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 border="0" cellpadding="0" cellspacing="0" width="813">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">4% Paraformaldehyde</td>
			<td style="width:175px;">Lugen SCI&#160;</td>
			<td style="width:201px;">LGB-1175-4B</td>
			<td style="width:191px;">sample fixation&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Acrylamide</td>
			<td style="width:175px;">Affymetrix</td>
			<td style="width:201px;">75820</td>
			<td style="width:191px;">Hydrogel monomer solution&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">2,2&#8217;-Azobis[2-(2-imidazolin-2-yl) propane] dihydrochloride</td>
			<td style="width:175px;">Wako Pure Chemical Industries&#160;&#160;</td>
			<td style="width:201px;">VA-044</td>
			<td style="width:191px;">Hydrogel monomer solution&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Boric acid</td>
			<td style="width:175px;">Affymetrix</td>
			<td style="width:201px;">76324</td>
			<td>ETC buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Sodium Dodecyl Sulfate (SDS)</td>
			<td style="width:175px;">Affymetrix</td>
			<td style="width:201px;">18220</td>
			<td>ETC buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Sodium hydroxide pellets</td>
			<td style="width:175px;">Junsei chemical</td>
			<td style="width:201px;">1310-73-2</td>
			<td>ETC buffer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:175px;">Santa Cruz Biotechnology</td>
			<td style="width:201px;">sc-2323A</td>
			<td>Immunolabeling</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Triton X-100</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:201px;">T8787</td>
			<td>Immunolabeling</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Sodium azide</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:201px;">S2002</td>
			<td>Immunolabeling</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Collagen type &#8547;</td>
			<td style="width:175px;">Abcam</td>
			<td style="width:201px;">AB6586</td>
			<td>Antibody/immunolabeling</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Tyrosine hydroxylase (TH)</td>
			<td style="width:175px;">Millipore</td>
			<td style="width:201px;">AB152</td>
			<td>Antibody/immunolabeling</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Alexa Fluor Cy3 Donkey anti-Rabbit IgG (H+L)</td>
			<td style="width:175px;">Jackson ImmunoResearch</td>
			<td style="width:201px;">711-165-152</td>
			<td style="width:191px;">Secondary antibody/ immunolabeling</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Alexa Fluor 488 Donkey anti-Rabbit IgG (H+L)</td>
			<td style="width:175px;">Life Technologies - Molecular Probes</td>
			<td>A21206</td>
			<td style="width:191px;">Secondary antibody/ immunolabeling</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Confocal dish</td>
			<td style="width:175px;">SPL lifesciences</td>
			<td style="width:201px;">101350</td>
			<td>Imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Confocal microscope</td>
			<td style="width:175px;">Leica</td>
			<td style="width:201px;">SP8</td>
			<td>Imaging</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Sucrose</td>
			<td style="width:175px;">Junsei chemical</td>
			<td style="width:201px;">31365-0301</td>
			<td>CUBIC-mount solution&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Urea</td>
			<td style="width:175px;">Affymetrix</td>
			<td style="width:201px;">23036</td>
			<td>CUBIC-mount solution&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">N,N,N&#8217;,N&#8217;-tetrakis(2-hydroxypropyl)ethylenediamine</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:201px;">122262</td>
			<td>CUBIC-mount solution&#160;</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:247px;">ECT chamber</td>
			<td style="width:175px;">Logos Biosystems, Inc.</td>
			<td style="width:201px;">C10101</td>
			<td>ETC system</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">ECT chamber controller</td>
			<td style="width:175px;">Logos Biosystems, Inc.</td>
			<td style="width:201px;">C10201</td>
			<td>ETC system</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Temperature probe</td>
			<td style="width:175px;">Logos Biosystems, Inc.</td>
			<td style="width:201px;">C12101</td>
			<td>ETC system</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:247px;">Peristatic pump</td>
			<td style="width:175px;">Baoding longer precision pump Co., Ltd</td>
			<td style="width:201px;">YZ1515X</td>
			<td>ETC system</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Buffer reservoir</td>
			<td style="width:175px;">Logos Biosystems, Inc.</td>
			<td style="width:201px;">C10401</td>
			<td>ETC system</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Tissue container&#160;</td>
			<td style="width:175px;">Logos Biosystems, Inc.</td>
			<td style="width:201px;">C12001</td>
			<td>ETC system</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Container holder for 1 tissue container</td>
			<td style="width:175px;">Logos Biosystems, Inc.</td>
			<td style="width:201px;">C12002</td>
			<td>ETC system</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Mouse brain slice holder</td>
			<td style="width:175px;">Logos Biosystems, Inc.</td>
			<td style="width:201px;">C12004</td>
			<td>ETC system</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Whole rat brain holder</td>
			<td style="width:175px;">Logos Biosystems, Inc.</td>
			<td style="width:201px;">C12007</td>
			<td>ETC system</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Peristaltic pump tubing</td>
			<td style="width:175px;">Logos Biosystems, Inc.</td>
			<td style="width:201px;">C12104</td>
			<td>ETC system</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Tabletop-centrifuge</td>
			<td style="width:175px;">Hanil science industrial Co., Ltd</td>
			<td style="width:201px;">MICRO 12</td>
			<td>c-PRESTO</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:247px;">Syringe pump</td>
			<td style="width:175px;">Baoding longer precision pump Co., Ltd</td>
			<td style="width:201px;">LSP02-1B</td>
			<td>s-PRESTO</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Syringe (20 ml)</td>
			<td style="width:175px;">Korea vaccine Co., Ltd.</td>
			<td style="width:201px;">KV-S20</td>
			<td>s-PRESTO</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">3-way stopcock</td>
			<td style="width:175px;">Hyupsung medical Co.,Ltd.</td>
			<td style="width:201px;">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="https://ecsource-jove-com.remotexs.ntu.edu.sg/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="https://ecsource-jove-com.remotexs.ntu.edu.sg/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="https://ecsource-jove-com.remotexs.ntu.edu.sg/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="https://ecsource-jove-com.remotexs.ntu.edu.sg/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="https://ecsource-jove-com.remotexs.ntu.edu.sg/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

