specimen. A growing list of tissue clearing protocols including 3DISCO, CLARITY, Sca/e, ClearT2, and SeeDB provide new ways for researchers to image their tissue of interest as a whole. Among them, 3DISCO is a highly reproducible and straightforward method, which can clear different types of tissues and can be utilized with various microscopy techniques. This protocol describes this straightforward procedure and presents its various applications. It also discusses the limitations and possible difficulties and how to overcome them.

To obtain high-resolution images of fluorescently labeled cells within large tissues, ideally, the biological samples should be imaged without sectioning. 3DISCO is a straightforward tissue clearing procedure based on sequential incubation with organic solvents. Upon clearing, the organs become transparent allowing an end-to-end laser scan of the specimen.

成像在细胞水平上通过3DISCO清除完整生物系统

Tissue clearing and subsequent imaging of transparent organs is a powerful method to analyze fluorescently labeled cells and molecules in 3D, in intact ...

神经科学

High-Resolution 3D Imaging of Rabies Virus Infection in Solvent-Cleared Brain Tissue

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and study the distribution, tropism, and abundance of pathogens inside of organ tissues provides pivotal data on disease development and progression. Using conventional microscopy methods, immunolabeling is mostly restricted to thin sections obtained from paraffin-embedded or frozen samples. However, the limited 2D image plane of these thin sections may lead to the loss of crucial information on the complex structure of an infected organ and the cellular context of the infection. Modern multicolor, immunostaining-compatible tissue clearing techniques now provide a relatively fast and inexpensive way to study high-volume 3D image stacks of virus-infected organ tissue. By exposing the tissue to organic solvents, it becomes optically transparent. This matches the sample&#8217;s refractive indices and eventually leads to a significant reduction of light scattering. Thus, in combination with long free working distance objectives, large tissue sections up to 1 mm in size can be imaged by conventional confocal laser scanning microscopy (CLSM) at high resolution. Here, we describe a protocol to apply deep-tissue imaging after tissue clearing to visualize rabies virus distribution in infected brains in order to study topics like virus pathogenesis, spread, tropism, and neuroinvasion.

Novel, immunostaining-compatible tissue clearing techniques like the ultimate 3D imaging of solvent-cleared organs allow the 3D visualization of rabies virus brain infection and its complex cellular environment. Thick, antibody-labeled brain tissue slices are made optically transparent to increase imaging depth and to enable 3D analysis by confocal laser scanning microscopy.

溶剂清除脑组织狂犬病病毒感染的高分辨率3D成像

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and ...

Optical Clearing and Imaging of Immunolabeled Kidney Tissue

Optical clearing techniques render tissue transparent by equilibrating the refractive index throughout a sample for subsequent three-dimensional (3-D) imaging. They have received great attention in all research areas for the potential to analyze microscopic multicellular structures that extend over macroscopic distances. Given that kidney tubules, vasculature, nerves, and glomeruli extend in many directions, which have been only partially captured by traditional two-dimensional techniques so far, tissue clearing also opened up many new areas of kidney research. The list of optical clearing methods is rapidly growing, but it remains difficult for beginners in this field to choose the best method for a given research question. Provided here is a simple method that combines antibody labeling of thick mouse kidney slices; optical clearing with cheap, non-toxic and ready-to-use chemical ethyl cinnamate; and confocal imaging. This protocol describes how to perfuse kidneys and use an antigen-retrieval step to increase antibody- binding without requiring specialized equipment. Its application is presented in imaging different multicellular structures within the kidney, and how to troubleshoot poor antibody penetration into tissue is addressed. We also discuss the potential difficulties of imaging endogenous fluorophores and acquiring very large samples and how to overcome them. This simple protocol provides an easy-to-setup and comprehensive tool to study tissue in three dimensions.

The combination of antibody labeling, optical clearing, and advanced light microscopy allows three-dimensional analysis of complete structures or organs. Described here is a simple method to combine immunolabeling of thick kidney slices, optical clearing with ethyl cinnamate, and confocal imaging that enables visualization and quantification of three-dimensional kidney structures.

免疫标记肾组织的光学清除与成像

Optical clearing techniques render tissue transparent by equilibrating the refractive index throughout a sample for subsequent three-dimensional (3-D) ...

