Name: novel passive clearing methods for the rapid production of optical transparency in whole cns tissue
Uploaded: 2018-05-08T14:00:00.000+00:00
Duration: 6 min 14 s
Description: Watch this Scientific Journal Video about Novel Passive Clearing Methods for the Rapid Production of Optical Transparency in Whole CNS Tissue at JoVE.com

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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+brain+imaging:+A+powerful+tool+for+neuroscience.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+molecular+interrogation+of+intact+biological+systems.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+CLARITY+for+rapid+and+high-resolution+imaging+of+intact+tissues.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CLARITY+reveals+dynamics+of+ovarian+follicular+architecture+and+vasculature+in+three-dimensions.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+application+of+the+electrophoretic+tissue+clearing+technology,+CLARITY,+to+intact+solid+organs+including+brain,+pancreas,+liver,+kidney,+lung,+and+intestine.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+and+perspectives+in+tissue+clearing+using+CLARITY.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-body+tissue+stabilization+and+selective+extractions+via+tissue-hydrogel+hybrids+for+high-resolution+intact+circuit+mapping+and+phenotyping.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+phenotyping+within+transparent+intact+tissue+through+whole-body+clearing.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+tissue+clearing+(PACT):+Technical+evaluation+and+new+perspectives+in+immunofluorescence,+histology,+and+ultrastructure.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+the+optical+transparency+of+rodent+tissues+by+modified+PACT-based+passive+clearing.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+animal+perfusion+fixation+for+rodents.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+clearing+of+the+mouse+central+nervous+system+using+passive+CLARITY.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SeeDB:+A+simple+and+morphology-preserving+optical+clearing+agent+for+neuronal+circuit+reconstruction.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+expression+of+the+receptor+for+advanced+glycation+end+products+in+neurons+and+astrocytes+in+a+triple+transgenic+mouse+model+of+Alzheimer's+disease.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contralaterally+transplanted+human+embryonic+stem+cell-derived+neural+precursor+cells+(ENStem-A)+migrate+and+improve+brain+functions+in+stroke-damaged+rats.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+differential+plaque+depositions+in+the+brains+of+Tg2576+and+Tg-APPswe/PS1dE9+transgenic+mouse+models+of+Alzheimer+disease.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prdm+proto-oncogene+transcription+factor+family+expression+and+interaction+with+the+Notch-Hes+pathway+in+mouse+neurogenesis.">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, 大大减少了组织清理所需的时间, 同时仍然保持组织完整性和结构6,10。此外, 我们首次展示了组织清除协议在整个中枢神经系统组织中的应用, 以及在一个高阶啮齿动物模型中, 大鼠10。
具体地说, psPACT 涉及在水凝胶形成过程中的两个单独步骤中的样品处理4% 丙烯酰胺和 0.25% VA-044, 而 mPACT 进一步建立在 psPACT 和补充 8% SDS 清除解决方案与0.5% α thioglycerol10。分离丙烯酰胺和随后的 VA-044 步骤, 避免需要去除残留的 unpolymerized 水凝胶单体, 周围的组织, 这可能危及最终组织完整性后, 实现透明度与协议;此外, 这大大减少了产生最大光学间隙所需的时间。在 mPACT 方法中添加α thioglycerol 进一步加速了清除过程13, 因为α thioglycerol 是一种不褐变剂, 它帮助清除组织中仅使用原始协议方法中的试剂而无法访问的区域。7,8,9。
对啮齿动物组织的协议、psPACT 和 mPACT 的比较表明, 虽然 psPACT 提高了相对于原始协定协议的光学透明度, mPACT 最有效地允许清除和随后的高分辨率成像的整体哺乳动物器官和切片片, 如图 2中所示,图 3 , 图 4。与其他两种清除方法相比, mPACT 法处理后的 thioglycerol 的脂质清除对透明、效率和组织稳定性的差异是关键。这可以通过免疫荧光和共聚焦显微镜 (图 5D, E) 对加工器官进行快速3D 分析, 特别是关于血液血管形态学。
虽然在基于 SDS 的清除解决方案中添加α thioglycerol 对于加速光学透明度是至关重要的, 但它也略微增加了组织的脆弱性, 使其更容易受到后处理的损害。mPACT 在效率方面的明显优越性超过了这一限制;然而, 目前正在做的工作, 以确保最大限度的清洁可以达到最小, 不损害组织完整性。
因此, 我们的结果建立了 mPACT 作为一个强大的调查工具, 提供了3D 分析哺乳动物器官的功能和解剖特征, 包括但不限于神经网络, 血液血管, 和组织基质结构。向前迈进, 我们相信 mPACT 在调查导致疾病病理生理学的高阶生物机制方面尤其有用, 例如组织损伤、发育畸形、癌症和神经变性。疾病14,15,16,17。结合对疾病的更精细的细胞机制的研究, mPACT 有可能提供一个更完整, 多尺度的理解如何不同的疾病机制相互作用, 引起观察表型。