Nanyang Technological University

Research

JoVE Journal

Biology

12.0K 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

Methods for the Study of the Zebrafish Maxillary Barbel

Barbels are skin sensory appendages found in fishes, reptiles and amphibians. The zebrafish, Danio rerio, develops two pairs of barbels- a short nasal pair and a longer maxillary pair. Barbel tissue contains cells of ectodermal, mesodermal and neural crest origin, including skin cells, glands, taste buds, melanocytes, circulatory vessels and sensory nerves. Unlike most adult tissue, the maxillary barbel is optically clear, allowing us to visualize the development and maintenance of these tissue types throughout the life cycle. 
This video shows early development of the maxillary barbel (beginning approximately one month post-fertilization) and demonstrates a surgical protocol to induce regeneration in the adult appendage (&gt;3 months post-fertilization). Briefly, the left maxillary barbel of an anesthetized fish is elevated with sterile forceps just distal to the caudal edge of the maxilla. A fine, sterile spring scissors is positioned against the forceps to cut the barbel shaft at this level, establishing an anatomical landmark for the amputation plane. Regenerative growth can be measured with respect to this plane, and in comparison to the contralateral barbel. Barbel tissue regenerates rapidly, reaching maximal regrowth within 2 weeks of injury.
Techniques for analyzing the regenerated barbel include dissecting and embedding matched pairs of barbels (regenerate and control) in the wells of a standard DNA electrophoresis gel. Embedded specimens are conveniently photographed under a stereomicroscope for gross morphology and morphometry, and can be stored for weeks prior to downstream applications such as paraffin histology, cryosectioning, and/or whole mount immunohistochemistry. These methods establish the maxillary barbel as a novel in vivo tissue system for studying the regenerative capacity of multiple cell types within the genetic context of zebrafish.

The zebrafish maxillary barbel is an integumentary sense organ containing ectodermal, mesodermal and neural crest derivatives. Importantly, the adult barbel can regenerate after proximal amputation. This video introduces maxillary barbel development and demonstrates a surgical protocol to induce regeneration, followed by collection, embedding and downstream imaging of barbel specimens.

上颌Barbel斑马鱼的研究方法

Barbels are skin sensory appendages found in fishes, reptiles and amphibians.  The zebrafish, Danio rerio, develops two pairs of barbels- a short nasal ...

