Name: three-dimensional rendering and analysis of immunolabeled, clarified human placental villous vascular networks
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这项工作得到了纽约国家办事处的支持, 为发展中残疾人士和胎盘分析有限责任公司, 新的拉罗谢尔, 纽约。

本议定书遵循了纽约国家发展残疾基础研究研究所人类研究伦理学委员会的指导方针。
1. 长柔毛树解剖
<ol><li>用磷酸盐抛光生理盐水 (PBS)14冲洗福尔马林固定胎盘组织, 在解剖显微镜的舞台上取出福尔马林并放置在培养皿中。使用手术刀和细钳, 以逗胎盘组织分开寻找长柔毛树 (白色丝状分支结构)。解剖出绒毛树 (几毫米长) 的碎片, 将组织转移到含有1毫升 PBS 的微管上。</li>
</ol>2. 免疫荧光染色
<ol><li>用吸管, 取出 pbs, 换成新鲜的 pbs。让我们站10分钟, 偶尔旋转。再重复一次。 注意: 这一步和所有进一步的步骤都是在室温下进行的, 除了主要的抗体孵化。</li>
	<li>使用吸管, 去除和取代 pbs +0.1% Triton-X100 + 2% 山羊血清 (pbs t GS) permeabilize 组织和阻断非特异性的二级抗体结合。孵育30分钟。</li>
	<li>用2% 山羊血清、小鼠单克隆 anti-CK7 和兔 anti-CK7 的 pbs 取代 pbs T GS。<ol>
<li>孵化组织过夜在4摄氏度。</li>
		<li>清除其余的抗体, 用 PBS-GS 清洗10分钟, 偶尔旋转。</li>
		<li>移除 pbs gs 并替换为包含山羊抗小鼠 igg 的 pbs gs, 再加上一个绿色发射 (520 nm) 荧光和山羊抗兔 igg 结合红外发射 (652 nm) 荧光。</li>
		<li>重复步骤2.1 删除抗体。</li>
		<li>将 immunolabeled 组织在室温下存放在黑暗中。</li>
	</ol>
</li>
</ol>3. 组织的澄清
<ol><li>用 PBS 在10分钟间隔内冲洗样品3次。</li>
	<li>通过用吸管去除 PBS 并用50% 乙醇取代它 (甲醇也可用) 来脱水组织。孵育10分钟, 用新鲜的50% 乙醇代替, 孵育10分钟. 再重复一次, 总共有3孵化。重复孵化与70%。然后用100% 乙醇代替, 孵育15分钟 (或者甲醇可以使用)。</li>
	<li>用一个细尖钳, 转移组织到一个新鲜的微管含有〜1毫升 Solution-1 4 小时。</li>
	<li>用细尖钳, 将组织转移到含有 Solution-2 的新鲜微管中4小时。 注意: 当储存在黑暗中时, 免疫荧光在室温下稳定至少6月。</li>
	<li>不要与任何水溶液混合, (例如安装介质), 因为清除将丢失。</li>
</ol>4. 显微镜安装
<ol><li>不要在标准的玻璃滑梯和盖玻片之间安装组织, 因为组织是软的, 容易变形。</li>
	<li>请参阅图 5B并在赛克斯摩尔会议厅中安装该组织, 如下所示;<ol>
<li>用细尖钳放置一个25毫米圆盖玻片在房间基地。</li>
		<li>用细尖钳把橡胶垫片放在底座上的盖玻片上。</li>
		<li>用吸管放置一滴 (~ 40 µL) 的 Solution-2 在盖玻片的中心。</li>
		<li>用细尖钳将一块组织从 Solution-2 转移到盖玻片中心的下落。</li>
		<li>在垫片顶部放置第二盖玻片。</li>
		<li>将锁定环放入底座顶部, 压缩盖玻片和垫片直至牢固。 注意: 小心不要过度收紧或盖玻片会粉碎。</li>
		<li>将22口径的针插入底座和垫片的端口。</li>
		<li>用1.5 毫升的 Solution-2 填充3毫升的注射器。</li>
		<li>在注射器上安装一个25口径的针头。</li>
		<li>将注射器针插入到相反的端口并通过垫片。</li>
		<li>确保室内空气通过其他25口径针, 轻轻注入 solution-2and 填充腔内。</li>
		<li>取出针头和注射器, 将房间放在共焦显微镜舞台上的大玻璃滑梯上。</li>
	</ol>
</li>
	<li>或者, 从硅烷板上切割自定义井。<ol>
<li>用剪刀切割 25.4 x 25.4 毫米2件从该板。</li>
		<li>用手术刀切开 ~ 12.7 x 12.7 毫米2在片断。</li>
		<li>把一块牢牢地压在显微镜滑梯上。</li>
		<li>用 Solution-2 填满井顶。</li>
		<li>将组织片从微管转移到井中心。</li>
		<li>用细尖的镊子把盖玻片放在井顶, 这样盖玻片的底部就会被 Solution-2 浸湿。</li>
		<li>在共焦显微镜阶段安装与组织的克斯克摩尔室组件。</li>
	</ol>
</li>
</ol>5. 共焦显微术
