这项研究得到了中国博士后 (No. 2014T70392 至 YF)、中国国家自然科学基金 (No. 81673766 至 YF)、新教师启动基金、复旦大学 Zuoxue 基金会的赠款和发展上海市高峰学科综合医学项目 (20150407)。

所有涉及动物的程序均遵循复旦大学上海医学院动物伦理委员会的指导方针 (批准编号 20160225-013)。
1. 透明小鼠卵巢的制备
<ol><li>
解决方案的准备
	<ol>
<li>准备磷酸盐缓冲盐水 (PBS) 溶液 (1 米, pH 7.6) 与0.1% 海卫 X-100 (PBST)。制作 1 L 10x PBS 库存溶液, 混合87克氯化钠, 3.1 克 NaH2PO4 ·2H2O, 28.7 克 Na2HPO4 · 12H2o 添加 dH2至 1 L, 并搅拌〜 2 h. 调整到 pH 7.4 与 1 N 氢氧化钠和室温贮存。使用 dH2O 将此10x 库存解决方案稀释 1/10, 然后在 PBS 中制作0.1% 海卫 X-100。</li>
		<li>制备水凝胶溶液 (pH 值 7.5), 由 mixings 4% (重量/卷) 甲醛 (粉煤灰) 溶液, 4% (重/卷) 丙烯酰胺, 0.05% (重量/卷) bisacrylamide, 和 0.25% (重量/卷) VA-044 启动器在双蒸馏水冰。</li>
		<li>在双蒸馏水中, 将含有 4% (重/卷) 十二烷基硫酸钠 (SDS) 的 200 mM 硼酸钠缓冲液混合, 准备清除溶液 (pH 值 8.5)。</li>
	</ol>
</li>
	<li>
Transcardial 灌注和卵巢准备
	<ol>
<li>在所有的过程中保持所有的解决方案。</li>
		<li>麻醉成年雌性野生型 C57BL/6 小鼠腹腔注射戊巴比妥 (0.35 毫克/千克) 溶液。一旦它显示没有脚趾夹紧的反应, 老鼠是正确地被麻醉的。</li>
		<li>用粘胶带固定在扁平泡沫板上的四肢伸展的老鼠。</li>
		<li>通过胸、皮、骨和剪刀在剑过程中切开心脏。</li>
		<li>在动物的右心房做一个切口, 然后用20毫升的冰冷 1x PBS 在10毫升/分钟灌注左心室。</li>
		<li>灌注至少20毫升的水凝胶溶液在10毫升/分钟的动物。</li>
		<li>用剪刀切开 medioventral 线上的腹部皮肤, 露出整个腹腔, 并切除肠道。收集卵巢和子宫, 以保持完整的卵泡形态学。</li>
		<li>在显微镜下去除卵巢周围的脂肪组织。</li>
	</ol>
</li>
	<li>
气
	<ol>
<li>将卵巢放在10毫升水凝胶溶液中, 在4° c 内固定3天。</li>
		<li>将卵巢移植到5毫升的管子中, 用水凝胶溶液填充管。伸展膜在顶部的管, 然后包裹膜周围的颈部的管。 注意: 确保膜和水凝胶溶液之间没有气泡。</li>
		<li>在37° c 的恒温箱中放置一根管子, 最多3小时, 使水凝胶聚合。</li>
		<li>用刮刀将卵巢从凝胶中取出, 用纸巾小心地从卵巢周围移除多余的凝胶。用 1x PBS 冲洗卵巢四次。</li>
	</ol>
</li>
	<li>
被动结算
	<ol>
<li>将卵巢浸入10毫升的清除溶液中。轻轻摇动在 80 rpm 在37° c 开始清算过程。</li>
		<li>在第一周, 每3天更改一次解决方案。一周后, 每周刷新一次清除解决方案, 直到卵巢变得清晰。通常这个过程大约需要2-3 星期。 注: 卵巢可直接 immunostained 或可在4° c 的 PBS 中以叠氮化钠 (0.02%) 的形式储存, 免疫前数周。</li>
	</ol>
</li>
</ol>2. 免疫
<ol><li>洗卵巢两次在 PBST 在1天在37° c 在一个振动筛。</li>
	<li>孵育在主抗体/PBST 溶液 (表 1) 的一个振动筛上的卵巢在天2-3 在37° c。在这里的工作中, 主要的抗体是血小板-内皮细胞黏附分子 1 (CD31) 和酪氨酸羟化酶稀释1:10 和1:50 分别在 PBST。 注意: 如果使用两种或多种初级抗体, 请确保有不同的动物来源。</li>
	<li>洗掉的主要抗体与 PBST 缓冲在37° c 天4在一个振动筛。</li>
	<li>孵育卵巢与所需的第二抗体在 PBST 在5-6 天在37° c 在一个振动筛。在这里介绍的工作中, 次要抗体是 alexa 面粉488为酪氨酸羟化酶和 alexa 面粉594为 CD31 稀释在1:50 在 PBST。</li>
	<li>在37° c 的7天, 在一个振动筛上, 用 PBST 缓冲液冲洗第二抗体。</li>