科研

生物学

Synthesis and Calibration of Phosphorescent Nanoprobes for Oxygen Imaging in Biological Systems

Oxygen measurement by phosphorescence quenching [1, 2] consists of the following steps: 1) the probe is delivered into the medium of interest (e.g. blood or interstitial fluid); 2) the object is illuminated with light of appropriate wavelength in order to excite the probe into its triplet state; 3) the emitted phosphorescence is collected, and its time course is analyzed to yield the phosphorescence lifetime, which is converted into the oxygen concentration (or partial pressure, pO2). The probe must not interact with the biological environment and in some cases to be 4) excreted from the medium upon the measurement completion. Each of these steps imposes requirements on the molecular design of the phosphorescent probes, which constitute the only invasive component of the measurement protocol. Here we review the design of dendritic phosphorescent nanosensors for oxygen measurements in biological systems. The probes consist of Pt or Pd porphyrin-based polyarylglycine (AG) dendrimers, modified peripherally with polyethylene glycol (PEG's) residues. For effective two-photon excitation, termini of the dendrimers may be modified with two-photon antenna chromophores, which capture the excitation energy and channel it to the triplet cores of the probes via intramolecular FRET (F&ouml;rster Resonance Energy Transfer). We describe the key photophysical properties of the probes and present detailed calibration protocols.

We present principles of oxygen measurements by phosphorescence quenching and review design of porphyrin-based dendritic nanosensors for oxygen imaging in biological systems.

氧成像在生物系统中的磷光纳米探针的合成和校准

Oxygen measurement by phosphorescence quenching [1, 2] consists of the following steps: 1) the probe is delivered into the medium of interest (e.g. blood ...

Differential Imaging of Biological Structures with Doubly-resonant Coherent Anti-stokes Raman Scattering (CARS)

Coherent Raman imaging techniques have seen a dramatic increase in activity over the past decade due to their promise to enable label-free optical imaging with high molecular specificity 1. The sensitivity of these techniques, however, is many orders of magnitude weaker than fluorescence, requiring milli-molar molecular concentrations 1,2. Here, we describe a technique that can enable the detection of weak or low concentrations of Raman-active molecules by amplifying their signal with that obtained from strong or abundant Raman scatterers. The interaction of short pulsed lasers in a biological sample generates a variety of coherent Raman scattering signals, each of which carry unique chemical information about the sample. Typically, only one of these signals, e.g. Coherent Anti-stokes Raman scattering (CARS), is used to generate an image while the others are discarded. However, when these other signals, including 3-color CARS and four-wave mixing (FWM), are collected and compared to the CARS signal, otherwise difficult to detect information can be extracted 3. For example, doubly-resonant CARS (DR-CARS) is the result of the constructive interference between two resonant signals 4. We demonstrate how tuning of the three lasers required to produce DR-CARS signals to the 2845 cm-1 CH stretch vibration in lipids and the 2120 cm-1 CD stretching vibration of a deuterated molecule (e.g. deuterated sugars, fatty acids, etc.) can be utilized to probe both Raman resonances simultaneously. Under these conditions, in addition to CARS signals from each resonance, a combined DR-CARS signal probing both is also generated. We demonstrate how detecting the difference between the DR-CARS signal and the amplifying signal from an abundant molecule's vibration can be used to enhance the sensitivity for the weaker signal. We further demonstrate that this approach even extends to applications where both signals are generated from different molecules, such that e.g. using the strong Raman signal of a solvent can enhance the weak Raman signal of a dilute solute.

Differential Imaging of Biological Structures with Doubly-resonant Coherent Anti-stokes Raman Scattering CARS

A combination of three single wavelength short-pulsed lasers is used to generate coherent anti-Stokes Raman scattering (CARS) and doubly-resonant CARS (DR-CARS). The difference between these signals provides enhanced sensitivity for otherwise difficult to detect coherent Raman signals, enabling imaging of weak Raman scatterers.

差分成像生物结构与双共振相干反斯托克斯拉曼散射（CARS）

Coherent Raman imaging techniques have seen a dramatic increase in activity over the past decade due to their promise to enable label-free optical imaging ...

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or&#160;photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.