<ol><li>将细胞中的组织集中在10x 的目标上。</li>
	<li>在焦距平面上上下移动显微镜, 测量组织的顶部和底部之间的距离。</li>
	<li>移动舞台在2.5 µm 步骤收集一个共焦图像文件, 其中包含一组图像通过组织从上到下。</li>
</ol>6. 逆转清算
<ol><li>通过将组织转移到含有 > 20x 的微管中以100% 乙醇的体积来逆转清除。在1小时间隔替换与70% 乙醇, 然后50% 乙醇和最后 PBS。存放在4摄氏度在黑暗中。 注: 现在处理组织的标准石蜡嵌入, 切片和组织学或免疫组织化学染色。</li>
</ol>7. 反褶积
注意: 对于以下步骤, 软件命令、选项卡和下拉菜单是斜体的。
<ol><li>启动反褶积软件并打开图像文件。</li>
	<li>输入 "夏天y" 窗口中列出的参数。<ol>
<li>对于 "间距" , 在µm 中输入体素 x、y 和 z 维度。</li>
		<li>对于每个颜色通道, 从下拉菜单中选择用于染色的荧光染料。</li>
		<li>对于 "情态", 请从下拉菜单中选择 "激光扫描共焦"。</li>
		<li>对于 "物镜", 请从下拉菜单中选择 "10x"。</li>
		<li>对于 "浸入介质", 从下拉菜单中选择 "空气"。</li>
		<li>单击 "应用"按钮。</li>
	</ol>
</li>
	<li>单击 "Deconvolution" 选项卡。</li>
	<li>在下拉菜单中单击 "3D 反褶积" 。</li>
	<li>在 "3D-卷积"窗口中, 确保设置为: 1) 自适应的 psf 盲、2) 理论的 psf、3) 使用 (默认自适应的 psf、10迭代、中等噪声)、4) 基名称。</li>
	<li>单击 "绿色复选按钮"启动程序。</li>
	<li>完成后, 重复步骤 7.3-7.6 以重新启动程序。当完成第三次和最后一次重新启动它。</li>
	<li>在 "数据管理器"窗口中, 单击以 "10-10-10-" 为前缀的文件。</li>
	<li>单击 "文件"选项卡, 然后单击 "另存为"。</li>
	<li>在 "保存s" 窗口中, 从 "数据类型"下拉菜单中选择 "8bit 无符号整数" , 然后单击 "保存"。</li>
</ol>8. 图像处理
<ol><li>启动斐济软件。<ol>
<li>单击 "文件", 单击 "打开"并选择 Deconvolved 图像文件, 然后单击 "打开" 窗口中的 "打开"按钮。</li>
		<li>单击 "图像/颜色/分割通道" , 然后将三色图像文件转换为仅包含一种颜色的三个新文件。保存包含 immunolabeled 长柔毛毛细管图像集的红色图像文件。删除绿色和蓝色图像文件。</li>
		<li>用以下方法减去红色图像文件中的背景荧光: "处理/减去背景"。</li>
		<li>在下拉菜单中, 将滚动球半径设置为10像素。</li>
		<li>保留未选中的4菜单选项, 然后单击 "确定"。</li>
		<li>单击 "进程堆栈" 菜单上的 "是" 。</li>
		<li>通过打开 "图像/调整/亮度对比度"菜单并调整最小、最大、亮度和对比度设置, 删除固有的组织荧光 (自发荧光)。注意不要去除任何毛细血管免疫荧光。</li>
		<li>通过打开 "图像/调整/阈值"菜单并调整滑块以突出显示毛细管免疫荧光, 以最小化剩余的无关荧光。检查 "深色背景"框, 然后单击 "应用"。在 "转换为二进制下拉"菜单上, 选中 "黑色背景"框, 然后单击 "确定"。保存黑白 (二进制) 图像文件以进行分析。</li>
	</ol>
</li>
</ol>9. 分析
<ol><li>对象计数和骨架。<ol>
<li>选择在步骤8.1.8 中创建的二进制图像文件。</li>
		<li>单击 "分析/3D OC 选项" , 然后在下拉菜单中只检查 "卷"和 "存储结果..."框。确保未选中"显示数字"框。单击 "确定" 按钮.
</li>
		<li>"Analyze/3D 对象计数器"上的 Click。在下拉菜单中, 将 "大小筛选: 最小值"设置为5000。确保未选中 "在边缘上排除对象"框。检查是否选中了 "对象的、统计信息和"摘要 "框。单击 "确定" 按钮。 注意: 这可能需要几分钟时间, 具体取决于文件大小和计算机配置。</li>
		<li>保存统计数据表和对象映射文件。</li>
		<li>选择对象映射文件。单击 "插件/骨架/缩略2维-3 d"以缩略二进制文件中的毛细管网络。单击 "文件/另存为"。在下拉菜单中选择 "Tiff......" 。在 "另存为 TIFF" 窗口中, 将 "Skel" 添加到对象映射文件名中, 然后单击 "保存"按钮.
</li>
		<li>单击 "分析/骨架/分析骨架 2 d-3 d"。在下拉菜单中接受默认设置, 然后单击 "确定" 按钮。保存分支"信息"表、"结果"表和两个输出文件 (Skel 对象标记为骨架和带标记的骨架)。</li>
	</ol>
</li>
</ol>