医学

Immunostaining of Whole-Mount Retinas with the CLARITY Tissue Clearing Method

The tissue hydrogel delipidation method (CLARITY), originally developed by the Deisseroth laboratory, has been modified and widely used for immunostaining and imaging of thick brain samples. However, this advanced technology has not yet been used for whole-mount retinas. Although the retina is partially transparent, its thickness of approximately 200 &#181;m (in mice) still limits the penetration of antibodies into the deep tissue as well as reducing light penetration for high-resolution imaging. Here, we adapted the CLARITY method for whole-mount mouse retinas by polymerizing them with an acrylamide monomer to form a nanoporous hydrogel and then clearing them in sodium dodecyl sulfate to minimize protein loss and avoid tissue damage. CLARITY-processed retinas were immunostained with antibodies for retinal neurons, glial cells, and synaptic proteins, mounted in a refractive index matching solution, and imaged. Our data demonstrate that CLARITY&#160;can improve the quality of standard immunohistochemical staining and imaging for retinal neurons and glial cells in whole-mount preparation. For instance, 3D resolution of fine axon-like and dendritic structures of dopaminergic amacrine cells were much improved by CLARITY. Compared to non-processed whole-mount retinas, CLARITY can reveal immunostaining for synaptic proteins such as postsynaptic density protein 95. Our results show that CLARITY renders the retina more optically transparent after the removal of lipids and preserves fine structures of retinal neurons and their proteins, which can be routinely used for obtaining high-resolution imaging of retinal neurons and their subcellular structures in whole-mount preparation.

Here we present a protocol to adapt the CLARITY method of the brain tissues for whole-mount retinas to improve the quality of standard immunohistochemical staining and high-resolution imaging of retinal neurons and their subcellular structures.

使用清晰组织清除方法对全山视网膜进行免疫染色

The tissue hydrogel delipidation method (CLARITY), originally developed by the Deisseroth laboratory, has been modified and widely used for immunostaining ...

Imaging and Quantification of Intact Neuronal Dendrites via CLARITY Tissue Clearing

Brain activity, the electrochemical signals passed between neurons, is determined by the connectivity patterns of neuronal networks, and from the morphology of processes and substructures within these neurons. As such, much of what is known about brain function has arisen alongside developments in imaging technologies that allow further insight into how neurons are organized and connected in the brain. Improvements in tissue clearing have allowed for high-resolution imaging of thick brain slices, facilitating morphological reconstruction and analyses of neuronal substructures, such as dendritic arbors and spines. In tandem, advances in image processing software provide methods of quickly analyzing large imaging datasets. This work presents a relatively rapid method of processing, visualizing, and analyzing thick slices of labeled neural tissue at high-resolution using CLARITY tissue clearing, confocal microscopy, and image analysis. This protocol will facilitate efforts toward understanding the connectivity patterns and neuronal morphologies that characterize healthy brains, and the changes in these characteristics that arise in diseased brain states.

Neuronal dendritic morphology often underlies function. Indeed, many disease processes that affect the development of neurons manifest with a morphological phenotype. This protocol describes a simple and powerful method for analyzing intact dendritic arbors and their associated spines.

通过 CLARITY 组织清除对完整神经元树突进行成像和定量

Brain activity, the electrochemical signals passed between neurons, is determined by the connectivity patterns of neuronal networks, and from the ...

A Tissue Clearing Method for Neuronal Imaging from Mesoscopic to Microscopic Scales

A detailed protocol is provided here to visualize neuronal structures from mesoscopic to microscopic levels in brain tissues. Neuronal structures ranging from neural circuits to subcellular neuronal structures are visualized in mouse brain slices optically cleared with ScaleSF. This clearing method is a modified version of ScaleS and is a hydrophilic tissue clearing method for tissue slices that achieves potent clearing capability as well as a high-level of preservation of fluorescence signals and structural integrity. A customizable three dimensional (3D)-printed imaging chamber is designed for reliable mounting of cleared brain tissues. Mouse brains injected with an adeno-associated virus vector carrying enhanced green fluorescent protein gene were fixed with 4% paraformaldehyde and cut into slices of 1-mm thickness with a vibrating tissue slicer. The brain slices were cleared by following the clearing protocol, which include sequential incubations in three solutions, namely, ScaleS0 solution, phosphate buffer saline (&#8211;), and ScaleS4 solution, for a total of 10.5&#8211;14.5 h. The cleared brain slices were mounted on the imaging chamber and embedded in 1.5% agarose gel dissolved in ScaleS4D25(0) solution. The 3D image acquisition of the slices was carried out using a confocal laser scanning microscope equipped with a multi-immersion objective lens of a long working distance. Beginning with mesoscopic neuronal imaging, we succeeded in visualizing fine subcellular neuronal structures, such as dendritic spines and axonal boutons, in the optically cleared brain slices. This protocol would facilitate understanding of neuronal structures from circuit to subcellular component scales.

