这项工作得到了大脑韩国21加 Yonsei 大学医学科学项目的支持。此外, 这项工作得到了韩国国家研究基金会 (NRF-2017R1D1A1B03030315) 的赠款的支持。

所有程序均经 Yonsei 大学医学院适当的研究伦理委员会批准。所有实验动物都是按照 Yonsei 大学医学院实验动物护理委员会的指导方针而牺牲的。
1. 试剂的制备
注意: 多聚甲醛 (粉煤灰)、丙烯酰胺和月桂酸钠 (SDS) 是有毒刺激物, 因此应在通风罩中处理, 并配备适当的个人防护设备 (PPE; 实验室大衣, 手套, 防护眼镜)。
<ol><li>4% 丙烯酰胺 (a) 溶液 (A4P0): 将20毫升40% 丙烯酰胺溶液添加到180毫升的0.1 米磷酸盐缓冲盐水 (PBS) 中。</li>
	<li>0.25% VA-044 溶液: 添加0.5 克 2,2´-偶 [2-(2-imidazolin 2 基) 丙烷] 二盐酸盐 (VA-044) 粉到200毫升的0.1 米磷酸盐缓冲盐水 (PBS)。虽然 VA-044 粉可以储存在室温下, 它必须储存在4摄氏度后, 在 PBS 的增溶。为取得最佳效果, 请只准备实验所需的解决方案量。</li>
	<li>协议鸡尾酒解决方案: 添加10克丙烯酰胺到225毫升4% 多聚甲醛 (粉煤灰) 溶液, 并调整体积为200毫升与4% 粉煤灰。使用前, 在50毫升圆锥管中加入100毫克的 VA-044 粉, 40 毫升的混合物。</li>
	<li>协议清除解决方案缓冲区: 添加40克月桂酸钠 (SDS) 到350毫升0.1 米 pbs 和调整体积为500毫升与0.1 米 pbs。对于 mPACT 清除解决方案, 将0.5% α thioglycerol 添加到协议清除解决方案缓冲区。</li>
	<li>本研究使用的小鼠为2周大的 BALB 雄性小鼠;这项研究中使用的老鼠是2周大的雄性 SD 大鼠。</li>
</ol>2. 麻醉和灌注手术
注意: 粉煤灰和丙烯酰胺是有毒的刺激物, 因此应在通风罩内使用适当的 PPE。
<ol><li>麻醉动物在无病原体的房间, 30 毫克/千克的 zoletil 使用1毫升注射器与26口径针。监视动物 5-10 分钟。</li>
	<li>一旦动物到达麻醉的手术平面, 使用脚趾捏反应法确定麻醉深度。确认对停止响应。 注意: 以下步骤涉及鼠标和大鼠手术遵循类似的协议在以前的研究中使用11。</li>
	<li>通过外壳和腹壁, 在肋笼下面做一个5-6 厘米的切口。</li>
	<li>使用弯曲, 钝剪刀在隔膜切口, palpating 胸区预先定位切口部位。</li>
	<li>将切口延伸至胸腔的整个长度, 以暴露胸腔。</li>
	<li>小心地置换肺部。把胸腔切开到锁骨上。使用虹膜剪刀, 对左心室后端进行小切口。将18口径钝或橄榄尖的灌注针插入升主动脉, 穿过切口心室。</li>
	<li>用止血夹紧心脏, 确保针头的安全, 防止渗漏。或者, 使用修改过的止血在针尖周围夹住主动脉。</li>
	<li>在右心房做一个大切口, 注意确保对降主动脉的最小损伤。老鼠现在已经准备好灌注了。</li>
</ol>3. 大鼠全身灌注和解剖
注意: 粉煤灰和丙烯酰胺是有毒的刺激物, 因此应在通风罩内使用适当的 PPE。
注: 以下整个灌注步骤类似于前一项研究中使用的一种协议, 即 "宇et"。(2016)10,11。