<ol>
	<li>Thornburg, K. L., Kolahi, K., Pierce, M., Valent, A., Drake, R., Louey, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+features+of+placental+programming.">Biological features of placental programming.</a> Placenta. 48, 47-53 (2016).</li><li>Misra, D. P., Salafia, C. M., Charles, A. K., Miller, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Birth+weights+smaller+or+larger+than+the+placenta+predict+BMI+and+blood+pressure+at+age+7+years.">Birth weights smaller or larger than the placenta predict BMI and blood pressure at age 7 years.</a> J Dev Orig Health Dis. 1 (2), 123-130 (2010).</li><li>Burton, G. J., Fowden, A. L., Thornburg, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Placental+Origins+of+Chronic+Disease.">Placental Origins of Chronic Disease.</a> Physiol Rev. 96 (4), 1509-1565 (2016).</li><li>Srinivasan, A. P., Omprakash, B. O. P., Lavanya, K., Subbulakshmi Murugesan, P., Kandaswamy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+prospective+study+of+villous+capillary+lesions+in+complicated+pregnancies.">A prospective study of villous capillary lesions in complicated pregnancies.</a> J Pregnancy. 2014, 193925 (2014).</li><li>Jones, C. J. P., Desoye, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+possible+function+for+placental+pericytes.">A new possible function for placental pericytes.</a> Cells Tissues Organs. 194 (1), 76-84 (2011).</li><li>Maly, A., Goshen, G., Sela, J., Pinelis, A., Stark, M., Maly, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histomorphometric+study+of+placental+villi+vascular+volume+in+toxemia+and+diabetes.">Histomorphometric study of placental villi vascular volume in toxemia and diabetes.</a> Hum Pathol. 36 (10), 1074-1079 (2005).</li><li>Ellery, P. M., Cindrova-Davies, T., Jauniaux, E., Ferguson-Smith, A. C., Burton, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+transcriptional+activity+in+the+syncytiotrophoblast+of+the+human+placenta.">Evidence for transcriptional activity in the syncytiotrophoblast of the human placenta.</a> Placenta. 30 (4), 329-334 (2009).</li><li>Jirkovsk&#225;, M., Kub&#237;nov&#225;, L., Jan&#225;&#269;ek, J., Kal&#225;b, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3-D+study+of+vessels+in+peripheral+placental+villi.">3-D study of vessels in peripheral placental villi.</a> Image Anal Stereol. 26 (3), 165-168 (2011).</li><li>Pearce, P., 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=Image-Based+Modeling+of+Blood+Flow+and+Oxygen+Transfer+in+Feto-Placental+Capillaries.">Image-Based Modeling of Blood Flow and Oxygen Transfer in Feto-Placental Capillaries.</a> PLOS ONE. 11 (10), 0165369 (2016).</li><li>Plitman Mayo, R., Olsthoorn, J., Charnock-Jones, D. S., Burton, G. J., Oyen, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computational+modeling+of+the+structure-function+relationship+in+human+placental+terminal+villi.">Computational modeling of the structure-function relationship in human placental terminal villi.</a> J Biomech. 49 (16), 3780-3787 (2016).</li><li>Mayhew, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+stereological+perspective+on+placental+morphology+in+normal+and+complicated+pregnancies.">A stereological perspective on placental morphology in normal and complicated pregnancies.</a> J Anat. 215 (1), 77-90 (2009).</li><li>Teasdale, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gestational+changes+in+the+functional+structure+of+the+human+placenta+in+relation+to+fetal+growth:+a+morphometric+study.">Gestational changes in the functional structure of the human placenta in relation to fetal growth: a morphometric study.</a> Am J Obstet Gynecol. 137 (5), 560-568 (1980).</li><li>Richardson, D. S., Lichtman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clarifying+Tissue+Clearing.">Clarifying Tissue Clearing.</a> Cell. 162 (2), 246-257 (2015).</li><li>Dulbecco, R., Vogt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plaque+formation+and+isolation+of+pure+lines+with+poliomyelitis+viruses.">Plaque formation and isolation of pure lines with poliomyelitis viruses.</a> J Exp Med. 99 (2), 167-182 (1954).</li><li>Gill, J. S., Salafia, C. M., Grebenkov, D., Vvedensky, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+oxygen+transport+in+human+placental+terminal+villi.">Modeling oxygen transport in human placental terminal villi.</a> J Theor Biol. 291, 33-41 (2011).</li><li>Plitman Mayo, R., Charnock-Jones, D. S., Burton, G. J., Oyen, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+modeling+of+human+placental+terminal+villi.">Three-dimensional modeling of human placental terminal villi.</a> Placenta. 43, 54-60 (2016).</li></ol>

作者没有什么可透露的。

机构审查委员会批准了收集胎盘绒毛组织选择性终止妊娠的福尔马林固定。在执行这些程序之前, 对医疗记录作了简要的回顾, 指出了孕产妇年龄、均等, 并确认没有任何潜在的医疗 (如例如、高血压、糖尿病和狼疮) 或胎儿 (结构或染色体异常,异常生长) 进行。数据表和任何收集的标本只用研究 ID 标记。因此, 所有标本都在离开操作套件之前被取消标识。绒毛由训练有素的观察者收集;去除了...

