Name: refined clarity-based tissue clearing for three-dimensional fibroblast organization in healthy and injured mouse hearts
Uploaded: 2021-05-16T14:00:00
Description: Watch this Scientific Journal Video about Refined CLARITY-Based Tissue Clearing for Three-Dimensional Fibroblast Organization in Healthy and Injured Mouse Hearts at JoVE.com

作者要感谢CCHMC康福克斯成像核心在开发该模型的帮助和指导，以及来自临床工程的马特·巴蒂对所有3D打印部件的设计。德米特里亚·菲切瑟得到了国家卫生研究院（NHLBI，T32 HL125204）的培训补助金的支持，杰弗瑞·莫尔肯廷得到了霍华德·休斯医学研究所的支持。

所有涉及小鼠的实验都得到了辛辛那提儿童医院医疗中心机构动物护理和使用委员会（IACUC）的批准。该机构还通过了AALAC（美国实验室动物护理认证协会）认证。老鼠通过颈椎脱位被安乐死，接受生存外科手术的小鼠得到疼痛缓解（见下文）。用于疼痛管理和安乐死的所有方法都基于美国兽医医学协会安乐死小组的建议。所有的老鼠都被安置在玉米棒床上用品单元里，随时有水和食物。老鼠被安...

<ol>
	<li>Nagaraju, C. 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=Myofibroblast+phenotype+and+reversibility+of+fibrosis+in+patients+With+end-stage+heart+failure.">Myofibroblast phenotype and reversibility of fibrosis in patients With end-stage heart failure.</a> Journal of the American College of Cardiology. 73 (18), 2267-2282 (2019).</li><li>Yoon, S., Eom, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+failure+with+preserved+ejection+fraction:+present+status+and+future+directions.">Heart failure with preserved ejection fraction: present status and future directions.</a> Experimental and Molecular Medicine. 51 (12), 1-9 (2019).</li><li>Borlaug, B. A., Redfield, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diastolic+and+systolic+heart+failure+are+distinct+phenotypes+within+the+heart+failure+spectrum.">Diastolic and systolic heart failure are distinct phenotypes within the heart failure spectrum.</a> Circulation. 123, 2006-2014 (2011).</li><li>Ivey, M. J., Tallquist, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+the+cardiac+fibroblast.">Defining the cardiac fibroblast.</a> Circulation Journal. 80 (11), 2269-2276 (2016).</li><li>Fu, X., 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=Specialized+fibroblast+differentiated+states+underlie+scar+formation+in+the+infarcted+mouse+heart.">Specialized fibroblast differentiated states underlie scar formation in the infarcted mouse heart.</a> Journal of Clinical Investigation. 128 (5), 2127-2143 (2018).</li><li>Kanisicak, O., 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=Genetic+lineage+tracing+defines+myofibroblast+origin+and+function+in+the+injured+heart.">Genetic lineage tracing defines myofibroblast origin and function in the injured heart.</a> Nature Communications. 7, 12260 (2016).</li><li>Sadeghi, A. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+3D+cardiac+fibrotic+tissue+to+study+fibrotic+remodeling.">Engineered 3D cardiac fibrotic tissue to study fibrotic remodeling.</a> Advanced Healthcare Materials. 6 (11), 1601434 (2017).</li><li>Nam, Y. 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=Induction+of+diverse+cardiac+cell+types+by+reprogramming+fibroblasts+with+cardiac+transcription+factors.">Induction of diverse cardiac cell types by reprogramming fibroblasts with cardiac transcription factors.</a> Development. 141 (22), 4267-4278 (2014).</li><li>Bruns, D. 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=The+right+ventricular+fibroblast+secretome+drives+cardiomyocyte+dedifferentiation.">The right ventricular fibroblast secretome drives cardiomyocyte dedifferentiation.</a> PLoS One. 14 (8), 0220573 (2019).</li><li>Ski&#246;ldebrand, 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=Inflammatory+activation+of+human+cardiac+fibroblasts+leads+to+altered+calcium+signaling,+decreased+connexin+43+expression+and+increased+glutamate+secretion.">Inflammatory activation of human cardiac fibroblasts leads to altered calcium signaling, decreased connexin 43 expression and increased glutamate secretion.