	<li>8天将组织转化为折射率均匀化的折射率匹配解。通过检查网格线是否可见, 将组织放在带有网格线的纸上以检查其视觉清晰度。一旦组织得到充分的澄清, 它可以成像。</li>
	<li>做一个腻子缸, 可以只是周围的组织, 然后塑造成马蹄形的形状, 并按外脊紧紧地在一个玻璃幻灯片, 以使一个密封的空间。腻子的高度应该比卵巢稍高一些。</li>
	<li>将被澄清的卵巢小心地放入腻子的中心, 并加入折射率匹配溶液。把一个玻璃底的盘子放在腻子上, 用腻子来固定盘子。</li>
</ol>3. 共焦显微镜
<ol><li>为成像与共聚焦显微镜, 在 "镍 E" 面板选择的镜头 (在这种情况下, 水浸泡25X 物镜, 1.1 NA, 2 毫米 WD) 与适当的工作距离 (在这种情况下 2.0 mm)。</li>
	<li>
在 "购置" 面板中, 选择频道数。这里, 使用三通道。
	<ol>
<li>首先, 使用 "眼睛端口" 标签和 XY 控制器操纵杆通过显微镜目镜将组织移到场中心。</li>
		<li>关闭快门灯后, 单击 "实时" 选项卡, 分别调整 "激光电源"、"高压 (增益)" 和 "偏移"。较高的激光功率和高压可导致较高的信号, 较高的偏移量可以降低背景噪声。</li>
		<li>选择正确的 "扫描大小" 和 "计数"。使用 1024年 x 1024 µm2的扫描大小, 计数为4到8。</li>
	</ol>
</li>
	<li>
在设置图像质量后, 在面板 "Z 强度校正" 中定义组织的深度, 然后在子房的最顶层 (在组织的中心) 上定位镜片, 然后调整顶层的采集, 单击 "加号"按钮来定义顶层。
	<ol>
<li>将镜片向下移动, 并为组织底部设置适当的高压和激光功率, 并将最低层信息添加到 Z 强度校正表中。单击 "到 nd" 以将设置与 "nd 购置" 面板同步。</li>
	</ol>
</li>
	<li>
在 "ND 获取" 面板中, 首先设置扫描步骤的数量。然后, 勾选标签 "Z" 和 "大图像"。
	<ol>
<li>转到 "扫描大图像" 窗口, 根据组织的边界, 通过目镜的组织可视化来定义字段的大小。返回 "ND 获取" 面板, 输入扫描区域 ("字段") 的大小, 并选择10% 和15% 之间的重叠。</li>
		<li>单击 "运行 Z 校正", 而不是 "立即运行", 以自动设置每个图层的适当的高压、激光功率和偏移量, 并等待图像处理。</li>
	</ol>
</li>
</ol>4. 3D 重建单个卵泡及其 Vasculatures
<ol><li>首先, 使用 ImageJ5打开原始 NIS 文件。单击 "图像" 按钮, 然后选择 "颜色" 和 "拆分通道"。然后, 单击 "文件", 并使用 "另存为" 中的 "图像序列" 选项将通道单独保存为 tiff 文件系列。</li>
	<li>使用 Imaris 打开一个 tiff 文件系列。单击 "编辑" 按钮, 然后选择 "添加频道" 以添加其余的频道。可以在 "显示调整" 选项卡中更改通道的名称和颜色。在 "编辑" 下的下拉列表中, 可能需要在 "图像属性" 面板中更正组织的厚度。</li>
	<li>对于最终图像的3D 裁剪和多余部件的去除, 单击 "编辑" 按钮, 然后选择 "裁剪 3D"。可以通过拖动边框来调整字段的大小。</li>
	<li>
在超越面板中用灯丝算法重构容器信号, 单击 "花丝" 按钮以创建新的灯丝, 然后单击 "下一步"。
	<ol>
<li>为了确定最大和最薄的直径, 去 "切片" 面板, 找到追踪船。距离将自动显示在 "测量" 面板上, 选择两个点的最大宽度的船只。</li>
		<li>返回 "超过" 面板并输入测量的宽度, 然后单击 "下一步"。</li>
	</ol>
</li>
	<li>调整起始和种子点阈值。在 "相机" 面板中, 选择 "选择"。如果某些自动产生的球体需要被移除, 请按 shift 键并点击点。通过选择 "导航" 并移动鼠标, 可以旋转结构以确保保留了所有正确的点, 然后单击 "下一步"。</li>
	<li>选择本地对比度的最高阈值, 然后单击 "下一步"。不要选择 "检测棘", 并继续完成血管缸的重建。可以通过单击 "颜色" 选项卡来编辑颜色。统计数据可以提取在 "统计" 面板的重建灯丝。多余的部件可以在 "编辑" 面板中删除。 注: 其他算法可用于测量总卵泡或卵泡壁的体积。在这里所提出的工作中, 卵母细胞是由两种二次抗体的自发荧光标记的。由于数据量大, 工作站服务器被用于数据分析。</li>
</ol>5. 统计分析
注: 图像分析数据为标准误差。
<ol><li>使用独立样本 t 检验对卵泡进行比较, 用皮尔逊相关试验比较滤泡壁与毛细管长度的相关系数。设置 P < 0.05 的值作为统计意义的极限。</li>
</ol>