我们对发育中胎盘及其病理的理解, 在很大程度上局限于推断相邻绒毛之间的空间关系以及从组织学切片中提取的毛细血管。在这项研究中, 我们通过开发方法来生成三维 (3D) 的人胎盘毛细管网络的效果图, 这些结果适合于分析毛细管网络特征 (例如分支、固态)。为此, 我们将免疫荧光染色与两种商业组织清洁产品、Visikol-1 和 Visikol-2 (以下简称 Solution-1 和 Solution-2) 相结合。
人胎盘是一个巨大的复杂的血管位于母亲的 intervillous 血液和发育中的胎儿之间的接口。脐带从插入到绒毛膜板中延伸出来, 在动脉和静脉网络中 ramify, 用精心制作的血管网络覆盖绒毛膜表面。他们的末端然后击穿入胎盘盘的内部或深度, 在那里他们接受几个分支世代并且结束在末端绒毛和他们包含的毛细管网络, 气体交换的地点, 营养素和新陈代谢胎儿和母体血液之间的浪费。
对胎盘毛细管网络的侮辱在发展期间有持久的后果为胎儿、新生儿和紧急成人1,2,3的健康。鉴于妊娠相关的病症, 如流产, 宫内生长限制, 先兆子痫和孕妇糖尿病4,5,6有一个很高的价值, 制定测量方法和胎盘绒毛毛细管网络的表征。一个主要的障碍是胎盘血管网络包含广泛的范围。表面血管网络的直径可达4-5 毫米。末端具长柔毛毛细血管的顺序为 10-20 µm 直径;胎盘含有超过300公里的血管7。目前, 很少有易于使用和快速的技术, 可以捕捉这些极端的船只规模。到目前为止, 只有少量的绒毛可以通过显微镜来呈现。例如, Jirkovska et将重点放在足月胎盘绒毛上, 将共焦显微术与连续光学切片结合, 从120µm 厚剖面获得1µm 间隔;未提供所研究的样本数和统计数据, 8。对毛细管结构进行了鉴定, 并对绒毛和毛细血管的轮廓进行了手工绘制, 并将追查导出用于图像分析。虽然作者们讨论了他们的发现对 "生长的长柔毛血管网" 的影响, 但这样的结论是有问题的, 只有 "期限" (36 + 周孕龄) 组织的研究。类似地, 梅奥et al 和皮尔斯et等. 依赖于同一年龄的组织, 用于模拟血液流动和氧转运, 但其分析仅限于几个术语, 终端绒毛9,10.视学也被应用于研究绒毛状血管的结构。但同样的, 重点通常是以后怀孕分娩 liveborn 婴儿有一个或多个妊娠并发症11,12。
直到最近, 共焦显微术被限制为100-200 µm 的组织深度的成像, 因为吸收的激发和发射荧光的覆盖组织13 。虽然组织清除和3D 组织学已被广泛描述的文献和有许多方法, 组织清除许多不适合使用与组织一般, 因为他们不可逆转地破坏细胞形态学通过 hyperhydration蛋白质或脂肪的去除。因此, 无法验证这些结果是否表明了组织本身, 而不是加工工件, 而我们的组织清除过程是一种可逆的技术, 能够验证传统的组织学。组织清除一般涉及三种主要方法之一: 1) 在 RI 匹配解中, 对组织成分的折射率 (ri) 进行均匀匹配, 消除由于连续而引起的透镜的累积光散射。低 ri (细胞质) 和高 ri (蛋白质/脂) 成分的波动;2) 将脂质成分嵌入水凝胶中, 利用电泳/扩散法去除脂质成分;3) 蛋白质结构的扩张/变性, 以增加溶剂的穿透力, 以鼓励 RI 13的均匀性。虽然这些方法可以使组织透明, 并允许生成生物标志物的3D 表示, 但这些3D 表示有疑问的临床价值, 因为它是挑战, 以确定这些图像是否预示着组织的性质或组织清除过程。另一方面, 由于我们的组织清除是可逆的标准组织学和/或免疫组化可以应用到同一组织, 以评估是否有临床意义的改变。
本研究对47例从23例正常和选择性终止妊娠到9-23 个完成周妊娠期和两次足月正常分娩的远端长柔毛标本进行了分析。免疫荧光标记滋养层和内皮已经使对长柔毛血管网络的变化及其复杂性进行了定量和自动化的分析。
通过该协议, 我们对终端绒毛及其毛细管网络进行了前所未有的分离和分析。这种方法, 当应用到长柔毛和毛细管网络发展横跨妊娠将确定这些属性是出生的健康儿童的基础。当应用于复杂妊娠的研究时, 它还将阐明胎盘病变何时以及如何改变其鞘的绒毛状树木和毛细血管网络, 以及这些如何影响胎儿的福祉。