</a> Heliyon. 3 (10), 00406 (2017).</li><li>Jing, D., 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=Tissue+clearing+of+both+hard+and+soft+tissue+organs+with+the+pegasos+method.">Tissue clearing of both hard and soft tissue organs with the pegasos method.</a> Cell Research. 28, 803-818 (2018).</li><li>Kolesov&#225;, H., &#268;apek, M., Radochov&#225;, B., Jan&#225;&#269;ek, J., Sedmera, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+different+tissue+clearing+methods+and+3D+imaging+techniques+for+visualization+of+GFP-expressing+mouse+embryos+and+embryonic+hearts.">Comparison of different tissue clearing methods and 3D imaging techniques for visualization of GFP-expressing mouse embryos and embryonic hearts.</a> Histochemistry and Cell Biology. 146 (2), 141-152 (2016).</li><li>Salamon, R. J., Zhang, Z., Mahmoud, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capturing+the+cardiac+injury+response+of+targeted+cell+populations+via+cleared+heart+three-dimensional+imaging.">Capturing the cardiac injury response of targeted cell populations via cleared heart three-dimensional imaging.</a> Journal of Visualized Experiments. (157), (2020).</li><li>Wang, Z., 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=Imaging+transparent+intact+cardiac+tissue+with+single-cell+resolution.">Imaging transparent intact cardiac tissue with single-cell resolution.</a> Biomedical Optics Express. 9 (2), 423-436 (2018).</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>Yokoyama, T., 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=Quantification+of+sympathetic+hyperinnervation+and+denervation+after+myocardial+infarction+by+three-dimensional+assessment+of+the+cardiac+sympathetic+network+in+cleared+transparent+murine+hearts.">Quantification of sympathetic hyperinnervation and denervation after myocardial infarction by three-dimensional assessment of the cardiac sympathetic network in cleared transparent murine hearts.</a> PLoS One. 12 (7), 0182072 (2017).</li><li>Perbellini, F., 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=Free-of-Acrylamide+SDS-based+Tissue+Clearing+(FASTClear)+for+three+dimensional+visualization+of+myocardial+tissue.">Free-of-Acrylamide SDS-based Tissue Clearing (FASTClear) for three dimensional visualization of myocardial tissue.</a> Scientific Reports. 7, 5188 (2017).</li><li>Tainaka, K., Kuno, A., Kubota, S. I., Murakami, T., Ueda, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+principles+in+tissue+clearing+and+staining+protocols+for+whole-body+cell+profiling.">Chemical principles in tissue clearing and staining protocols for whole-body cell profiling.</a> Annual Review of Cell and Developmental Biology. 32, 713-741 (2016).</li><li>Tainaka, 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=Whole-body+imaging+with+single-cell+resolution+by+tissue+decolorization.">Whole-body imaging with single-cell resolution by tissue decolorization.</a> Cell. 159, 911-924 (2014).</li><li>Nehrhoff, I., 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=3D+imaging+in+CUBIC-cleared+mouse+heart+tissue:+going+deeper.">3D imaging in CUBIC-cleared mouse heart tissue: going deeper.</a> Biomedical Optics Express. 7 (9), 3716-3720 (2016).</li><li>Yamamoto, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multifunctional+reporter+mouse+line+for+Cre-+and+FLP-dependent+lineage+analysis.">A multifunctional reporter mouse line for Cre- and FLP-dependent lineage analysis.</a> Genesis. 47 (2), 107-114 (2009).</li><li>Turner, P. V., Brabb, T., Pekow, C., Vasbinder, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Administration+of+substances+to+laboratory+animals:+Routes+of+administration+and+factors+to+consider.">Administration of substances to laboratory animals: Routes of administration and factors to consider.