<ol>
	<li>Brown, H. M., Russell, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+and+lymphatic+vasculature+in+the+ovary:+development,+function+and+disease.">Blood and lymphatic vasculature in the ovary: development, function and disease.</a> Hum Reprod Update. 20 (1), 29-39 (2014).</li><li>McFee, R. 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=Inhibition+of+vascular+endothelial+growth+factor+receptor+signal+transduction+blocks+follicle+progression+but+does+not+necessarily+disrupt+vascular+development+in+perinatal+rat+ovaries.">Inhibition of vascular endothelial growth factor receptor signal transduction blocks follicle progression but does not necessarily disrupt vascular development in perinatal rat ovaries.</a> Biol Reprod. 81 (5), 966-977 (2009).</li><li>Coveney, D., Cool, J., Oliver, T., Capel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Four-dimensional+analysis+of+vascularization+during+primary+development+of+an+organ,+the+gonad.">Four-dimensional analysis of vascularization during primary development of an organ, the gonad.</a> Proc Natl Acad Sci U S A. 105 (20), 7212-7217 (2008).</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>Schindelin, 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=Fiji:+an+Open+Source+platform+for+biological+image+analysis.">Fiji: an Open Source platform for biological image analysis.</a> Nat Methods. 9 (7), 676-682 (2012).</li><li>Cao, G., Fehrenbach, M. L., Williams, J. T., Finklestein, J. M., Zhu, J. X., Delisser, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+in+platelet+endothelial+cell+adhesion+molecule-1-null+mice.">Angiogenesis in platelet endothelial cell adhesion molecule-1-null mice.</a> Am J Pathol. 175 (2), 903-915 (2009).</li><li>Manni, L., Holm&#228;ng, A., Lundeberg, T., Aloe, L., Stener-Victorin, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+expression+of+alpha+(1)-+and+beta+(2)-adrenoceptors+and+p75+neurotrophin+receptors+in+rats+with+steroid-induced+polycystic+ovaries.">Ovarian expression of alpha (1)- and beta (2)-adrenoceptors and p75 neurotrophin receptors in rats with steroid-induced polycystic ovaries.</a> Auton Neurosci. 118 (1 - 2), 79-87 (2005).</li><li>Chourasia, T. K., Chaube, R., Singh, V., Joy, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Annual+and+periovulatory+changes+in+tyrosine+hydroxylase+activity+in+the+ovary+of+the+catfish+Heteropneustes+fossilis.">Annual and periovulatory changes in tyrosine hydroxylase activity in the ovary of the catfish Heteropneustes fossilis.</a> Gen Comp Endocrinol. 166 (1), 111-116 (2010).</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>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> Annu Rev Cell Dev Biol. 32, 713-741 (2016).</li><li>Liang, H., Schofield, E., Paxinos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+Serotonergic+Fibers+in+the+Mouse+Spinal+Cord+Using+the+CLARITY/CUBIC+Technique.">Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique.</a> J Vis Exp. (108), e53673 (2016).</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>Phillips, J., Laude, A., Lightowlers, R., Morris, C. M., Turnbull, D. M., Lax, N. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+passive+CLARITY+and+immunofluorescent+labelling+of+multiple+proteins+in+human+cerebellum:+understanding+mechanisms+of+neurodegeneration+in+mitochondrial+disease.">Development of passive CLARITY and immunofluorescent labelling of multiple proteins in human cerebellum: understanding mechanisms of neurodegeneration in mitochondrial disease.</a> Sci Rep. 6, 26013 (2016).</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>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), e274 (2016).</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>Chung, A. S., Ferrara, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+and+pathological+angiogenesis.">Developmental and pathological angiogenesis.</a> Annu Rev Cell Dev Biol. 27, 563-584 (2011).</li><li>Rodgers, R. J., Irving-Rodgers, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+the+ovarian+follicular+antrum+and+follicular+fluid.">Formation of the ovarian follicular antrum and follicular fluid.</a> Biol Reprod. 82 (6), 1021-1029 (2010).</li><li>Siu, M. K. Y., Cheng, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+blood-follicle+barrier+(BFB)+in+disease+and+in+ovarian+function.">The blood-follicle barrier (BFB) in disease and in ovarian function.</a> Adv Exp Med Biol. 763, 186-192 (2014).</li><li>Dodt, H. U., 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=Ultramicroscopy:+three-dimensional+visualization+of+neuronal+networks+in+the+whole+mouse+brain.">Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain.</a> Nat Methods. 4, 331-336 (2007).</li><li>Hama, 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=Scale:+a+chemical+approach+for+fluorescence+imaging+and+reconstruction+of+transparent+mouse+brain.">Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain.</a> Nat Neurosci. 14, 1481-1488 (2011).</li><li>Erturk, A., 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=Three-dimensional+imaging+of+solvent-cleared+organs+using+3DISCO.">Three-dimensional imaging of solvent-cleared organs using 3DISCO.</a> Nat Protoc. 7, 1983-1995 (2012).</li><li>Kuwajima, 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=Clear(T):+a+detergent-+and+solvent-free+clearing+method+for+neuronal+and+non-neuronal+tissue.">Clear(T): a detergent- and solvent-free clearing method for neuronal and non-neuronal tissue.</a> Development. 140, 1364-1368 (2013).</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, 1154-1161 (2013).</li><li>Lai, H. 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=Rationalisation+and+validation+of+an+acrylamide-free+procedure+in+three-dimensional+histological+imaging.">Rationalisation and validation of an acrylamide-free procedure in three-dimensional histological imaging.</a> PLOS ONE. 11, e0158628 (2016).</li><li>Susaki, E. A., 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-brain+imaging+with+single-cell+resolution+using+chemical+cocktails+and+computational+analysis.">Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis.</a> Cell. 157, 726-739 (2014).</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>Liu, A. K. L., Lai, H. M., Chang, R. C. C., Gentleman, S. M. <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):+A+novel+protocol+of+tissue+clearing+for+three-dimensional+visualisation+of+human+brain+tissues.">Free-of-acrylamide SDS-based tissue clearing (FASTClear): A novel protocol of tissue clearing for three-dimensional visualisation of human brain tissues.</a> Neuropathol Appl Neurobiol. 43, 346-351 (2016).</li><li>Xu, N., 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=Fast+free-of-acrylamide+clearing+tissue+(FACT)-an+optimized+new+protocol+for+rapid,+high-resolution+imaging+of+three-dimensional+brain+tissue.">Fast free-of-acrylamide clearing tissue (FACT)-an optimized new protocol for rapid, high-resolution imaging of three-dimensional brain tissue.</a> Sci Rep. 7, 9895 (2017).</li><li>Migone, F. F., Cowan, R. G., Williams, R. M., Gorse, K. J., Zipfel, W. R., Quirk, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+reveals+an+essential+role+of+vasoconstriction+in+rupture+of+the+ovarian+follicle+at+ovulation.">In vivo imaging reveals an essential role of vasoconstriction in rupture of the ovarian follicle at ovulation.</a> Proc Natl Acad Sci U S A. 113, 2294-2299 (2016).</li><li>Malki, S., Tharp, M. E., Bortvin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+whole-mount+approach+for+accurate+quantitative+and+spatial+assessment+of+fetal+oocyte+dynamics+in+mice.">A whole-mount approach for accurate quantitative and spatial assessment of fetal oocyte dynamics in mice.</a> Biol Reprod. 93 (113), (2015).</li></ol>