<table><tbody><tr><td>10% Formaldehyde solution (w/v) in aqueous phosphate buffer</td><td>Macron Fine Chemicals</td><td>H-121-08</td><td>General fixation agent, ready to use formula, use caution as vapors are toxic</td></tr><tr><td>Scalpel blades</td><td>ThermoFisher Scientific</td><td>08-916-5B No. 11</td><td></td></tr><tr><td>Scalpel</td><td>ThermoFisher Scientific</td><td>08-913-5</td><td></td></tr><tr><td>Fine forceps</td><td>Electron Microscopy Services</td><td>78354-119</td><td></td></tr><tr><td>Micro Tube (1.7 mL)</td><td>PGC Scientifics</td><td>505-201</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Sigma</td><td>D8537</td><td>PBS</td></tr><tr><td>Pipette</td><td>VWR</td><td>52947-948</td><td>disposable, 3ml transfer pipette</td></tr><tr><td>Triton X-100</td><td>Boehriner Mannheim</td><td>789 704</td><td>Dilute to 0.1% from stock</td></tr><tr><td>Goat Serum</td><td>Gibco</td><td>16210-064</td><td>Dilute to 2% in PBS solution</td></tr><tr><td>Mouse monoclonial anti-ck7 Keratin 7 Ab-2 (Clone OV-TL 12/30)</td><td>ThermoFisher Scientific</td><td>MS-1352-RQ</td><td></td></tr><tr><td>Rabbit Anti-CD31 antibody</td><td>Abcam</td><td>ab28364</td><td></td></tr><tr><td>green emitting (520 nm) fluorochrome&amp;nbsp;</td><td>Invitrogen</td><td>A11017</td><td>Alexa-Fluor 488</td></tr><tr><td>infrared emitting (652 nm) fluorochrome</td><td>Invitrogen</td><td>A21072</td><td>Alexa Fluor 633</td></tr><tr><td>Ethanol alcohol 200 proof</td><td>Pharmco-Aaper</td><td>111000200</td><td>Dilute down to lower concentrations using PBS as needed</td></tr><tr><td>Solution-1</td><td>Visikol Inc.</td><td></td><td>Visikol Histo-1</td></tr><tr><td>Solution-2</td><td>Visikol Inc.</td><td></td><td>Visikol Histo-2</td></tr><tr><td>Skyes-Moore chambers</td><td>BellCo Glass Inc.</td><td>P/N 1943-11111</td><td></td></tr><tr><td>25 gauge needle</td><td>ThermoFisher Scientific</td><td>14-826AA</td><td>BD Precision Glide Needes</td></tr><tr><td>3 mL syringe</td><td>ThermoFisher Scientific</td><td>14-823-40</td><td>BD disposable syringe</td></tr><tr><td>PDMS silicon sheets</td><td>McMaster-Carr</td><td>P/N 578T31</td><td></td></tr><tr><td>confocal microscope</td><td>Nikon Inc.</td><td></td><td>Nikon C1 Confocal Microscope</td></tr><tr><td>Deconvolution software</td><td>Media Cybernetics</td><td></td><td>AutoQuant X22</td></tr><tr><td>Fiji image processing software</td><td></td><td></td><td>free, Open source&amp;nbsp; software available at https://fiji.sc</td></tr><tr><td>Hematoxylin</td><td>Leica Biosystems</td><td>3801570</td><td>Component 1 of SelecTech H&amp;amp;E staining system</td></tr><tr><td>Alcoholic Eosin</td><td>Leica Biosystems</td><td>3801615</td><td>Component 2 of SelecTech H&amp;amp;E staining system</td></tr><tr><td>Blue Buffer</td><td>Leica Biosystems</td><td>3802918</td><td>Component 3 of SelecTech H&amp;amp;E staining system</td></tr><tr><td>Aqua Define MCX</td><td>Leica Biosystems</td><td>3803598</td><td>Component 4 of SelecTech H&amp;amp;E staining system</td></tr><tr><td>Immunohistochemistry detection system</td><td>ThermoFisher Scientific</td><td>TL-125-QHD</td><td>UltraVision Quanto Detection System HRP DAB</td></tr></tbody></table>

three-dimensional rendering and analysis of immunolabeled, clarified human placental villous vascular networks