<ol><li>将心脏的右心房连接到18口径的针头上, 注意不要引入任何气泡。</li>
	<li>使用50毫升注射器, 快速和均匀地泵送50毫升冷0.1 米的 PBS 溶液含肝素 (10 单位/毫升)。</li>
	<li>将18口径的针连接到蠕动泵的管上。</li>
	<li>用200毫升的冷0.1 米 PBS 溶液冲洗, 含肝素 (10 单位/毫升), 循环速度为10毫升/分。</li>
	<li>固定与250毫升冷4% 粉煤灰溶液的循环速度为10毫升/分钟。这只老鼠在这个阶段应该是僵硬的。</li>
	<li>收集并储存剩余的粉煤灰溶液进行处理。</li>
	<li>对于协议, 灌注组织与冷冻协议鸡尾酒解决方案的4% 粉煤灰, 4% 丙烯酰胺, 0.25% VA-044 粉与18口径针, 然后取出头部和脊柱。通过从颈部到鼻子的中线切开使头骨暴露出来。这种额外的灌注步骤是不必要的 psPACT 和 mPACT 方法;固定后, 立即隔离头部和脊柱, 并暴露头骨。</li>
	<li>通过除去残余的颈部和脊柱肌肉来揭露头骨的基底。</li>
	<li>用 rongeurs 和剪刀把头骨剥开。切除大脑和脊髓。</li>
	<li>将组织在4% 粉煤灰中贮存4摄氏度;组织最多可储存1周。</li>
</ol>4. 大鼠和小鼠中枢神经系统凝胶单体的注入和聚合
注意: 丙烯酰胺、SDS、α thioglycerol 和粉煤灰是刺激物, 因此应在通风罩中处理, 并适当小便。
<ol><li>
协议 (被动清算技术)
	<ol>
<li>将固定的小鼠和大鼠的整个中枢神经系统 (大脑和脊髓) 分离和培养成50毫升管, 其中含有冷冻公约鸡尾酒溶液, 4% 粉煤灰, 4% 丙烯酰胺, 0.25% VA-044 粉, 并贮存在4摄氏度为 24 h. 确保组织完全沉浸在 s 中解决方案.</li>
		<li>用与氮气罐相连的组织凝胶杂交系统将样品嵌入氮气中10分钟: 将系统设置为37摄氏度。将组织转移到含有新鲜冷 (4 °c) 协议的50毫升管, 并与管帽连接, 然后打开真空。</li>
		<li>要聚合水凝胶, 将包含样品的管放在震动孵化器 (150 rpm, 37 °c) 3 小时, 或直到聚合完成。</li>
		<li>使用印迹纸 (请参阅材料表), 移除围绕组织的剩余聚合水凝胶。</li>
		<li>将组织转移到含有清除溶液的50毫升管 (8% SDS 在0.1 米 PBS, pH 8.0)。</li>
		<li>将样品放在摇晃的孵化器中, 设置为37摄氏度和150转, 直到组织被清除。平均来说, 鼠标 CNS 需要大约20天的时间才能实现完全的清晰度。</li>
	</ol>
</li>
	<li>
psPACT (过程分离被动清除技术)
	<ol>
<li>用固定的小鼠和老鼠在干净的长凳上, 将整个中枢神经系统 (大脑和脊髓) 与骨刀和剪刀隔离。</li>
		<li>将组织转移到50毫升管, 含4% 个粉煤灰, 并贮存在4摄氏度, 24 小时. 确保组织完全浸入固定体中。</li>
		<li>在0.1 米 pbs 中清洗固定组织1小时, 然后转到 A4P0 溶液 (0.1 米 pbs 中的4% 丙烯酰胺)。存储在37°c 为24小时。</li>
		<li>在0.1 米 PBS 中清洗组织5分钟。</li>