作者没有什么可透露的。

在目前的研究中, 我们提出3D 成像来评估毛细血管与个体生长卵泡之间的关系。在我们以前使用相同的协议9的工作中, 我们研究了大血管、卵泡之间的相互作用以及完整的小鼠卵巢中卵泡的位置。被动清晰的方法使我们能够研究微 vasculatures、卵泡、黄和卵泡之间的相互关系以及在不同发育阶段重建卵巢结构。
为了获得完整的组织的3D 图像数据, 清除过程是第一个重要步骤。有两种主要的策略-脱水方法和亲水性试剂的方法, 如被动清晰度10。由于脱水方法往往导致荧光信号减少, 并经常产生有毒的有机溶液废物, 因此我们将注意力集中在简化被动透明方法的一些步骤上, 用于各种组织类型。
清除过程中的第一个关键步骤是 transcardial 灌注。在灌注过程中, 心脏抽吸在 PBS 和水凝胶溶液的全身循环中起着重要作用, 可以冲洗血液, 降低背景荧光。因此, 在水凝胶完全分布于整个动物之前, 应准确、快速地进行灌注手术, 以避免动物过早死亡。在整个实验过程中, 所有的药剂必须保持在冰上, 如梁et al.所述11, 否则在灌注完成之前, 它们将开始聚合。下一步的重要步骤是去除水凝胶, 并有必要删除尽可能多的多余的凝胶, 因为它会保留抗体。一旦组织清除, 它应该从清除解决方案, 并储存在 PBS 在4° c, 因为太多的时间在清除解决方案将导致太多的蛋白质失去。在共焦显微镜成像中, Z 设置应始终设置在 X-y 范围之前。如果首先设置了 x-y 区域, 则 Z 设置不能与整个 x-y 字段同步。
阳et al.首次引入了无源清晰度12在他们的协议中, 他们没有执行灌注步骤, 而是用氮气脱。此外, 他们的结算解决方案没有包括 bisacrylamide 在水凝胶溶液中, 他们使用 8% SDS 在结算解决方案, 他们使用轮辋的折射率匹配媒体。该方法通过增加 SDS 浓度13, 增加了灌注步骤和无氮气脱气程序14, 并将该方法与其他清除方法 (如 mPACT15) 进行了改进。在这里, 我们进一步发展的协议, 结合 transcardial 灌注和无氮脱气, 减少 SDS 在结算解决方案到 4%, 并使用 FocusClear 作为折射率匹配媒体, 这提供更好的质量显微镜图像。
在被动清晰之后, 可以进行共焦多显微镜和图像分析, 以获得完整的卵巢滤泡血管的3D 结构。该协议将非光渗透性的完整子房转化为光学透明的水凝胶-杂交纳米孔形式, 适合由大分子如抗体16渗透。与传统的2D 断层组织学分析不同, 被动的清晰度和染色的特定标记允许3D 映射的关系之间的毛细血管和个体生长卵泡。
在成人组织中, 主动血管重塑和血管生成主要发生在病理过程, 如伤口愈合, 骨折修复, 肿瘤和转移形成, retinopathies, fibroses, 和类风湿关节炎17。然而, 卵泡期间的周期性变化代表了一种独特的血管重塑能力。卵泡液在次级卵泡中形成后, 在卵母细胞中填充并包围, 为卵母细胞成熟和卵泡发育提供了合适的微环境18。先前的2D 成像研究表明, 卵巢卵泡是由一个网状的毛细血管在膜的19。颗粒层是且的, 每个卵泡被它自己的毛细血管所包围。与 CD31 在血管6中的基本作用一致, 我们在卵泡中发现了从初级到次级卵泡 (图 2G和2H) 过渡期间毛细血管长度的增加。而不是随机分布的毛细血管周围的个别初级或次级卵泡, 3D 重建毛细血管表明, 毛细血管分支分布在卵泡是可检测的大卵泡, 并在大多数情况下, 这些毛细管分支形成了一个中空的空间 (图 3)。
一个主要的问题是, 适应方法需要较长的时间来清除卵巢, 但需要很长的时间, 以最佳的清除效果。为了节省一些时间, 我们建议一次收集所有需要的样品, 并将它们一起清除, 因为清除的组织可以储存在 PBS 和0.02% 的叠氮化钠在4° c 的几个月。另一个限制是共焦显微镜的工作距离。高倍率物镜的工作距离较短, 这可能是厚样品成像的障碍。
已为透明和完整组织的成像引入了若干协议, 包括巴布20、缩放21、3DISCO22、ClearT23、SeeDB24、清晰度16、被动清晰度 (与4或没有水凝胶25), 约定12, 立方26,27, FASTClear28, 和事实29, 但只有三个已用于整个卵巢组织清除和评估9, 30,31。SeeDB30和 ScaleA231已用于整个卵巢组织的成像, 但这些协议只允许单个免疫步骤。然而, 被动的清晰度允许多种蛋白质标记反应, 如本研究和我们以前的研究9所示。
最后, 我们的适应协议, 以确定3D 结构关系的滤泡层和血管的整体澄清卵巢组织提供了一种新的方法来研究血管生成在不同的卵巢疾病, 如多囊卵巢综合征和卵巢癌。