</a> Journal of the American Association for Laboratory Animal Science. 50 (5), 600-613 (2011).</li><li>Means, C. 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=Sphingosine+1-phosphate+S1P2+and+S1P3+receptor-mediated+Akt+activation+protects+against+in+vivo+myocardial+ischemia-reperfusion+injury.">Sphingosine 1-phosphate S1P2 and S1P3 receptor-mediated Akt activation protects against in vivo myocardial ischemia-reperfusion injury.</a> American Journal of Physiology - Heart and Circuatory Physiology. 292 (6), 2944-2951 (2007).</li><li>Michael, L. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+ischemia+and+reperfusion:+A+murine+model.">Myocardial ischemia and reperfusion: A murine model.</a> American Journal of Physiology - Heart and Circuatory Physiology. 269 (6), 2147-2154 (1995).</li><li>Ahn, D., 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=Induction+of+myocardial+infarcts+of+a+predictable+size+and+location+by+branch+pattern+probability-assisted+coronary+ligation+in+C57BL/6+mice.">Induction of myocardial infarcts of a predictable size and location by branch pattern probability-assisted coronary ligation in C57BL/6 mice.</a> American Joural of Physiology: Circulatory Physiology. 286 (3), 1201-1207 (2004).</li><li>Patten, R. D., 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=Ventricular+remodeling+in+a+mouse+model+of+myocardial+infarction.">Ventricular remodeling in a mouse model of myocardial infarction.</a> American Journal of Physiology - Heart and Circuatory Physiology. 274 (5), 1812-1820 (1998).</li><li>Gao, X. M., Dart, A. M., Dewar, E., Jennings, G., Du, X. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serial+echocardiographic+assessment+of+left+ventricular+dimensions+and+function+after+myocardial+infarction+in+mice.">Serial echocardiographic assessment of left ventricular dimensions and function after myocardial infarction in mice.</a> Cardiovascular Research. 45 (2), 330-338 (2000).</li><li>Vagnozzi, R. 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=An+acute+immune+response+underlies+the+benefit+of+cardiac+stem+cell+therapy.">An acute immune response underlies the benefit of cardiac stem cell therapy.</a> Nature. 577, 405-409 (2020).</li><li>Sengupta, P. P., Tajik, A. J., Chandrasekaran, K., Khandheria, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Twist+mechanics+of+the+left+ventricle.+Principles+and+application.">Twist mechanics of the left ventricle. Principles and application.</a> Journal of the American College of Cardiology: Cardiovascular Imaging. 1 (3), 366-376 (2008).</li><li>Arts, T., 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=Macroscopic+three-dimensional+motion+patterns+of+the+left+ventricle.">Macroscopic three-dimensional motion patterns of the left ventricle.</a> Advances in Experimental Medicine and Biology. 346, 383-392 (1993).</li><li>Willems, I. E. M. G., Havenith, M. G., De Mey, J. G. R., Daemen, M. J. A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+muscle+actin-positive+cells+in+healing+human+myocardial+scars.">The muscle actin-positive cells in healing human myocardial scars.</a> American Journal of Pathology. 145 (4), 868-875 (1994).</li><li>Hashmi, S., Al-Salam, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+myocardial+infarction+and+myocardial+ischemia-reperfusion+injury:+A+comparison.">Acute myocardial infarction and myocardial ischemia-reperfusion injury: A comparison.</a> International Journal of Clinical Experimental Pathology. 8, 8786-8796 (2015).</li></ol>