		<li>在0.1 米的 PBS 中浸泡 0.25% VA-044 的组织。贮存在37°c 为6-24 小时, 然后转移到新鲜 0.25% VA-044/PBS 溶液。</li>
		<li>使用组织凝胶杂交系统 (参见材料表) 将样品嵌入氮气中, 并将其与氮气罐连接: 将组织转移到含有新鲜冷 (4 °c) 0.25% VA-044/PBS 溶液的50毫升管中, 并与管帽连接。打开真空。</li>
		<li>将组织转移到清除溶液中 (8% SDS 在0.1 米 PBS, pH 8.0)。</li>
		<li>在震动孵化器中孵化样品, 设置为37摄氏度和150转, 直到组织被清除。平均来说, 老鼠中枢神经系统需要大约17天的时间才能达到完全的清晰度。</li>
	</ol>
</li>
	<li>
mPACT (改进的被动清除技术)
	<ol>
<li>用固定的小鼠和老鼠在干净的长凳上, 将整个中枢神经系统 (大脑和脊髓) 与骨刀和剪刀隔离。</li>
		<li>按照 psPACT 协议的步骤 4.2.2 4.2.8。</li>
		<li>将组织转移到清除液中。请注意, 与协议和 psPACT 协议不同, mPACT 需要一个由0.5% α thioglycerol 组成的清除解决方案, 除0.1 米 PBS 中的8% 个 SDS 外, pH 为8.0。α thioglycerol 是一种不褐变剂, 有助于清除组织更迅速和有效。确保组织完全沉浸在溶液中。</li>
		<li>将样品放在摇晃的孵化器中, 设置为37摄氏度和150转, 直到组织被清除。平均来说, 老鼠中枢神经系统需要大约2周的时间才能达到完全的清晰度。</li>
	</ol>
</li>
</ol>5. 染色的折射率匹配和清除
注: n轮辋 (基于 Nycodenz 的折射率匹配解决方案) 由30毫升基缓冲器中溶解的0.8 克/毫升 Nycodenz 粉末 (0.01% 叠氮化钠和 Tween-20, 0.1 米 PBS, pH 8.0) 组成。建议将该溶液放置在37摄氏度的振动孵化箱中, 以便适当地对粉末进行溶剂化。
<ol><li>在0.1 米 pbs 中孵育 0.1% X-100 的组织为2小时, 然后在0.1 米 pbs 中用2% 牛血清白蛋白 (BSA) 阻断 6 h。</li>
	<li>洗涤部分三次在 PBST (0.1% Tween-20 在 0.1 M PBS) 为 2 h. 着色部分以主要抗体 (在这种情况下, 抗兔 PECAM-CD31 抗体, 染色血管) 2 天。</li>
	<li>染色切片与二次抗体 (在这种情况下, 山羊抗兔 IgG Cy3 荧光共轭) 在 2% BSA 2 天。</li>
	<li>在 PBST 中清洗标记的组织三次, 2 小时, 并在15毫升n轮辋中存储 2-10 天。</li>
	<li>在成像之前, 将标记的组织移动到35或60毫米组织培养皿上的少量n轮辋上。修复与硅胶周围的菜的底部边缘。</li>
	<li>添加 1.5-2 毫升新鲜的n轮辋。</li>
</ol>6. 图像处理
<ol><li>使用共焦激光扫描 microscopewith 10x 放大器获取已清除组织的图像 (请参阅材料表)。对于 Cy3-labeled 组织, 使用波长为 550-600 nm。</li>
</ol>