卵泡是卵巢的基本结构和功能单位, 其发育与卵巢内的血管高度相关。血管为卵泡提供营养和荷尔蒙, 从而在卵泡的生长和成熟中发挥重要作用1。
包括选择性血管标记、转基因小鼠模型和药物开发在内的各种技术的结合, 增加了我们对卵巢血管网络、血管生成和血管功能的认识。卵泡.卵巢被称为活动器官, 因为它在卵泡和排卵期间改造各种组织和血管网络。在血管的大小和结构的这种积极重塑是需要的生物功能的发展和招募卵泡。
传统的组织学和计量方法使用的卵巢切片和 immunolabeling 的血管是有限的二维 (2D) 图像2。随着三维 (3D) 重建技术的发展, 2D 图像的组织切片可以重叠, 使一个3D 的结构, 但这种方法仍然有一些局限性-切片的组织可以破坏的显微构造, 部分组织往往是缺掉的, 重要的劳动参与了从切片获得的图像进行3D 重建。全组织3D 成像与共聚焦显微镜可以克服许多这些限制, 但这些方法仅限于对胚胎卵巢血管生成的评价3。使用完整的组织清除方法, 如清晰的4可以增加可视化体积, 以解决产后和成人卵巢中的这些问题, 这些方法提供无任何结构变形的卵巢的光清除。对完整子房的3D 结构进行成像, 为图像分析软件提供了一个精确的图像数据库, 如本工作中使用的 Imaris 软件包。
整个成年期的卵巢重塑是一个动态生理系统的一部分, 这使得卵巢成为一个很好的模型来研究血管生成的调节。此外, 评估卵巢血管在女性生殖系统的病理条件, 如多囊卵巢综合征或卵巢癌的作用, 可以通过整个卵巢组织成像进行研究。被动清晰度法的发展和先进的图像分析软件的使用, 提供了详细的空间信息的关系之间的血管和卵巢结构, 如卵泡。