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-044 发起者解答在二个单独步骤为了形成水凝胶混合物。mPACT (修改后的公约) 进一步改善 psPACT 通过补充 8% SDS 清除解决方案与0.5% α thioglycerol。在14天, 通过协议、psPACT 和 mPACT 处理的示例进行比较, 并在图 2B中显示。
在先前的一项研究中, 我们报告说, 虽然最初的协议协议在23天内实现了光清除, psPACT 和 mPACT 在短短20和14天内清除了整个鼠标 CNS, 分别为10。在本研究中, 当我们通过 mPACT 协议处理小鼠大脑样本时, 我们发现它们在14天后达到了光学透明性 (图 2B)。通过 psPACT 和 mPACT 处理的大鼠全中枢神经系统样品也表明 mPACT 达到最大间隙, 效率最高, 因为组织分别在30天和20日内被清除 (图 3A, B)。与整个中枢神经系统相比, 成年大鼠大脑仅在5天内被 mPACT 清除 (图 4A)。psPACT 和 mPACT 处理后通过免疫荧光分析清除大鼠脑血管形态, 证明这些清除程序对大脑的解剖和功能分析 (图 4B, mPACT 数据) 的效用显示在 2016)10中。
利用这些优化的无源组织清除协议, 我们能够可视化完整的、透明的整个中枢神经系统模型, 尽管三协议中的清除时间不同。优化的被动清除方法在较短的时间内实现了器官清晰度。这些结果表明, mPACT 清除方法比以往的被动清除方法更稳定、快速地生成清晰的中枢神经系统。因此, mPACT 在未来哺乳动物器官的结构和解剖分析中具有很大的潜力, 并且在疗效和安全性方面与现有方法有着显著的优势。
利用 mPACT 生成光学上清除的内脏和胚胎
除全中枢神经组织外, 成年大鼠脾脏和肾脏分别在19天和23日 mPACT 清除 (图 5A, B)。通过 mPACT 协议处理3天后, E10.5 和 E13.5 的小鼠胚胎也被清除。图 5C-E显示了整个胚胎血管形态的可视化, 以及小鼠胚胎、马尾马和大鼠脾脏尾部区域的微血管。这些结果表明, mPACT 可以用来产生透明模型的各种组织和器官的3D 解剖分析。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57123/57123fig1.jpg"> 图 1: 优化的被动清除技术 (契约) 的示意图表示形式.每个步骤所需的试剂旁边都提供了每个三公约协议中涉及的各个步骤的概要。最初的契约协议是从水凝胶形成的组织聚合与4% 粉煤灰, 4% 丙烯酰胺 (a) 溶液和 0.25% VA-044 引发剂溶液的冷混合物。组织用氮气聚合后, 用 8% SDS 清除液被动清除。psPACT (过程分离的协议) 使用单独处理过程 4% a. 溶液和 0.25% VA-044 引发剂溶液为凝胶形成的组织;这些条件与原始公约议定书的情况不同。mPACT (修改后的公约) 额外增加了0.5% α thioglycerol 的 psPACT 协议的 8% SDS 清除解决方案。对于所有三个协议, 完整的组织在 > = 37 °c 孵化在一个震动孵化器, 直到最大的清除 iss 达到。<a href="/files/ftp_upload/57123/57123fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57123/57123fig2.jpg"> 图 2: 无源组织清除对小鼠整个中枢神经系统 (CNS) 的光学透明性。(A)在一段时间内通过基于契约的方法, 规划整个 CNS 组织清除。(B)在处理后的14天内, 对小鼠全中枢神经系统中的三种被动清除协议 (原始协议、psPACT、mPACT) 进行比较。(C) 14 天内 mPACT 处理的鼠标大脑的光清除。此数字已从宇et 201610修改。2C 的字母是3毫米高。<a href="/files/ftp_upload/57123/57123fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57123/57123fig3.jpg"> 图 3: psPACT 和 mPACT 后大鼠全中枢神经组织的光学透明度。(A)成年大鼠全中枢神经组织在30天后达到最大的光间隙与 psPACT。(B)成年大鼠全中枢神经组织在20天后达到了 mPACT 的最大光学间隙。前后图显示了治疗前后整个中枢神经系统的大小, 用眼睛测量。<a href="/files/ftp_upload/57123/57123fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57123/57123fig4.jpg"> 图 4: 切片成年大鼠脑的 mPACT 处理和微血管可视化。(A)在 mPACT 处理3和5天后, 对4毫米厚的成年大鼠脑部分进行比较。(B) 3D psPACT 处理后用 anti-CD31 抗体对成年大鼠脑内血管形态进行投影。缩放条, 1500 µm 和100µm。此数字已从宇et 201610修改。<a href="/files/ftp_upload/57123/57123fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57123/57123fig5.jpg"> 图 5: mPACT 处理啮齿动物器官和整个小鼠胚胎.mPACT 在成年鼠(A)脾脏和(B)肾脏分别19和23天实现了光学透明度。(C) mPACT 在3天后产生透明 E10.5 和 E13.5 老鼠胚胎。(D)在清除与 mPACT 后, 在小鼠胚胎尾区 anti-CD31 微血管的可视化。(E) 3D 经 mPACT 处理后, 对大鼠马尾和(F)大鼠脾 anti-CD31 进行血管投射。此数字已从宇et 201610修改。<a href="/files/ftp_upload/57123/57123fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