<ol><li>Zhu, X., Xia, Y., Wang, X., Si, K., Gong, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optical+brain+imaging%3A+A+powerful+tool+for+neuroscience%5BTitle%5D)+AND+%22Neurosci+Bull%22%5BJournal%5D)">Optical brain imaging: A powerful tool for neuroscience.</a> Neurosci Bull. 33 (1), 95-102 (2017).</li>
<li>Chung, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structural+and+molecular+interrogation+of+intact+biological+systems%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Structural and molecular interrogation of intact biological systems.</a> Nature. 497 (7449), 332-337 (2013).</li>
<li>Tomer, R., Ye, L., Hsueh, B., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Advanced+CLARITY+for+rapid+and+high-resolution+imaging+of+intact+tissues%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">Advanced CLARITY for rapid and high-resolution imaging of intact tissues.</a> Nat Protoc. 9 (7), 1682-1697 (2014).</li>
<li>Feng, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CLARITY+reveals+dynamics+of+ovarian+follicular+architecture+and+vasculature+in+three-dimensions%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">CLARITY reveals dynamics of ovarian follicular architecture and vasculature in three-dimensions.</a> Sci Rep. 7, 44810(2017).</li>
<li>Lee, H., Park, J. H., Seo, I., Park, S. H., Kim, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Improved+application+of+the+electrophoretic+tissue+clearing+technology%2C+CLARITY%2C+to+intact+solid+organs+including+brain%2C+pancreas%2C+liver%2C+kidney%2C+lung%2C+and+intestine%5BTitle%5D)+AND+%22BMC+Dev+Biol%22%5BJournal%5D)">Improved application of the electrophoretic tissue clearing technology, CLARITY, to intact solid organs including brain, pancreas, liver, kidney, lung, and intestine.</a> BMC Dev Biol. 14, 48(2014).</li>
<li>Jensen, K. H. R., Berg, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Advances+and+perspectives+in+tissue+clearing+using+CLARITY%5BTitle%5D)+AND+%22J+Chem+Neuroanat%22%5BJournal%5D)">Advances and perspectives in tissue clearing using CLARITY.</a> J Chem Neuroanat. 86, 19-34 (2017).</li>
<li>Treweek, J. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Whole-body+tissue+stabilization+and+selective+extractions+via+tissue-hydrogel+hybrids+for+high-resolution+intact+circuit+mapping+and+phenotyping%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">Whole-body tissue stabilization and selective extractions via tissue-hydrogel hybrids for high-resolution intact circuit mapping and phenotyping.</a> Nat Protoc. 10 (11), 1860-1896 (2015).</li>
<li>Yang, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-cell+phenotyping+within+transparent+intact+tissue+through+whole-body+clearing%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Single-cell phenotyping within transparent intact tissue through whole-body clearing.</a> Cell. 158 (4), 945-958 (2014).</li>
<li>Neckel, P. H., Mattheus, U., Hirt, B., Just, L., Mack, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Large-scale+tissue+clearing+%28PACT%29%3A+Technical+evaluation+and+new+perspectives+in+immunofluorescence%2C+histology%2C+and+ultrastructure%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Large-scale tissue clearing (PACT): Technical evaluation and new perspectives in immunofluorescence, histology, and ultrastructure.</a> Sci Rep. 6, 34331(2016).</li>
<li>Woo, J., Lee, M., Seo, J. M., Park, H. S., Cho, Y. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimization+of+the+optical+transparency+of+rodent+tissues+by+modified+PACT-based+passive+clearing%5BTitle%5D)+AND+%22Exp+Mol+Med%22%5BJournal%5D)">Optimization of the optical transparency of rodent tissues by modified PACT-based passive clearing.</a> Exp Mol Med. 48 (12), 274(2016).</li>
<li>Gage, G. J., Kipke, D. R., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Whole+animal+perfusion+fixation+for+rodents%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Whole animal perfusion fixation for rodents.</a> J Vis Exp. (65), (2012).</li>
<li>Roberts, D. G., Johnsonbaugh, H. B., Spence, R. D., MacKenzie-Graham, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optical+clearing+of+the+mouse+central+nervous+system+using+passive+CLARITY%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Optical clearing of the mouse central nervous system using passive CLARITY.</a> J Vis Exp. (112), (2016).</li>
<li>Ke, M. T., Fujimoto, S., Imai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((SeeDB%3A+A+simple+and+morphology-preserving+optical+clearing+agent+for+neuronal+circuit+reconstruction%5BTitle%5D)+AND+%22Nat+Neurosci%22%5BJournal%5D)">SeeDB: A simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction.</a> Nat Neurosci. 16 (8), 1154-1161 (2013).</li>
<li>Choi, B. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Increased+expression+of+the+receptor+for+advanced+glycation+end+products+in+neurons+and+astrocytes+in+a+triple+transgenic+mouse+model+of+Alzheimer%27s+disease%5BTitle%5D)+AND+%22Exp+Mol+Med%22%5BJournal%5D)">Increased expression of the receptor for advanced glycation end products in neurons and astrocytes in a triple transgenic mouse model of Alzheimer's disease.</a> Exp Mol Med. 46, 75(2014).</li>
<li>Chang, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Contralaterally+transplanted+human+embryonic+stem+cell-derived+neural+precursor+cells+%28ENStem-A%29+migrate+and+improve+brain+functions+in+stroke-damaged+rats%5BTitle%5D)+AND+%22Exp+Mol+Med%22%5BJournal%5D)">Contralaterally transplanted human embryonic stem cell-derived neural precursor cells (ENStem-A) migrate and improve brain functions in stroke-damaged rats.</a> Exp Mol Med. 45, 53(2013).</li>
<li>Kim, T. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+differential+plaque+depositions+in+the+brains+of+Tg2576+and+Tg-APPswe%2FPS1dE9+transgenic+mouse+models+of+Alzheimer+disease%5BTitle%5D)+AND+%22Exp+Mol+Med%22%5BJournal%5D)">Analysis of differential plaque depositions in the brains of Tg2576 and Tg-APPswe/PS1dE9 transgenic mouse models of Alzheimer disease.</a> Exp Mol Med. 44 (8), 492-502 (2012).</li>
<li>Kinameri, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prdm+proto-oncogene+transcription+factor+family+expression+and+interaction+with+the+Notch-Hes+pathway+in+mouse+neurogenesis%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Prdm proto-oncogene transcription factor family expression and interaction with the Notch-Hes pathway in mouse neurogenesis.</a> PLoS One. 3 (12), e3859(2008).</li>
</ol>