本文提出了一种精炼的组织清除方法，允许在内在心脏成纤维细胞的可视化，无论是在基线和受伤后，以特征和更好地了解成纤维细胞在小鼠的心脏。这个增强的协议解决了现有的组织清除协议的限制，试图确定特定的细胞类型在成人或新生儿心脏12，13，14，16，17，20。...

虽然心肌细胞是心脏中体积最大的部分，但心脏成纤维细胞更丰富，并且严重参与调节该器官的基线结构和修复特征。心脏成纤维细胞具有高度的移动性、机械反应能力和表型范围，具体取决于其激活的程度。心脏成纤维细胞是维持细胞外基质（ECM）正常水平所必需的，这些细胞产生太少或过多的ECM可能导致疾病1、2、3。鉴于心脏成纤维细胞在疾病中的重要性，在确定新的治疗策略方面，特别是试图限制过度纤维化4、5、6、7方面，已成为研究的日益重要的课题。受伤后，成纤维细胞激活并分化成一种更合成的细胞类型，称为肌纤维细胞，它可以是增殖和分泌丰富的 ECM，以及具有收缩活性，有助于改造心室。
虽然心脏成纤维细胞在二维文化6、8、9、10中对其特性进行了广泛的评估，但更不了解其在三维活心脏中的特性和动态，无论是基线还是疾病刺激。在这里，描述了一种精致的方法，组织清除成年小鼠的心脏，同时保持与Roas26-loxP-eGFP x Tcf21-MerCreMer遗传报告系统标记的成纤维细胞的荧光。在心脏内，Tcf21是静默成纤维细胞4的相对特定的标记。在给予塔莫西芬以激活不可受诱惑的MerCreMer蛋白后，基本上所有静默成纤维细胞都会永久表达来自Roas26轨迹的增强绿色荧光蛋白（eGFP），从而允许它们在体内跟踪。
有许多完善的组织清除协议存在，其中一些已经应用于心脏11，12，13，14，15，16，17。然而，许多用于不同组织清除协议的试剂被发现淬灭内源性荧光信号18。此外，成人心脏是很难清除，由于丰富的血红蛋白组含有蛋白质，产生自流19。因此，本协议的目标是保持成纤维细胞标记荧光与同时抑制血红细胞自身荧光在受伤的成人心脏，以最佳的三维可视化体内12，13，14，16，17，20。
先前的研究试图检查心脏成纤维细胞在体内使用香水抗体来标记这些细胞，虽然这样的研究受到抗体渗透和心脏血管结构14，16，17，20的限制。虽然萨拉蒙等人已经显示出组织清除与维持新生儿心脏局部神经元荧光，和Nehrhoff等人已显示维持荧光标记骨髓细胞，维持内源性荧光通过整个心室壁尚未证明，包括成人心脏成纤维细胞在基线或受伤后13，20的可视化。此组织清除协议根据 CLARITY 方法（透明脂质交换丙烯酰胺-杂交硬成像/免疫染色/原位混合兼容组织水凝胶）和 PEGASOS（聚乙二醇 （PEG） 相关溶剂系统）优化了先前协议的混合物。这一精炼的协议允许对处于基线的老鼠心脏中的心脏成纤维细胞以及它们对不同类型的损伤的反应进行更有力的检查。该协议是直接和可重复的，将有助于描述心脏成纤维细胞在体内的行为。