We demonstrate the use of fluorescence photo activation localization microscopy (FPALM) to simultaneously image multiple types of fluorescently labeled molecules&#160;within cells. The techniques described yield the localization of thousands to hundreds of thousands of individual fluorescent labeled proteins, with a precision of tens of nanometers&#160;within single cells.

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled ...

Methods for Skin Wounding and Assays for Wound Responses in C. elegans

The C. elegans epidermis and cuticle form a simple yet sophisticated skin layer that can repair localized damage resulting from wounding. Studies of wound responses and repair in this model have illuminated our understanding of the cytoskeletal and genomic responses to tissue damage. The two most commonly used methods to wound the C. elegans adult skin are pricks with microinjection needles, and local laser irradiation. Needle wounding locally disrupts the cuticle, epidermis, and associated extracellular matrix, and may also damage internal tissues. Laser irradiation results in more localized damage. Wounding triggers a succession of readily assayed responses including elevated epidermal Ca2+ (seconds-minutes), formation and closure of an actin-containing ring at the wound site (1-2 hr), elevated transcription of antimicrobial peptide genes (2-24 hr), and scar formation. Essentially all wild type adult animals survive wounding, whereas mutants defective in wound repair or other responses show decreased survival. Detailed protocols for needle and laser wounding, and assays for quantitation and visualization of wound responses and repair processes (Ca dynamics, actin dynamics, antimicrobial peptide induction, and survival) are presented.

Methods for Skin Wounding and Assays for Wound Responses in C. elegans

The adult C. elegans skin is a tractable model for studies of epithelial wound responses, including wound closure, scar formation, and innate immunity.

对于皮肤伤人方法和测定的伤口响应<em&gt;温度。线虫</em

The C. elegans epidermis and cuticle form a simple yet sophisticated skin layer that can repair localized damage resulting from wounding. Studies of wound ...

Volume Segmentation and Analysis of Biological Materials Using SuRVoS (Super-region Volume Segmentation) Workbench

Segmentation is the process of isolating specific regions or objects within an imaged volume, so that further study can be undertaken on these areas of interest. When considering the analysis of complex biological systems, the segmentation of three-dimensional image data is a time consuming and labor intensive step. With the increased availability of many imaging modalities and with automated data collection schemes, this poses an increased challenge for the modern experimental biologist to move from data to knowledge. This publication describes the use of SuRVoS Workbench, a program designed to address these issues by providing methods to semi-automatically segment complex biological volumetric data. Three datasets of differing magnification and imaging modalities are presented here, each highlighting different strategies of segmenting with SuRVoS. Phase contrast X-ray tomography (microCT) of the fruiting body of a plant is used to demonstrate segmentation using model training, cryo electron tomography (cryoET) of human platelets is used to demonstrate segmentation using super- and megavoxels, and cryo soft X-ray tomography (cryoSXT) of a mammalian cell line is used to demonstrate the label splitting tools. Strategies and parameters for each datatype are also presented. By blending a selection of semi-automatic processes into a single interactive tool, SuRVoS provides several benefits. Overall time to segment volumetric data is reduced by a factor of five when compared to manual segmentation, a mainstay in many image processing fields. This is a significant savings when full manual segmentation can take weeks of effort. Additionally, subjectivity is addressed through the use of computationally identified boundaries, and splitting complex collections of objects by their calculated properties rather than on a case-by-case basis.

Volume Segmentation and Analysis of Biological Materials Using SuRVoS Super-region Volume Segmentation Workbench

Segmentation of three-dimensional data from many imaging techniques is a major bottleneck in analysis of complex biological systems. Here, we describe the use of SuRVoS Workbench to semi-automatically segment volumetric data at various length-scales using example datasets from cryo-electron tomography, cryo soft X-ray tomography, and phase contrast X-ray tomography techniques.