作者没有什么可透露的。

尽管在协议中使用的被动、非电泳提取方法大大提高了与以前的组织清除方法一致的一致性, 如清晰度2、3、4、7,8, 该技术仍然存在几个缺点, 其中最紧迫的是实现最大组织清晰度12所需的时间长度。在当前的研究中, 我们提出了修改的协议协议, 大大减少了?...

科学和临床调查的一个基本目标是全面了解器官结构和功能;然而, 哺乳动物器官极其复杂的性质往往是完全实现这一目标的障碍1。清晰度 (透明脂交换丙烯酰胺-杂交刚性成像兼容 Tisssue-水凝胶)2,3,4, 其中包括建立一个基于丙烯酰胺的水凝胶混合从完整的组织, 达到各种器官的光学清除, 包括大脑、肝脏和脾脏, 同时保持其结构完整性5。因此, 清晰度不仅使可视化, 而且还有机会精细解剖复杂的细胞网络和组织形态, 而不需要切片。
为了达到组织间隙, 清晰度采用电泳方法去除手上样品的脂质含量。虽然已注意到生产物理稳定的组织-水凝胶杂交种, 研究表明, 其使用电泳组织清除 (等) 方法产生可变的结果, 在组织质量方面, 包括褐变, 表位损害, 和蛋白质损失5,6。为了解决这些问题, 已开发了7、8、9等修改的协议 (被动清晰度技术), 取代了被动的、基于离子洗涤剂的 delipidation 技术等处理。然而, 尽管在结果上取得了更大的一致性, 但条约需要更多的时间来获得最大的清除。此外, 这些技术尚未应用到整个中枢神经系统的形式, 或大鼠模型, 如老鼠和豚鼠。
本研究试图通过提出新的方法、psPACT (过程分离的公约) 和 mPACT (修改后的公约) 来解决这些限制, 以促进在老鼠和老鼠模型中的整个中枢神经系统和内脏器官的快速清除10。具体地说, psPACT 在水凝胶形成过程中分别在4% 丙烯酰胺和 0.25% VA-044 的两个步骤中处理组织;mPACT 基本上涉及相同的步骤, 但补充了基于 SDS 的清除解决方案与0.5% α thioglycerol 作为关键试剂。这两种技术都利用了内源系统和脑脊液循环系统, 大大减少了产生光间隙所需的时间。作为一项原则的证明, 我们展示了使用共焦显微镜分析在清除组织中的血管模式10。