<table>
	<tbody>
		<tr>
			<td>4-0 braided silk</td>
			<td>Ethicon</td>
			<td>K871H</td>
			<td></td>
		</tr>
		<tr>
			<td>8-0 prolene</td>
			<td>Ethicon</td>
			<td>8730H</td>
			<td></td>
		</tr>
		<tr>
			<td>40% Acrylamide Solution</td>
			<td>Bio-Rad</td>
			<td>1610140</td>
			<td></td>
		</tr>
		<tr>
			<td>Angiotensin II</td>
			<td>Sigma</td>
			<td>A9525-50G</td>
			<td></td>
		</tr>
		<tr>
			<td>Artificial Tear Ointment</td>
			<td>Covetrus</td>
			<td>048272</td>
			<td></td>
		</tr>
		<tr>
			<td>DABCO (1,4-diazabicyclo[2.2. 2]octane)</td>
			<td>Millipore Sigma</td>
			<td>D27802-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>GLUture topical tissue adhesive</td>
			<td>World Precision Instruments</td>
			<td>503763</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma</td>
			<td>H0777</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaris Start Analysis Software</td>
			<td>Oxford Instruments</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-osmotic pumps</td>
			<td>Alzet</td>
			<td>Model 1002</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Elements Analysis Software</td>
			<td>Nikon</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon A1R HD upright microscope</td>
			<td>Nikon</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal autoclaved chow</td>
			<td>Labdiet</td>
			<td>5010</td>
			<td></td>
		</tr>
		<tr>
			<td>Nycodenz, 5- (N-2, 3-dihydroxypropylacetamido)-2, 4, 6-tri-iodo-N, 
			N&#39;-bis (2, 3 dihydroxypropyl) isophthalamide</td>
			<td>CosmoBio</td>
			<td>AXS-1002424</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenylephrine Hydrochloride</td>
			<td>Sigma</td>
			<td>P6126-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>Photoinitiator</td>
			<td>Wako Chemicals</td>
			<td>VA-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Rosa26-nLacZ [FVB.Cg-Gt(ROSA)26Sortm1 (CAG-lacZ,-EGFP)Glh/J]</td>
			<td>Jackson Laboratories</td>
			<td>Jax Stock No:012429</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Azide</td>
			<td>Sigma Aldrich</td>
			<td>S2002-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride solution</td>
			<td>Hospira, Inc.</td>
			<td>NDC 0409-4888-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Tamoxifen</td>
			<td>Sigma Aldrich</td>
			<td>T5648</td>
			<td></td>
		</tr>
		<tr>
			<td>Tamoxifen food</td>
			<td>Envigo</td>
			<td>TD.130860</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Thermo Fisher Scientific</td>
			<td>BP337-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Quadrol, N,N,N&#8242;,N&#8242;-Tetrakis(2-Hydroxypropyl)ethylenediamine, decolorizing agent</td>
			<td>Millipore Sigma</td>
			<td>122262-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>X-Clarity electrophoretic clearing chamber</td>
			<td>Logos Biosystems</td>
			<td>C30001</td>
			<td></td>
		</tr>
		<tr>
			<td>X-Clarity electrophoretic clearing solution</td>
			<td>Logos Biosystems</td>
			<td>C13001</td>
			<td></td>
		</tr>
		<tr>
			<td>X-Clarity electrophoresis tissue basket</td>
			<td>Logos Biosystems</td>
			<td>C12001</td>
			<td></td>
		</tr>
		<tr>
			<td>X-Clarity electrophoresis tissue basket holder</td>
			<td>Logos Biosystems</td>
			<td>C12002</td>
			<td></td>
		</tr>
	</tbody>
</table>

refined clarity-based tissue clearing for three-dimensional fibroblast organization in healthy and injured mouse hearts

心血管疾病是全世界最常见的死亡原因，通常以心脏纤维化加剧为标志，这种纤维化会导致心室僵硬性随着心脏功能的改变而增加。心脏心室纤维化的增加是由于活化了常驻成纤维细胞，尽管这些细胞在三维（3-D）心脏内、基线或激活后如何运作，尚不清楚。为了检查成纤维细胞如何导致心脏病及其在三维心脏中的动力学，开发了一种精致的基于CLARITY的组织清除和成像方法，显示整个小鼠心脏内荧光标记的心脏成纤维细胞。组织常驻成纤维细胞是使用Roas26-loxP-eGFP荧光记者小鼠与表达Tcf21-MerCreMer敲门线的心脏成纤维细胞交叉的基因标记。该技术用于观察健康小鼠和心脏病纤维小鼠模型中整个成人左心室的成纤维细胞定位动力学。有趣的是，在一个损伤模型中，在受伤的小鼠心脏中观察到心脏成纤维细胞的独特模式，这些模式跟随包裹的纤维带向收缩方向。在缺血伤害模型中，成纤维细胞死亡发生，随后从梗塞边界区重新填充。总的来说，这种精炼的心脏组织澄清技术和数字成像系统允许心脏成纤维细胞的三维可视化，而不受抗体穿透失败或以前因组织处理而失去荧光的问题的限制。