<table border="0" cellpadding="0" cellspacing="0" width="1664">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Acrylamide</td>
			<td style="width:189px;">Vetec</td>
			<td style="width:127px;">v900845</td>
			<td style="width:861px;">http://www.sigmaaldrich.com/catalog/product/vetec/v900845</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Alexa Flour 488 (Dilution 1:50)&#160;</td>
			<td style="width:189px;">Life Technologies</td>
			<td style="width:127px;">A11039</td>
			<td style="width:861px;">https://www.thermofisher.com/antibody/product/Goat-anti-Chicken-IgY-H-L-Secondary-Antibody-Polyclonal/A-11039</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Alexa Flour 594 (Dilution 1:50)</td>
			<td style="width:189px;">Life Technologies</td>
			<td style="width:127px;">A11012</td>
			<td style="width:861px;">https://www.thermofisher.com/antibody/product/Goat-anti-Rabbit-IgG-H-L-Cross-Adsorbed-Secondary-Antibody-Polyclonal/A-11012</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Bisacrylamide</td>
			<td style="width:189px;">Amresco</td>
			<td style="width:127px;">172</td>
			<td style="width:861px;">http://www.amresco-inc.com/BIS-ACRYLAMIDE-0172.cmsx</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Black wall glass bottom dish (Willco-Dish)</td>
			<td style="width:189px;">Ted Pella</td>
			<td style="width:127px;">14032</td>
			<td style="width:861px;">http://www.tedpella.com/section_html/706dish.htm#black_wall</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Boric acid</td>
			<td style="width:189px;">Sinopharm Chemical Reagent</td>
			<td style="width:127px;">10004818</td>
			<td style="width:861px;">http://en.reagent.com.cn/enshowproduct.jsp?id=10004818</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:487px;">Disodium hydrogen phosphate dodecahydrate (Na2HPO4 12H2O)</td>
			<td style="width:189px;">Sinopharm Chemical Reagent</td>
			<td style="width:127px;">10020318</td>
			<td style="width:861px;">http://en.reagent.com.cn/enshowproduct.jsp?id=10020318</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">FocusClear</td>
			<td style="width:189px;">Celexplorer</td>
			<td style="width:127px;">FC-102</td>
			<td style="width:861px;">http://www.celexplorer.com/product_list.asp?MainType=107&#38;BRDarea=1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Parafilm</td>
			<td style="width:189px;">Bemis</td>
			<td style="width:127px;">PM996</td>
			<td style="width:861px;">http://www.parafilm.com/products</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Paraformaldehyde</td>
			<td style="width:189px;">Sinopharm Chemical Reagent</td>
			<td style="width:127px;">80096618</td>
			<td style="width:861px;">http://en.reagent.com.cn/enshowproduct.jsp?id=80096618</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">PECAM1/CD31, platelet-endothelial cell adhesion molecule 1 (Dilution 1:10)</td>
			<td style="width:189px;">Abcam</td>
			<td style="width:127px;">ab28364</td>
			<td style="width:861px;">http://www.abcam.com/cd31-antibody-ab28364.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Photoinitiator VA044</td>
			<td style="width:189px;">Wako</td>
			<td style="width:127px;">va-044/225-02111</td>
			<td style="width:861px;">http://www.wako-chem.co.jp/specialty/waterazo/VA-044.htm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Sodium azide</td>
			<td style="width:189px;">Sigma</td>
			<td style="width:127px;">S2002</td>
			<td style="width:861px;">http://www.sigmaaldrich.com/catalog/product/sial/s2002?lang=en&#38;region=US</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Sodium chloride (NaCl)</td>
			<td style="width:189px;">Sinopharm Chemical Reagent</td>
			<td style="width:127px;">10019318</td>
			<td style="width:861px;">http://en.reagent.com.cn/enshowproduct.jsp?id=10019318</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:487px;">Sodium dihydrogen phosphate dihydrate (NaH2PO4 2H2O)</td>
			<td style="width:189px;">Sinopharm Chemical Reagent</td>
			<td style="width:127px;">20040718</td>
			<td style="width:861px;">http://en.reagent.com.cn/enshowproduct.jsp?id=20040718</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Sodium dodecyl sulfate</td>
			<td style="width:189px;">Sinopharm Chemical Reagent</td>
			<td style="width:127px;">30166428</td>
			<td style="width:861px;">http://en.reagent.com.cn/enshowproduct.jsp?id=30166428</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Sodium hydroxide (NaOH)</td>
			<td style="width:189px;">Sinopharm Chemical Reagent</td>
			<td style="width:127px;">10019718</td>
			<td style="width:861px;">http://en.reagent.com.cn/enshowproduct.jsp?id=10019718</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Triton X-100</td>
			<td style="width:189px;">Sinopharm Chemical Reagent</td>
			<td style="width:127px;">30188928</td>
			<td style="width:861px;">http://en.reagent.com.cn/enshowproduct.jsp?id=30188928</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:487px;">Tyrosine hydroxylase (TH, Dilution 1:50)</td>
			<td style="width:189px;">Abcam</td>
			<td style="width:127px;">ab76442</td>
			<td style="width:861px;">http://www.abcam.com/tyrosine-hydroxylase-phospho-s40-antibody-ab51206.html</td>
		</tr>
	</tbody>
</table>

three-dimensional reconstruction of the vascular architecture of the passive clarity-cleared mouse ovary

卵巢是雌性生殖系统的主要器官, 对雌性配子的生产和内分泌系统的控制至关重要, 但复杂的结构关系和三维 (3D) 的血管结构卵巢不是很好描述。为了形象化3D 连接和结构的血管在完整的卵巢, 第一个重要的步骤是使卵巢光学清晰。为了避免组织萎缩, 我们使用了水凝胶固定的被动清晰度 (透明脂质交换丙烯酰胺-杂交刚性成像/免疫/原位杂交-相容组织水凝胶) 的协议方法清除完整的卵巢.免疫, 先进的多共聚焦显微镜, 和3D 图像重建, 然后用于可视化卵巢血管和卵泡毛细血管。采用这种方法, 我们发现卵泡毛细血管长度与卵泡壁容积之间存在显著的正相关 (P < 0.01)。

我们将无源清晰方法应用于无源卵巢清除的快速简便方法, 同时保留了卵泡和血管结构, 并从标记的血管和卵泡中获得最高的荧光信号。卵泡血管的3D 结构是由免疫为 CD31, 一个标记为内皮细胞6。用灯丝算法追踪成年小鼠卵巢中的 CD31 染色, 并显示卵泡毛细血管与初级和次级卵泡之间的相互关系 (图 1)。
利用斑点变换和表面算法检测酪氨酸羟化酶-immunostained 卵母细胞、颗粒和膜层, 并计算卵泡的总体积7,8。由于卵泡的压实, 一些部分的裁剪卵泡被确定为半手动。个体卵泡容积和卵母细胞的斑点转化和卵泡壁层的表面重建和血管的纤体重建后, 个体卵泡 vasculatures 与其他的透明关系观测到的指标, 特别是颗粒和膜层体积, (图 2)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56141/56141fig1.jpg"> 图 1: 在用适应的被动清晰度协议清除后, 完整的成年小鼠卵巢的高分辨率成像.(A-b)部分和 XYZ 大扫描获取。三维 (3D) 裁剪次 (C) 和主 (D) 卵泡 (顶行) 和3D 重构的毛细血管, 卵母细胞, 和颗粒/膜的相同卵泡 (底部排), 显示为识别的灯丝, 表面和点转换算法。刻度条 = 200 µm (a-b)、50µm (C)、30µm (D)。 <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56141/56141fig1large.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/56141/56141fig2.jpg"> 图 2: 初级和次级卵泡与 vasculatures 关系的比较指数.卵母细胞容积 (A) 是在3D 重建卵泡体积和卵母细胞后, 由斑点变换算法确定的, 滤泡壁容积 (B) 由表面确定算法和毛细丝是用灯丝算法跟踪的。卵泡容积 (C)、卵母细胞容积/毛细管长度比 (D)、滤泡壁容积/毛细管长度比 (E)、滤泡容积/毛细管长度比 (F)、毛细管长度 (H) 和使用软件包中的相应工具计算毛细管长度与滤泡壁容积 (G) 的相关性。误差线表示方法的标准误差。n = 6;* P < 0.05;** < 0.01。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56141/56141fig2large.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/56141/56141fig3.jpg"> 图 3:三维重建小鼠卵巢次级卵泡中的毛细血管.这将显示卵泡的卵母细胞一侧的中空空间 (白色方块显示空间的边框). <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56141/56141fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Three-dimensional Reconstruction of the Vascular Architecture of the Passive CLARITY-cleared Mouse Ovary at JoVE.com