母亲和胎儿之间的养分和气体交换发生在母体 intervillous 血的界面和巨大的长柔毛毛细血管网络中, 构成了人类胎盘的大部分实质。远端长柔毛毛细管网络是胎儿血液供应的终点后, 几代分支的血管延伸出脐带。这个网络有一个连续的细胞鞘, 合胞滋养屏障层, 防止胎儿血液和产妇血液的混合, 在其中持续沐浴。对胎盘毛细血管网络的完整性的侮辱, 发生在诸如孕产妇糖尿病、高血压和肥胖等疾病中, 会给胎儿、婴儿和成人带来严重的健康风险。为了更好地定义这些侮辱的结构效果, 为这项研究制定了一项协议, 它捕获毛细管网络结构的顺序为 1-2 毫米3 , 其中一个可以调查其拓扑特征的完全复杂性。为达到这一目的, 对胎盘端绒毛簇进行了解剖, 滋养层和毛细血管内皮 immunolabeled。然后用新的组织清理过程澄清这些样本, 从而可以获得共焦图像栈到 z 深度的 ~ 1 毫米。然后对这些堆栈的三维渲染进行处理和分析, 以生成基本的毛细管网络度量, 如体积、毛细管分支数和毛细管分支端点, 以验证这种方法是否适合毛细管网络特性。

从8周孕龄到足月分娩, 将人胎盘端绒毛簇中毛细血管的生成3D 效果计算为个体簇和骷髅进行网络分析。胎盘的功能单位 (图 1A) 是绒毛树, 是它们穿透胎盘实质的表面血管的延伸 (图 1B, 并在1C中放大)。然后 immunolabeled 解剖树的远端部分 (Figure1D和1E) 的碎片, 以显示绒?...

Watch this Scientific Journal Video about Three-dimensional Rendering and Analysis of Immunolabeled, Clarified Human Placental Villous Vascular Networks at JoVE.com

Three-dimensional Rendering and Analysis of Immunolabeled, Clarified Human Placental Villous Vascular Networks

本研究提出了一种可逆转组织清除、染色、3 d 渲染和分析人胎盘绒毛样品中血管网络的方法, 其顺序为 1-2 毫米3。

Immunolabeled, 澄清人胎盘长柔毛血管网络的三维渲染与分析

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that ...

three-dimensional-rendering-analysis-immunolabeled-clarified-human

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

9.6K 次观看.  The New York State Office for People with Developmental Disabilities. 本研究提出了一种可逆转组织清除、染色、3 d 渲染和分析人胎盘绒毛样品中血管网络的方法, 其顺序为 1-2 毫米3。

视频: Three-dimensional Rendering and Analysis of Immunolabeled, Clarified Human Placental Villous Vascular Networks

siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend diseases related to pregnancy. In this protocol, human primary villous cytotrophoblasts freshly isolated from placentas through a standard DNase/trypsin protocol are microporated with small interfering RNA (siRNA). This approach provided greater efficiency for siRNA transfection when compared to a lipofection-based method. Transfected cells can subsequently be analyzed by standard Western blot within a time frame of 3-4 days post-transfection. In addition, using cultured primary villous cytotrophoblasts, Electrophoretic Mobility Shift Assay (EMSA) analysis was optimized and performed on extracts from days 1 to 4. The use of these cultured primary cells and the protocol described allow for an evaluation of the implication of specific genes and transcription factors in the process of villous cytotrophoblast differentiation into a syncytiotrophoblast-like cell layer. However, the limited time span allowable in culture precludes the use of methods requiring more time, such as generation of a stable cell population. Therefore testing of this cell population requires highly optimized gene transfer protocols.

This protocol describes a method for efficiently transfecting siRNA in freshly isolated human villous cytotrophoblasts using microporation and identifying DNA-protein complexes in these cells. Transfected cells can be monitored by Western blot and EMSA analyses during the 4-day culture time.

在新鲜分离的人代绒毛滋养细胞转染和EMSA分析

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend ...

科研

遗传学

Assessment of Maternal Vascular Remodeling During Pregnancy in the Mouse Uterus

The placenta mediates the exchange of factors such as gases and nutrients between mother and fetus and has specific demands for supply of blood from the maternal circulation. The maternal uterine vasculature needs to adapt to this temporary demand and the success of this arterial remodeling process has implications for fetal growth. Cells of the maternal immune system, especially natural killer (NK) cells, play a critical role in this process. Here we describe a method to assess the degree of remodeling of maternal spiral arteries during mouse pregnancy. Hematoxylin and eosin-stained tissue sections are scanned and the size of the vessels analysed. As a complementary validation method, we also present a qualitative assessment for the success of the remodeling process by immunohistochemical detection of smooth muscle actin (SMA), which normally disappears from within the arterial vascular media at mid-gestation. Together, these methods enable determination of an important parameter of the pregnancy phenotype. These results can be combined with other endpoints of mouse pregnancy to provide insight into the mechanisms underlying pregnancy-related complications.

This protocol describes a technique to assess changes in the maternal vasculature during pregnancy in mice. Using stereological methods, remodeling of the decidual spiral arteries is assessed quantitatively and the results confirmed qualitatively using immunohistochemistry.

产妇血管重构的评估在怀孕期间在小鼠子宫

The placenta mediates the exchange of factors such as gases and nutrients between mother and fetus and has specific demands for supply of blood from the ...