心脏成纤维细胞对于心脏的基线功能以及心脏损伤的反应至关重要。以前试图了解这些细胞的排列和形态，主要是在二维设置中进行的。然而，一个精致的心脏组织清除（图2）和三维成像技术已经发布，这使得心脏成纤维细胞的先进，更详细的可视化。通过这种成像技术，发现成纤维细胞密集，在未受伤的心脏中有一个旋转形态（图3，补充视频...

Watch this Scientific Journal Video about Refined CLARITY-Based Tissue Clearing for Three-Dimensional Fibroblast Organization in Healthy and Injured Mouse Hearts at JoVE.com

Refined CLARITY-Based Tissue Clearing for Three-Dimensional Fibroblast Organization in Healthy and Injured Mouse Hearts

开发并应用于成年小鼠心脏的组织清除方法。这种方法旨在清除密集的，自动荧光的心脏组织，同时保持标记成纤维细胞荧光归因于遗传报告器策略。

在健康和受伤的老鼠心脏中为三维纤维细胞组织进行精炼的清晰组织清除

Cardiovascular disease is the most prevalent cause of mortality worldwide and is often marked by heightened cardiac fibrosis that can lead to increased ...

这种新型组织清除协议改进了现有技术，使体内对心脏特定细胞类型及其在损伤条件下的行为有更准确的观察。此协议是快速和相当简单的。它允许维持马卡荧光与最小的干扰，从背景组织自动荧光，而无需细胞或组织染色。首先使用70%乙醇浸泡纱布垫清洁实验鼠的腹腔表面。使用手术剪刀做一个三厘米的横切口，大约三米以下的xiphoid过程和脱光小鼠从腹部到西菲德过程，以分离皮肤从底层腹部壁组织。在皮下腹部壁组织中切口两厘米，在 xiphoid 过程下方三厘米处切口，并通过肋骨笼从中线垂直切开。把肋骨笼钉回去露出心脏。并使用配备 27 号针头的 10 毫升注射器将冷 PBS 注射到高级静脉卡瓦和主动脉中。当所有的血液都从心脏冲洗，送10毫升的冷4%PFA到优越的静脉卡瓦和主动脉开始固定过程。当所有的固定剂都闪过，切除心脏，并使用直刀手术刀分离腹腔，右心室和隔膜和左心室。将心脏放入一个15毫升的冷4%PFA圆锥管中，在坚果上以4摄氏度进行夜间孵化。第二天早上，在刚准备好的水凝胶溶液中加入0.25%的光照启动器溶液，并将心脏转移到水凝胶中。将圆锥管包裹在铝箔中，在四摄氏度下进行夜间孵化，不会受到身体干扰。第二天早上，在37摄氏度的珠子浴中加热管子2.5小时，然后用钳子将心脏小心地转移到15毫升的PBS管中。在 PBS 中洗心脏三次，每次洗一小时，在 37 摄氏度的螺母上，并将心脏转移到有源电泳机的篮子中。将盖子固定到位，用电泳清除液填充活性电泳室储液。腔室完成后，将篮子浸入溶液中，并将电泳机的盖子拧紧到位。然后在 1.5 安培和 37 摄氏度下运行电泳机 1.5 小时。电泳后，在视觉上检查心脏，以确保没有不透明的组织残留。在15毫升的两个中洗清心三次，在新鲜的PBS中洗一个小时，每次洗37摄氏度。在将心脏浸入去色剂中以减少下黄体自动荧光之前。48 小时后，在 PBS 中洗涤心脏三次，以示效果，并在成像前 48 小时在新准备的 RIMS 中平衡心脏。对于已清除心脏的 3D 成像，首先，将一层薄薄的真空润滑脂放在 3D 打印底部储液层的底部，与正在成像的样品高度相同。将储液池装满RIMS，并使用移液器尖端去除任何气泡。小心地将心脏放在 RIMS 中，左心室壁朝上，注意解决方案中不会引入气泡。使用真空润滑脂将玻璃盖滑贴到 3D 打印的顶部储层片的底部表面，并将顶部储层盖滑落到底部储层上，注意避免气泡。然后用甘油填充顶部储层。使用协聚焦显微镜对被清除的心脏进行成像。将精密的心脏组织清除与 3D 成像相结合，可对心脏成纤维细胞进行高级详细可视化。使用这种成像技术，成纤维细胞被观察到密集包装，并在未受伤的心脏有一个旋转形态。缺血性输液损伤后，在缺血区发现心脏成纤维细胞在受伤后的头三天出现损失。到第7天和第14天，成纤维细胞已经迁移或增殖，以重新填充受伤的组织区域。到第28天，心脏受伤区域周围心脏成纤维细胞的密度最大。心肌梗塞受伤一天半后，受伤的左心室内几乎没有成纤维细胞。然而，到第三天，成纤维细胞扩大，并存在于大多数左心室。与缺血手术相比，在几周内输注二号血管紧张素和苯甲苯丙蛋白不会导致心脏组织损失和壁变薄。相反，成纤维细胞沿着右手螺旋收缩模式的轴线对齐，如使用精制组织清除协议时所见。此外，经过血管紧张素二和苯甲酸酯治疗后，成纤维细胞显得小而圆润，而不是在缺血性复发和心肌梗塞损伤模型中观察到的主轴形状。电泳清除需要小心执行，尤其是在与已接受伤害协议的心脏一起工作时。此协议可用于评估心脏成纤维细胞对体内损伤的反应。不同的荧光标记也可用于了解心脏在细胞水平上对损伤的反应。