利用 SuRVoS (超区域体积分割) 工作台进行生物材料的体积分割与分析

Segmentation is the process of isolating specific regions or objects within an imaged volume, so that further study can be undertaken on these areas of ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipo: 三维脂肪组织成像的组织清除方法

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the imaging modality in a focused ion beam scanning electron microscope (FIB-SEM), which is used to obtain stacks of sequential images for 3D reconstruction and modelling. High-pressure freezing (HPF) allows close to native structural preservation, but the subsequent freeze substitution often does not provide sufficient contrast, especially for a bigger specimen, which is needed for high-quality imaging in the SEM required for 3D reconstruction. Therefore, in this protocol, after the freeze substitution, additional contrasting steps are carried out at room temperature. Although these steps are performed in a microwave, it is also possible to follow traditional bench processing, which requires longer incubation times.The subsequent embedding in minimal amounts of resin allows for faster and more precise targeting and preparation inside the FIB-SEM. This protocol is especially useful for samples that require preparation by high-pressure freezing for a reliable ultrastructural preservation but do not gain enough contrast during the freeze substitution for volume imaging using FIB-SEM. In combination with the minimal resin embedding, this protocol provides an efficient workflow for the acquisition of high-quality volume data.

Here we present a protocol for combining two sample processing techniques, high-pressure freezing and microwave-assisted sample processing, followed by minimal resin embedding for acquiring data with a focused ion beam scanning electron microscope (FIB-SEM). This is demonstrated using a mouse tibial nerve sample and Caenorhabditis elegans.

高压冷冻、微波辅助对比度增强和最小树脂嵌入在体积成像中的生物样品制备

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the ...

Exploring Adipose Tissue Structure by Methylsalicylate Clearing and 3D Imaging

Obesity is a major worldwide public health issue that increases the risk to develop cardiovascular diseases, type-2 diabetes, and liver diseases. Obesity is characterized by an increase in adipose tissue (AT) mass due to adipocyte hyperplasia and/or hypertrophia, leading to profound remodeling of its three-dimensional structure. Indeed, the maximal capacity of AT to expand during obesity is pivotal to the development of obesity-associated pathologies. This AT expansion is an important homeostatic mechanism to enable adaptation to an excess of energy intake and to avoid deleterious lipid spillover to other metabolic organs, such as muscle and liver. Therefore, understanding the structural remodeling that leads to the failure of AT expansion is a fundamental question with high clinical applicability. In this article, we describe a simple and fast clearing method that is routinely used in our laboratory to explore the morphology of mouse and human white adipose tissue by fluorescent imaging. This optimized AT clearing method is easily performed in any standard laboratory equipped with a chemical hood, a temperature-controlled orbital shaker and a fluorescent microscope. Moreover, the chemical compounds used are readily available. Importantly, this method allows one to resolve the 3D AT structure by staining various markers to specifically visualize the adipocytes, the neuronal and vascular networks, and the innate and adaptive immune cells distribution.

Here, we describe a simple, inexpensive and fast clearing method to resolve the 3D structure of both mouse and human white adipose tissue using a combination of markers to visualize vasculature, nuclei, immune cells, neurons, and lipid-droplet coat proteins by fluorescent imaging.

通过甲基异硫清除和三D成像探索脂肪组织结构

Obesity is a major worldwide public health issue that increases the risk to develop cardiovascular diseases, type-2 diabetes, and liver diseases. Obesity ...

Xenopus laevis Egg Extract Preparation and Live Imaging Methods for Visualizing Dynamic Cytoplasmic Organization

Traditionally used for bulk biochemical assays, Xenopus laevis egg extracts have emerged as a powerful imaging-based tool for studying cytoplasmic phenomena, such as cytokinesis, mitotic spindle formation and assembly of the nucleus. Building upon early methods that imaged fixed extracts sampled at sparse time points, recent approaches image live extracts using time-lapse microscopy, revealing more dynamical features with enhanced temporal resolution. These methods usually require sophisticated surface treatments of the imaging vessel. Here we introduce an alternative method for live imaging of egg extracts that require no chemical surface treatment. It is simple to implement and utilizes mass-produced laboratory consumables for imaging. We describe a system that can be used for both wide-field and confocal microscopy. It is designed for imaging extracts in a 2-dimensional (2D) field, but can be easily extended to imaging in 3D. It is well-suited for studying spatial pattern formation within the cytoplasm. With representative data, we demonstrate the typical dynamic organization of microtubules, nuclei and mitochondria in interphase extracts prepared using this method. These image data can provide quantitative information on cytoplasmic dynamics and spatial organization.

Xenopus laevis Egg Extract Preparation and Live Imaging Methods for Visualizing Dynamic Cytoplasmic Organization

We describe a method for the preparation and live imaging of undiluted cytoplasmic extracts from Xenopus laevis eggs.