Three-dimensional Reconstruction of the Vascular Architecture of the Passive CLARITY-cleared Mouse Ovary

在这里, 我们提出了被动清晰度和3D 重建方法的适应性, 可视化卵巢血管和卵泡毛细血管在完整的小鼠卵巢。

无源清清除小鼠卵巢血管构筑的三维重建

The ovary is the main organ of the female reproductive system and is essential for the production of female gametes and for controlling the endocrine ...

three-dimensional-reconstruction-vascular-architecture-passive

Nanyang Technological University

Research

JoVE Journal

Biology

8.6K 次观看.  Fudan University. 在这里, 我们提出了被动清晰度和3D 重建方法的适应性, 可视化卵巢血管和卵泡毛细血管在完整的小鼠卵巢。

视频: Three-dimensional Reconstruction of the Vascular Architecture of the Passive CLARITY-cleared Mouse Ovary

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

嗨 - C：一个方法研究基因组的三维架构。

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

科研

生物学

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

诱导多能干细胞来源于小鼠的生成

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for inclusion and exclusion of data points, and computer-aided pattern recognition to support the interpretation and findings of the analysis. It has become increasingly important to be able to measure fluorescence images constructed from three-dimensional (3D) datasets in order to be able to capture the complexity of cellular dynamics and understand the basis of cellular plasticity within biological systems. Sophisticated microscopy instruments have permitted the visualization of 3D fluorescence images through the acquisition of multispectral fluorescence images and powerful analytical software that reconstructs the images from confocal stacks that then provide a 3D representation of the collected 2D images. Advanced design-based stereology methods have progressed from the approximation and assumptions of the original model-based stereology1 even in complex tissue sections2. Despite these scientific advances in microscopy, a need remains for an automated analytic method that fully exploits the intrinsic 3D data to allow for the analysis and quantification of the complex changes in cell morphology, protein localization and receptor trafficking. 
Current techniques available to quantify fluorescence images include Meta-Morph (Molecular Devices, Sunnyvale, CA) and Image J (NIH) which provide manual analysis. Imaris (Andor Technology, Belfast, Northern Ireland) software provides the feature MeasurementPro, which allows the manual creation of measurement points that can be placed in a volume image or drawn on a series of 2D slices to create a 3D object. This method is useful for single-click point measurements to measure a line distance between two objects or to create a polygon that encloses a region of interest, but it is difficult to apply to complex cellular network structures. Filament Tracer (Andor) allows automatic detection of the 3D neuronal filament-like however, this module has been developed to measure defined structures such as neurons, which are comprised of dendrites, axons and spines (tree-like structure). This module has been ingeniously utilized to make morphological measurements to non-neuronal cells3, however, the output data provide information of an extended cellular network by using a software that depends on a defined cell shape rather than being an amorphous-shaped cellular model. To overcome the issue of analyzing amorphous-shaped cells and making the software more suitable to a biological application, Imaris developed Imaris Cell. This was a scientific project with the Eidgen&ouml;ssische Technische Hochschule, which has been developed to calculate the relationship between cells and organelles. While the software enables the detection of biological constraints, by forcing one nucleus per cell and using cell membranes to segment cells, it cannot be utilized to analyze fluorescence data that are not continuous because ideally it builds cell surface without void spaces. To our knowledge, at present no user-modifiable automated approach that provides morphometric information from 3D fluorescence images has been developed that achieves cellular spatial information of an undefined shape (Figure 1). 
We have developed an analytical platform using the Imaris core software module and Imaris XT interfaced to MATLAB (Mat Works, Inc.). These tools allow the 3D measurement of cells without a pre-defined shape and with inconsistent fluorescence network components. Furthermore, this method will allow researchers who have extended expertise in biological systems, but not familiarity to computer applications, to perform quantification of morphological changes in cell dynamics.

We developed a software platform that utilizes Imaris Neuroscience, ImarisXT and MATLAB to measure the changes in morphology of an undefined shape taken from three-dimensional confocal fluorescence of single cells. This novel approach can be used to quantify changes in cell shape following receptor activation and therefore represents a possible additional tool for drug discovery.

细胞形态变化，从三维荧光图像量化的分析工具，

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for ...