<table><tbody><tr><td>Sodium Dodecyl Sulfate (SDS)</td><td>Affymetrix, Inc.</td><td>75819</td><td>Clearing solution</td></tr><tr><td>Nycodenz</td><td>Axia-Shield</td><td>1002424</td><td>nRIMS solution</td></tr><tr><td>40% Acrylamide Solution</td><td>Bio Rad Laboratories, Inc.</td><td>161-0140</td><td>Polymerization (A4P0)</td></tr><tr><td>2,2&amp;acute;-Azobis[2-(2-imidazolin-2-yl)propane] Dihydrochloride</td><td>Wako Pure Chemical Industries, Ltd.</td><td>017-19362</td><td>Polymerization (VA-044)</td></tr><tr><td>1-Thioglycerol</td><td>Sigma-Aldrich</td><td>M1753-100ML</td><td>Clearing solution (mPACT)</td></tr><tr><td>Tween-20</td><td>Georgiachem</td><td>9005-64-5</td><td>nRIMS solution</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T8787-50ML</td><td>Immuno Staining</td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Bovogen</td><td>BSA100</td><td>Immuno Staining</td></tr><tr><td>Heparin</td><td>Merck Millipore</td><td>375095</td><td>Perfusion (PBS)</td></tr><tr><td>Sodium azide</td><td>Sigma-Aldrich</td><td>S2002-25G</td><td>nRIMS solution</td></tr><tr><td>PECAM-CD31 antibody</td><td>Santa Cruz Biotechnology Inc.</td><td>sc-28188</td><td>Immuno Staining</td></tr><tr><td>Goat anti-rabbit-IgG Cy3 fluorescent conjugate</td><td>Jackson ImmunoResearch Inc.</td><td>111-165-003</td><td>Immuno Staining</td></tr><tr><td>4% Paraformaldehyde</td><td>Tech &amp;amp; Innovation</td><td>BPP-9004</td><td>Perfusion, Polymerization</td></tr><tr><td>20X Phosphate Buffered Saline (pH 7.4)</td><td>Tech &amp;amp; Innovation</td><td>BPB-9121</td><td>Perfusion, Buffer</td></tr><tr><td>10 mL stripette</td><td>Coatar</td><td>4488</td><td>Solution transfer</td></tr><tr><td>50 mL tube</td><td>Falcon</td><td>352070</td><td>Clearing tube</td></tr><tr><td>35 mm Cell culture dish</td><td>SPL</td><td>20035</td><td>Imaging</td></tr><tr><td>Confocal dish</td><td>SPL</td><td>211350</td><td>Imaging</td></tr><tr><td>1 mL syringe</td><td>Korea vaccine Co., Ltd</td><td>26G 1/2</td><td>Anesthetize&amp;nbsp;</td></tr><tr><td>50 mL syringe</td><td>Korea vaccine Co., Ltd</td><td>21G1 1/4</td><td>Perfusion</td></tr><tr><td>Acrylamide</td><td>Sigma-Aldrich</td><td>A3553</td><td>Polymerization (A4P0)</td></tr><tr><td>Whatman 3MM paper</td><td>Sigma-Aldrich</td><td>Z270849</td><td>Blotting paper for gel removal</td></tr><tr><td>Confocal microscope</td><td>Zeiss</td><td>LSM780</td><td>Imaging</td></tr><tr><td>ZEN lite Software</td><td>Zeiss</td><td>ZEN 2012</td><td>Imaging</td></tr><tr><td>Peristaltic pump</td><td>Longerpump</td><td>BT100-1F</td><td>Perfusion</td></tr><tr><td>EasyGel</td><td>Lifecanvas Technologies</td><td>EasyGel</td><td>Tissue gel hybridization system</td></tr></tbody></table>

novel passive clearing methods for the rapid production of optical transparency in whole cns tissue

由于清晰度的发展, 一种电化学清除技术, 允许在透明组织内的三维表型映射, 多种新的清除方法, 包括立方 (清晰, 无障碍的脑成像鸡尾酒和计算分析), 开关 (系统范围控制的交互时间和化学动力学的化学品), 地图 (放大分析的蛋白质组) 和协议 (被动清晰度技术), 以进一步扩大现有的工具箱生物组织的显微分析。本研究旨在改进和优化原始的协议程序, 为一系列完整的啮齿动物组织, 包括整个中枢神经系统 (CNS), 肾脏, 脾脏, 和整个小鼠胚胎。这些新的技术被称为 psPACT (过程分离协议) 和 mPACT (修改后的契约), 提供了非常有效的方法来映射细胞电路, 并在完整的正常和病理组织中可视化亚型结构。在下面的协议中, 我们提供了一个详细的, 分步概述如何实现最大的组织间隙, 最小的入侵他们的结构完整性通过 psPACT 和 mPACT。

利用优化的无源交换技术生成全中枢神经系统的透明模型
通过各种无源清除技术 (图 1), 快速实现了小鼠和全中枢神经组织的光间隙。图 2A显示了一段时间内组织清理的示意图。与原始的契约方法不同, psPACT (过程分离的协议) 包括处理样品与4% 丙烯酰胺 (A4P0) 和 0.25% VA-04...

Watch this Scientific Journal Video about Novel Passive Clearing Methods for the Rapid Production of Optical Transparency in Whole CNS Tissue at JoVE.com

Novel Passive Clearing Methods for the Rapid Production of Optical Transparency in Whole CNS Tissue

一种新的用于全中枢神经系统光透明快速生成的无源清除方法

在这里, 我们提出了两个新的方法, psPACT 和 mPACT, 以实现最大的光学透明度和随后显微分析的组织血管在完整的啮齿动物整个中枢神经系统。

Since the development of CLARITY, a bioelectrochemical clearing technique that allows for three-dimensional phenotype mapping within transparent tissues, ...

novel-passive-clearing-methods-for-rapid-production-optical

Research

JoVE Journal

Neuroscience

8.7K 次观看.  Yonsei University College of Medicine. 在这里, 我们提出了两个新的方法, psPACT 和 mPACT, 以实现最大的光学透明度和随后显微分析的组织血管在完整的啮齿动物整个中枢神经系统。