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

Nanyang Technological University

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

Rapid Optical Clearing for Semi-High-Throughput Analysis of Tumor Spheroids

Tumor spheroids are fast becoming commonplace in basic cancer research and drug development. Obtaining data regarding protein expression within the spheroid at the cellular level is important for analysis, yet existing techniques are often expensive, laborious, use non-standard equipment, cause significant size distortion, or are limited to relatively small spheroids. This protocol presents a new method of mounting and clearing spheroids that address these issues while allowing for confocal analysis of the inner structure of spheroids. In contrast to existing approaches, this protocol provides for rapid mounting and clearing of a large number of spheroids using standard equipment and laboratory supplies. Mounting spheroids in a pH-neutral agarose-PBS gel solution before introducing a refractive-index-matched clearing solution minimizes size distortion common to other similar techniques. This allows for detailed quantitative and statistical analysis where the accuracy of size measurements is paramount. Furthermore, compared to liquid clearing solutions, the agarose gel technique keeps spheroids fixed in place, allowing for the collection of three-dimensional (3D) confocal images. The present article elaborates how the method yields high-quality two- and 3D images that provide information about inter-cell variability and inner spheroid structure.

Tumor spheroids are becoming increasingly utilized to assess tumor cell-microenvironment interactions and therapy response. The present protocol describes a robust but simple method for semi-high-throughput imaging of 3D tumor spheroids using rapid optical clearing.

用于肿瘤球体半高通量分析的快速光学清除

Tumor spheroids are fast becoming commonplace in basic cancer research and drug development. Obtaining data regarding protein expression within the ...

癌症研究

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.

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.

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

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 ...

Investigation of Spatial Interaction Between Astrocytes and Neurons in Cleared Brains

Combining viral vector transduction and tissue clearing using the CLARITY method makes it possible to simultaneously investigate several types of brain cells and their interactions. Viral vector transduction enables the marking of diverse cell types in different fluorescence colors within the same tissue. Cells can be identified genetically by activity or projection. Using a modified CLARITY protocol, the potential sample size of astrocytes and neurons has grown by 2-3 orders of magnitude. The use of CLARITY allows the imaging of complete astrocytes, which are too large to fit in their entirety in slices, and the examination of the somata with all their processes. In addition, it provides the opportunity to investigate the spatial interaction between astrocytes and different neuronal cell types, namely, the number of pyramidal neurons in each astrocytic domain or the proximity between astrocytes and specific inhibitory neuron populations. This paper describes, in detail, how these methods are to be applied.

Combining viral vector transduction and brain clearing using the CLARITY method allows the investigation of a large number of neurons and astrocytes simultaneously.

研究清除大脑中星形胶质细胞和神经元之间的空间相互作用

Combining viral vector transduction and tissue clearing using the CLARITY method makes it possible to simultaneously investigate several types of brain ...

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 ...

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.

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 ...

生物学

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.