Slide Preparation Method to Preserve Three-dimensional Chromatin Architecture of Testicular Germ Cells

During testicular germ cell differentiation, the structure of nuclear chromatin dynamically changes. The following describes a method designed to preserve the three-dimensional chromatin arrangement of testicular germ cells found in mice; this method has been termed as the three-dimensional (3D) slide method. In this method, testicular tubules are directly treated with a permeabilization step that removes cytoplasmic material, followed by a fixation step that fixes nuclear materials. Tubules are then dissociated, the cell suspension is cytospun, and cells adhere to slides. This method improves sensitivity towards detection of subnuclear structures and is applicable for immunofluorescence, DNA, and RNA fluorescence in situ hybridization (FISH) and the combination of these detection methods. As an example of a possible application of the 3D slide method, a Cot-1 RNA FISH is shown to detect nascent RNAs. The 3D slide method will facilitate the detailed examination of spatial relationships between chromatin structure, DNA, and RNA during testicular germ cell differentiation.

The material here describes a method developed to preserve the three-dimensional chromatin structure of testicular germ cells. This has been termed the three-dimensional (3D) slide method. This method improves sensitivity for detection of subnuclear structures and is applicable for immunofluorescence, DNA, and RNA fluorescence in situ hybridization (FISH).

幻灯片制备方法，以保存睾丸细菌细胞的三维染色质结构

During testicular germ cell differentiation, the structure of nuclear chromatin dynamically changes. The following describes a method designed to preserve ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

隔离和鼠标主动脉内皮细胞原代培养

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

鼠冠状动脉血管平滑肌细胞的分离

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

FRET成像的三维水凝胶

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...

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

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

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

Adipo: 三维脂肪组织成像的组织清除方法

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

Quadruple-Checkerboard: A Modification of the Three-Dimensional Checkerboard for Studying Drug Combinations

The concept of drug-combination therapy is becoming very important mainly with the drastic increase in resistance to drugs. The Quadruple&#160;checkerboard, also called the Q-checkerboard, aims at maximizing the number of possible combinations that can be obtained between four drugs in one experiment to minimize the time and work needed to accomplish the same results with other protocols. This protocol is based on the simple micro dilution technique where the drugs are diluted and combined together in several 96-well plates.
In the first set of 96-well plates, Muller-Hinton broth is added followed by the first required drug (e.g., Cefotaxime here) to serially dilute it. After the first step is done, another set of 96-well plates is used to dilute the second drug (e.g., Amikaci), which will be transferred by removing a specific volume of drug 2 and put in the corresponding wells in the first set of 96-well plates that contains drug one. The third step is done by adding the required concentrations of the third drug (e.g., Levofloxacin), to the appropriate plates in the initial set containing combination of drug 1 and 2. The fourth step is done by adding the required concentrations of the fourth drug (e.g., Trimethoprim-sulfamethoxazol) into the appropriate plates in the first set. Then, E. coli ESBL bacterial inoculum will be prepared and added.
This method is important to evaluate all the possible combinations and has a wider range of possibilities to be tested furthermore for in vivo testing. Despite being a tiring technique requiring a lot of focus, the results are remarkable and time saving where a lot of combinations can be tested in a single experiment.

This protocol describes how to study all possible combinations that can be obtained between four drugs in one single experiment. This method is based on the standard 96-well plate micro dilution assay and the calculation of fractional inhibitory concentrations (FICs) to evaluate the results.

四重检查板:研究药物组合的三维棋盘的修改

The concept of drug-combination therapy is becoming very important mainly with the drastic increase in resistance to drugs. The ...

In Vitro Three-Dimensional Sprouting Assay of Angiogenesis Using Mouse Embryonic Stem Cells for Vascular Disease Modeling and Drug Testing

Recent advances in induced pluripotent stem cells (iPSC) and gene editing technologies enable the development of novel human cell-based disease models for phenotypic drug discovery (PDD) programs. Although these novel devices could predict the safety and efficacy of investigational drugs in humans more accurately, their development to the clinic still strongly rely on mammalian data, notably the use of mouse disease models. In parallel to human organoid or organ-on-chip disease models, the development of relevant in vitro mouse models is therefore an unmet need for evaluating direct drug efficacy and safety comparisons between species and in vivo and in vitro conditions. Here, a vascular sprouting assay that utilizes mouse embryonic stem cells differentiated into embryoid bodies (EBs) is described. Vascularized EBs cultured onto 3D-collagen gel develop new blood vessels that expand, a process called sprouting angiogenesis. This model recapitulates key features of in vivo sprouting angiogenesis-formation of blood vessels from a pre-existing vascular network-including endothelial tip cell selection, endothelial cell migration and proliferation, cell guidance, tube formation, and mural cell recruitment. It is amenable to screening for drugs and genes modulating angiogenesis and shows similarities with recently described three-dimensional (3D) vascular assays based on human iPSC technologies.

In Vitro Three-Dimensional Sprouting Assay of Angiogenesis Using Mouse Embryonic Stem Cells for Vascular Disease Modeling and Drug Testing

This assay utilizes mouse embryonic stem cells differentiated into embryoid bodies cultured in 3D-collagen gel to analyze the biological processes that control sprouting angiogenesis in vitro. The technique can be applied for testing drugs, modeling diseases, and for studying specific genes in the context of deletions that are embryonically lethal.

体外 使用小鼠胚胎干细胞进行血管疾病建模和药物测试的血管生成的三维发芽测定

Recent advances in induced pluripotent stem cells (iPSC) and gene editing technologies enable the development of novel human cell-based disease models for ...