Research

JoVE Journal

Medicine

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

异型三维<em在体外</em&gt;卵巢癌的发生和发展过程中的间质 - 上皮细胞相互作用模型

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical ...

科研

医学

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

三维组织培养模型研究人类原发性骨髓和恶性肿瘤

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

Three-dimensional Co-culture Model for Tumor-stromal Interaction

对于肿瘤基质相互作用的三维共培养模式

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Three-Dimensional 3D Tumor Spheroid Invasion Assay

三维（3D）肿瘤球体侵袭试验

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

人垂体腺瘤细胞的三维海藻酸钠珠文化

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from ...

Oral Biofilm Sampling for Microbiome Analysis in Healthy Children

口腔生物膜取样在健康儿童中的微生物分析

Oral biofilm and its molecular analysis provide a basis for investigating various dental research and clinical questions. Knowledge of biofilm composition ...

Optical Clearing and Imaging of Immunolabeled Kidney Tissue

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) ...

On-Site Sampling and Extraction of Brain Tumors for Metabolomics and Lipidomics Analysis

代谢组学和利皮多米分析脑肿瘤的现场采样与提取

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still ...

Advanced Cardiac Rhythm Management by Applying Optogenetic Multi-Site Photostimulation in Murine Hearts

通过在小鼠心脏中应用光遗传学多位点光刺激进行高级心律管理

Ventricular tachyarrhythmias are a major cause of mortality and morbidity worldwide. Electrical defibrillation using high-energy electric shocks is ...

Three-Dimensional Collagen Matrix Scaffold Implantation as a Liver Regeneration Strategy

三维胶原基质支架植入作为肝脏再生策略

Liver diseases are the leading cause of death worldwide. Excessive alcohol consumption, a high-fat diet, and hepatitis C virus infection promote fibrosis, ...