视频: Novel Passive Clearing Methods for the Rapid Production of Optical Transparency in Whole CNS Tissue

A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are characteristic to neurons in both normal and diseased brain. Towards this, a great deal of effort has been placed on building high-resolution neuroanatomical maps1-3. With the expansion of molecular genetics and advances in light microscopy has come the ability to query not only neuronal morphologies, but also the molecular and cellular makeup of individual neurons and their associated networks4. Major advances in the ability to mark and manipulate neurons through transgenic and gene targeting technologies in the rodent now allow investigators to 'program' neuronal subsets at will5-6. Arguably, one of the most influential contributions to contemporary neuroscience has been the discovery and cloning of genes encoding fluorescent proteins (FPs) in marine invertebrates7-8, alongside their subsequent engineering to yield an ever-expanding toolbox of vital reporters9. Exploiting cell type-specific promoter activity to drive targeted FP expression in discrete neuronal populations now affords neuroanatomical investigation with genetic precision.
Engineering FP expression in neurons has vastly improved our understanding of brain structure and function. However, imaging individual neurons and their associated networks in deep brain tissues, or in three dimensions, has remained a challenge. Due to high lipid content, nervous tissue is rather opaque and exhibits auto fluorescence. These inherent biophysical properties make it difficult to visualize and image fluorescently labelled neurons at high resolution using standard epifluorescent or confocal microscopy beyond depths of tens of microns. To circumvent this challenge investigators often employ serial thin-section imaging and reconstruction methods10, or 2-photon laser scanning microscopy11. Current drawbacks to these approaches are the associated labor-intensive tissue preparation, or cost-prohibitive instrumentation respectively.
Here, we present a relatively rapid and simple method to visualize fluorescently labelled cells in fixed semi-thick mouse brain slices by optical clearing and imaging. In the attached protocol we describe the methods of: 1) fixing brain tissue in situ via intracardial perfusion, 2) dissection and removal of whole brain, 3) stationary brain embedding in agarose, 4) precision semi-thick slice preparation using new vibratome instrumentation, 5) clearing brain tissue through a glycerol gradient, and 6) mounting on glass slides for light microscopy and z-stack reconstruction (Figure 1).
For preparing brain slices we implemented a relatively new piece of instrumentation called the 'Compresstome' VF-200 (http://www.precisionary.com/products_vf200.html). This instrument is a semi-automated microtome equipped with a motorized advance and blade vibration system with features similar in function to other vibratomes. Unlike other vibratomes, the tissue to be sliced is mounted in an agarose plug within a stainless steel cylinder. The tissue is extruded at desired thicknesses from the cylinder, and cut by the forward advancing vibrating blade. The agarose plug/cylinder system allows for reproducible tissue mounting, alignment, and precision cutting. In our hands, the 'Compresstome' yields high quality tissue slices for electrophysiology, immunohistochemistry, and direct fixed-tissue mounting and imaging. Combined with optical clearing, here we demonstrate the preparation of semi-thick fixed brain slices for high-resolution fluorescent imaging.

Here we describe a rapid and simple method to image fluorescently labeled cells in semi-thick brain slices. By fixing, slicing, and optically clearing brain tissue we describe how standard epifluorescent or confocal imaging can be used to visualize individual cells and neuronal networks within intact nervous tissue.

一个快速高分辨率的荧光成像的方法在半厚的脑片

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are ...

科研

神经科学

Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique non-linear deformations, dramatically hampering one's ability to evaluate cellular morphology, distribution and connectivity in the central nervous system (CNS). However, optical clearing techniques are changing the way tissues are visualized. These approaches permit one to probe deeply into intact organ preparations, providing tremendous insight into the structural organization of tissues in health and disease. Techniques such as Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) achieve this goal by providing a matrix that binds important biomolecules while permitting light-scattering lipids to freely diffuse out. Lipid removal, followed by refractive index matching, renders the tissue transparent and readily imaged in 3 dimensions (3D). Nevertheless, the electrophoretic tissue clearing (ETC) used in the original CLARITY protocol can be challenging to implement successfully and the use of a proprietary refraction index matching solution makes it expensive to use the technique routinely. This report demonstrates the implementation of a simple and inexpensive optical clearing protocol that combines passive CLARITY for improved tissue integrity and 2,2&#8242;-thiodiethanol (TDE), a previously described refractive index matching solution.