发育生物学

An In Vitro 3D Model and Computational Pipeline to Quantify the Vasculogenic Potential of iPSC-Derived Endothelial Progenitors

Induced pluripotent stem cells (iPSCs) are a patient-specific, proliferative cell source that can differentiate into any somatic cell type. Bipotent endothelial progenitors (EPs), which can differentiate into the cell types necessary to assemble mature, functional vasculature, have been derived from both embryonic and induced pluripotent stem cells. However, these cells have not been rigorously evaluated in three-dimensional environments, and a quantitative measure of their vasculogenic potential remains elusive. Here, the generation and isolation of iPSC-EPs via fluorescent-activated cell sorting are first outlined, followed by a description of the encapsulation and culture of iPSC-EPs in collagen hydrogels. This extracellular matrix (ECM)-mimicking microenvironment encourages a robust vasculogenic response; vascular networks form after a week of culture. The creation of a computational pipeline that utilizes open-source software to quantify this vasculogenic response is delineated. This pipeline is specifically designed to preserve the 3D architecture of the capillary plexus to robustly identify the number of branches, branching points, and the total network length with minimal user input.

Endothelial progenitors derived from induced pluripotent stem cells (iPSC-EPs) have the potential to revolutionize cardiovascular disease treatments and to enable the creation of more faithful cardiovascular disease models. Herein, the encapsulation of iPSC-EPs in three-dimensional (3D) collagen microenvironments and a quantitative analysis of these cells&#8217; vasculogenic potential are described.

用于量化 iPSC 衍生内皮孕激素的血管生成潜力的体外 3D 模型和计算管道

Induced pluripotent stem cells (iPSCs) are a patient-specific, proliferative cell source that can differentiate into any somatic cell type. Bipotent ...

Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is imperative to design experiments using human cells and tissues. An advantage of organ culture is maintenance of three-dimensional (3D) structural organization and extracellular matrix. The goal of the method described here is successful establishment of ex vivo human gestational tissue organ cultures and their healthy culture maintenance for 72-96 hr. The protocol details the immediate processing of research-consented, placental and decidual specimens fresh from the operating suite. These are abundant specimens that would otherwise be discarded. Detailed instructions on the sterile collection of these samples, including morphologic details on how to select appropriate tissues to establish 3D organ cultures, is provided. Placental villous and decidual tissues are microdissected into 2-3 mm3 pieces and placed separately on matrix-lined transwell filters and cultured for several days. Villous and decidual organ cultures are well suited for the study of human host-pathogen interaction. As compared to other model organisms, these human cultures are particularly advantageous to examine mechanism of infection for pathogens that demonstrate variable patterns of host specificity. As an example, we demonstrate infection of placental and decidual organ cultures with the clinically relevant, facultative intracellular bacterial pathogen Listeria monocytogenes.

A simple method to establish primary human placental (villous) and decidual organ cultures is described. Villous and decidual organ cultures are invaluable tools for studying pathogenesis at the human maternal-fetal interface. Infection with the facultative intracellular bacterium Listeria monocytogenes is demonstrated.

人胎盘及蜕膜器官培养研究在母胎界面感染

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is ...

免疫与感染

使用基于流式的方法改善离体胎盘外植体培养

Ex Vivo Placental Explant Flow Culture - Mimicking the Dynamic Conditions In Utero

The existing ex vivo placental explant culture models are primarily grounded in static culture systems using well plates. However, these models inadequately reflect the dynamic in utero setting, where the placenta encounters constant slight shear stress due to plasma or blood flow. To address this limitation, a flow culture system has been devised to bring ex vivo placental explant cultivation closer to the in utero flow conditions experienced within the maternal body. Within this approach, placental explants are cultivated in a sequence of five interconnected flow chambers. This setting&#160;maintains physiological oxygen concentrations and a consistent flow rate. The collected data reveals that under flow conditions, the preservation of tissue morphology exhibits notable enhancement compared to conventional static methods. This innovative technique introduces a straightforward means of ex vivo placental explant culture, offering a more faithful representation of the dynamic in vivo environment. Moreover, this study introduces new&#160;possibilities for investigating the functional dynamics of the feto-maternal interface. By embracing feasible dynamic methodologies, a deeper comprehension of placental biology is facilitated, underscoring its relevance for maternal-fetal health.

作者聚焦:通过胎盘组织中的单细胞测序和空间组织学推进研究 

Here is a protocol for culturing placental explants under constant flow conditions. This approach enhances traditional static villous culture systems by enabling the replication of dynamic physiological environments.

离体胎盘外植体流动培养 - 模拟子宫内的动态条件

The existing ex vivo placental explant culture models are primarily grounded in static culture systems using well plates. However, these models ...