The protocol provides a detailed method of neuronal imaging in brain slice using a tissue clearing method, ScaleSF. The protocol includes brain tissue preparation, tissue clarification, handling of cleared slices and confocal laser scanning microscopy imaging of neuronal structures from mesoscopic to microscopic levels.

一种从介观尺度到微观尺度的神经元成像组织清除方法

A detailed protocol is provided here to visualize neuronal structures from mesoscopic to microscopic levels in brain tissues. Neuronal structures ranging ...

Whole-Brain Single-Cell Imaging and Analysis of Intact Neonatal Mouse Brains Using MRI, Tissue Clearing, and Light-Sheet Microscopy

Tissue clearing followed by light-sheet microscopy (LSFM) enables cellular-resolution imaging of intact brain structure, allowing quantitative analysis of structural changes caused by genetic or environmental perturbations. Whole-brain imaging results in more accurate quantification of cells and the study of region-specific differences that may be missed with commonly used microscopy of physically sectioned tissue. Using light-sheet microscopy to image cleared brains greatly increases acquisition speed as compared to confocal microscopy. Although these images produce very large amounts of brain structural data, most computational tools that perform feature quantification in images of cleared tissue are limited to counting sparse cell populations, rather than all nuclei.
Here, we demonstrate NuMorph (Nuclear-Based Morphometry), a group of analysis tools, to quantify all nuclei and nuclear markers within annotated regions of a postnatal day 4 (P4) mouse brain after clearing and imaging on a light-sheet microscope. We describe magnetic resonance imaging (MRI) to measure brain volume prior to shrinkage caused by tissue clearing dehydration steps, tissue clearing using the iDISCO+ method, including immunolabeling, followed by light-sheet microscopy using a commercially available platform to image mouse brains at cellular resolution. We then demonstrate this image analysis pipeline using NuMorph, which is used to correct intensity differences, stitch image tiles, align multiple channels, count nuclei, and annotate brain regions through registration to publicly available atlases.
We designed this approach using publicly available protocols and software, allowing any researcher with the necessary microscope and computational resources to perform these techniques. These tissue clearing, imaging, and computational tools allow measurement and quantification of the three-dimensional (3D) organization of cell-types in the cortex and should be widely applicable to any wild-type/knockout mouse study design.

This protocol describes methods for conducting magnetic resonance imaging, clearing, and immunolabeling of intact mouse brains using iDISCO+, followed by a detailed description of imaging using light-sheet microscopy, and downstream analyses using NuMorph.

使用MRI，组织清除和光片显微镜对完整的新生小鼠大脑进行全脑单细胞成像和分析

Tissue clearing followed by light-sheet microscopy (LSFM) enables cellular-resolution imaging of intact brain structure, allowing quantitative analysis of ...

使用 SHORT 和 LSFM 对人脑进行高分辨率 3D 重建

Optical Clearing and Labeling for Light-sheet Fluorescence Microscopy in Large-scale Human Brain Imaging

Despite the numerous clearing techniques that emerged in the last decade, processing postmortem human brains remains a challenging task due to its dimensions and complexity, which make imaging with micrometer resolution particularly difficult. This paper presents a protocol to perform the reconstruction of volumetric portions of the human brain by simultaneously processing tens of sections with the SHORT (SWITCH - H2O2 - Antigen Retrieval - 2,2'-thiodiethanol [TDE]) tissue transformation protocol, which enables clearing, labeling, and sequential imaging of the samples with light-sheet fluorescence microscopy (LSFM). SHORT provides rapid tissue clearing and homogeneous multi-labeling of thick slices with several neuronal markers, enabling the identification of different neuronal subpopulations in both white and grey matter. After clearing, the slices are imaged via LSFM with micrometer resolution and in multiple channels simultaneously for a rapid 3D reconstruction. By combining SHORT with LSFM analysis within a routinely high-throughput protocol, it is possible to obtain the 3D cytoarchitecture reconstruction of large volumetric areas at high resolution in a short time, thus enabling comprehensive structural characterization of the human brain.

作者聚焦：推进 3D 细胞结构分析 - 人脑的快速体积重建 

The present protocol provides a step-by-step procedure for rapid and simultaneous optical clearing, muti-round labeling, and 3D volumetric reconstruction of tens of postmortem human brain sections by combining the (SWITCH - H2O2 - Antigen Retrieval - 2,2'-thiodiethanol [TDE]) SHORT tissue transformation technique with light-sheet fluorescence microscopy imaging in a routinely high-throughput protocol.