Optical clearing techniques are revolutionizing the way tissues are visualized. In this report we describe modifications of the original Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) protocol that yields more consistent and less expensive results.

鼠标中枢神经系统的光透明使用被动CLARITY

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique ...

Microsurgical Dissection and Tissue Clearing for High Resolution Intact Whole Retina and Vitreous Imaging

Neuronal and vascular structures of the retina in physiologic and pathologic conditions can be better visualized and characterized by using intact whole retina imaging techniques compared to conventional retinal flat mount preparations and sections. However, immunofluorescent imaging of intact whole retina is hindered by the opaque coatings of the eyeball, i.e., sclera, choroid, and retinal pigment epithelium (RPE) and the light scattering properties of retinal layers that prevent full thickness high resolution optical imaging. Chemical bleaching of the pigmented layers and tissue clearing protocols have been described to address these obstacles; however, currently described methods are not suitable for imaging endogenous fluorescent molecules such as green fluorescent protein (GFP) in intact whole retina. Other approaches bypassed this limitation by surgical removal of pigmented layers and the anterior segment of the eyeball allowing intact eye imaging, though the peripheral retina and hyaloid structures were disrupted. Presented here is an intact whole retina and vitreous immunofluorescent imaging protocol that combines surgical dissection of the sclera/choroid/retina pigment epithelium (RPE) layers with a modified tissue clearing method and light sheet fluorescent microscopy (LSFM). The new approach offers an unprecedented view of&#160;unperturbed vascular and neuronal elements of the retina as well as the vitreous and hyaloid vascular system in pathologic conditions.

<meta charset="utf8"></head><body>显微外科解剖和组织透明化，用于高分辨率完整全视网膜和玻璃体成像

Presented here is a protocol for intact whole retina imaging in which the outer opaque/pigmented layers of the eyeball are surgically removed, and optical clearing is applied to render retina transparent enabling the visualization of the peripheral retina and hyaloid vasculature in intact retina using light sheet fluorescent microscopy.

显微外科解剖和组织透明化，用于高分辨率完整全视网膜和玻璃体成像

Neuronal and vascular structures of the retina in physiologic and pathologic conditions can be better visualized and characterized by using intact whole ...

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 ...

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

The identification and exploration of the detailed organization of organs or of the whole body at the cellular level are fundamental challenges in biology. Transitional methods require a substantial amount of time and effort to obtain a 3D image and including sectioning the intact tissue, immunolabeling, and imaging serially-sectioned tissue, which produces a loss of information at each step of the process. In recently developed approaches for high-resolution imaging within intact tissue, molecular characterization has been restricted to the labeling of proteins. However, currently available protocols for organ clearing require a considerably long process time, making it difficult to implement tissue clearing techniques in the lab. We recently established a rapid and highly-reproducible protocol termed ACT-PRESTO (active clarity technique&#8211;pressure related efficient and stable transfer of macromolecules into organs), which allows for tissue clearance within several hours. Moreover, ACT-PRESTO enables rapid immunolabeling with conventional methods and accelerates antibody penetration into the deep layer of densely-formed, thick specimens by applying pressure or convection flow. We describe how to prepare tissues, how to clear by lipid removal using electrophoresis, and how to immuno-stain by a pressure-assisted delivery. The rapidity and consistency of the protocol will expedite the performance of 3D histological research and volume-based diagnoses.

ACT-PRESTO (active clarity technique&#8211;pressure related efficient and stable transfer of macromolecules into organs) enables rapid, efficient, but inexpensive tissue clearing and rapid antibody penetration into cleared tissues by passive diffusion or pressure-assisted delivery. Using this method, a wide range of tissues can be cleared, immunolabeled by multiple antibodies, and volume-imaged.

一种新的用于全中枢神经系统光透明快速生成的无源清除方法

摘要

探索更多视频

一个快速高分辨率的荧光成像的方法在半厚的脑片

鼠标中枢神经系统的光透明使用被动CLARITY

显微外科解剖和组织透明化，用于高分辨率完整全视网膜和玻璃体成像

显微外科解剖和组织透明化，用于高分辨率完整全视网膜和玻璃体成像

免疫标记肾组织的光学清除与成像

使用清晰组织清除方法对全山视网膜进行免疫染色

通过 CLARITY 组织清除对完整神经元树突进行成像和定量

一种从介观尺度到微观尺度的神经元成像组织清除方法

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

ACT-PRESTO:生物组织清算和体积成像方法免疫标记