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching morphogenesis. We have developed the limb skin vasculature model to study vascular development in which a pre-existing primitive capillary plexus is reorganized into a hierarchically branched vascular network. Whole-mount confocal microscopy with multiple labelling allows for robust imaging of intact blood vessels as well as their cellular components including endothelial cells, pericytes and smooth muscle cells, using specific fluorescent markers. Advances in this limb skin vasculature model with genetic studies have improved understanding molecular mechanisms of vascular development and patterning. The limb skin vasculature model has been used to study how peripheral nerves provide a spatial template for the differentiation and patterning of arteries. This video article describes a simple and robust protocol to stain intact blood vessels with vascular specific antibodies and fluorescent secondary antibodies, which is applicable for vascularized embryonic organs where we are able to follow the process of vascular development.

We introduce a whole-mount immunohistochemistry and laser scanning confocal microscopy with multiple labelling for analyzing intricate vascular network formation in mouse embryonic limb skin.

全安装胚胎肢体皮肤血管的免疫组织化学分析：一个模型系统，研究在胚胎血管分支形态发生

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching ...

生物学

Retinal Pericytes Visualization Using Tissue-Clearing and Fluorescent Labeling

3D Visualization of Retinal Vascular Pericytes in Mice by Immunostaining

Retinal pericytes are essential for vascular development and stability of the retina, playing a key role in maintaining the integrity of the retinal vasculature. To provide a detailed view of the morphological characteristics of pericytes, this study described a new approach combining the retro-orbital injection of a fluorescent agent, immunofluorescent-staining, and tissue-clearing treatment. Firstly, the fluorescent tomato lectin was injected into the retro-orbital sinus of the live mouse to label the retinal vasculature. After 5 min, the mouse was sacrificed, and its intact retina was carefully removed from the retinal cup and immunofluorescently stained with platelet-derived growth factor receptor &#946; to reveal the pericytes. Additionally, the stained retina was treated with tissue-clearing reagent and whole mounted on the microscope slide. Through these approaches, the retinal vasculature and pericytes were clearly observed in the transparent retina. Under a confocal microscope, we obtained more opportunities to take high-resolution images for further reconstructing and analyzing the morphological characteristics of pericytes along the retinal vascular tree in a three-dimensional view. Methodologically, this protocol offers an effective approach for visualizing pericytes within the retinal vasculature, providing valuable insights into their role under both physiological and pathological conditions.

The pericytes in retinal vasculature were examined by immunofluorescent staining with platelet-derived growth factor receptor &#946; after retro-orbital injection of fluorescent tomato lectin. The labeled retina was further treated with the tissue-clearing method and whole mounted for visualizing the three-dimensional views of pericytes surrounding retinal vasculature under a confocal microscope.

Retinal pericytes are essential for vascular development and stability of the retina, playing a key role in maintaining the integrity of the retinal ...

生物化学

The 4-vessel Sampling Approach to Integrative Studies of Human Placental Physiology In Vivo

The human placenta is highly inaccessible for research while still in utero. The current understanding of human placental physiology in vivo is therefore largely based on animal studies, despite the high diversity among species in placental anatomy, hemodynamics and duration of the pregnancy. The vast majority of human placenta studies are ex vivo perfusion studies or in vitro trophoblast studies. Although in vitro studies and animal models are essential, extrapolation of the results from such studies to the human placenta in vivo is uncertain. We aimed to study human placenta physiology in vivo at term, and present a detailed protocol of the method. Exploiting the intraabdominal access to the uterine vein just before the uterine incision during planned cesarean section, we collect blood samples from the incoming and outgoing vessels on the maternal and fetal sides of the placenta. When combining concentration measurements from blood samples with volume blood flow measurements, we are able to quantify placental and fetal uptake and release of any compound. Furthermore, placental tissue samples from the same mother-fetus pairs can provide measurements of transporter density and activity and other aspects of placental functions in vivo. Through this integrative use of the 4-vessel sampling method we are able to test some of the current concepts of placental nutrient transfer and metabolism in vivo, both in normal and pathological pregnancies. Furthermore, this method enables the identification of substances secreted by the placenta to the maternal circulation, which could be an important contribution to the search for biomarkers of placenta dysfunction.

The 4-vessel Sampling Approach to Integrative Studies of Human Placental Physiology In Vivo

We present a detailed method to study human placental physiology in vivo at term. The method combines blood sampling from the incoming and outgoing vessels on the maternal and fetal sides of the placenta with ultrasound measurements of volume blood flow and placental tissue sampling.

人体胎盘生理学综合研究的4血管取样方法<em&gt;体内</em

The human placenta is highly inaccessible for research while still in utero. The current understanding of human placental physiology in vivo is therefore ...

Immunolabeled, 澄清人胎盘长柔毛血管网络的三维渲染与分析

摘要

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此视频中的章节

在新鲜分离的人代绒毛滋养细胞转染和EMSA分析

产妇血管重构的评估在怀孕期间在小鼠子宫

用于量化 iPSC 衍生内皮孕激素的血管生成潜力的体外 3D 模型和计算管道

人胎盘及蜕膜器官培养研究在母胎界面感染

离体胎盘外植体流动培养 - 模拟子宫内的动态条件

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