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/pubmed?term=((Blood+and+lymphatic+vasculature+in+the+ovary%3A+development%2C+function+and+disease%5BTitle%5D)+AND+%22Hum+Reprod+Update%22%5BJournal%5D)">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/pubmed?term=((Inhibition+of+vascular+endothelial+growth+factor+receptor+signal+transduction+blocks+follicle+progression+but+does+not+necessarily+disrupt+vascular+development+in+perinatal+rat+ovaries%5BTitle%5D)+AND+%22Biol+Reprod%22%5BJournal%5D)">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/pubmed?term=((Four-dimensional+analysis+of+vascularization+during+primary+development+of+an+organ%2C+the+gonad%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">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/pubmed?term=((Advanced+CLARITY+for+rapid+and+high-resolution+imaging+of+intact+tissues%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">Advanced CLARITY for rapid and high-resolution imaging of intact tissues.</a> Nat Protoc. 9 (7), 1682-1697 (2014).</li>
<li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fiji%3A+an+Open+Source+platform+for+biological+image+analysis%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">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/pubmed?term=((Angiogenesis+in+platelet+endothelial+cell+adhesion+molecule-1-null+mice%5BTitle%5D)+AND+%22Am+J+Pathol%22%5BJournal%5D)">Angiogenesis in platelet endothelial cell adhesion molecule-1-null mice.</a> Am J Pathol. 175 (2), 903-915 (2009).</li>
<li>Manni, L., Holmäng, A., Lundeberg, T., Aloe, L., Stener-Victorin, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ovarian+expression+of+alpha+%281%29-+and+beta+%282%29-adrenoceptors+and+p75+neurotrophin+receptors+in+rats+with+steroid-induced+polycystic+ovaries%5BTitle%5D)+AND+%22Auton+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Annual+and+periovulatory+changes+in+tyrosine+hydroxylase+activity+in+the+ovary+of+the+catfish+Heteropneustes+fossilis%5BTitle%5D)+AND+%22Gen+Comp+Endocrinol%22%5BJournal%5D)">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/pubmed?term=((CLARITY+reveals+dynamics+of+ovarian+follicular+architecture+and+vasculature+in+three-dimensions%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">CLARITY reveals dynamics of ovarian follicular architecture and vasculature in three-dimensions.</a> Sci Rep. 7, 44810(2017).</li>
<li>Tainaka, K., Kuno, A., Kubota, S. I., Murakami, T., Ueda, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chemical+principles+in+tissue+clearing+and+staining+protocols+for+whole-body+cell+profiling%5BTitle%5D)+AND+%22Annu+Rev+Cell+Dev+Biol%22%5BJournal%5D)">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/pubmed?term=((Imaging+Serotonergic+Fibers+in+the+Mouse+Spinal+Cord+Using+the+CLARITY%2FCUBIC+Technique%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">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/pubmed?term=((Single-cell+phenotyping+within+transparent+intact+tissue+through+whole-body+clearing%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Single-cell phenotyping within transparent intact tissue through whole-body clearing.</a> Cell. 158 (4), 945-958 (2014).</li>
<li>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/pubmed?term=((Development+of+passive+CLARITY+and+immunofluorescent+labelling+of+multiple+proteins+in+human+cerebellum%3A+understanding+mechanisms+of+neurodegeneration+in+mitochondrial+disease%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">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/pubmed?term=((Optical+clearing+of+the+mouse+central+nervous+system+using+passive+CLARITY%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Optical clearing of the mouse central nervous system using passive CLARITY.</a> J Vis Exp. (112), (2016).</li>
<li>Woo, J., Lee, M., Seo, J. M., Park, H. S., Cho, Y. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimization+of+the+optical+transparency+of+rodent+tissues+by+modified+PACT-based+passive+clearing%5BTitle%5D)+AND+%22Exp+Mol+Med%22%5BJournal%5D)">Optimization of the optical transparency of rodent tissues by modified PACT-based passive clearing.</a> Exp Mol Med. 48 (12), e274(2016).</li>
<li>Chung, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structural+and+molecular+interrogation+of+intact+biological+systems%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Structural and molecular interrogation of intact biological systems.</a> Nature. 497 (7449), 332-337 (2013).</li>
<li>Chung, A. S., Ferrara, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Developmental+and+pathological+angiogenesis%5BTitle%5D)+AND+%22Annu+Rev+Cell+Dev+Biol%22%5BJournal%5D)">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/pubmed?term=((Formation+of+the+ovarian+follicular+antrum+and+follicular+fluid%5BTitle%5D)+AND+%22Biol+Reprod%22%5BJournal%5D)">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/pubmed?term=((The+blood-follicle+barrier+%28BFB%29+in+disease+and+in+ovarian+function%5BTitle%5D)+AND+%22Adv+Exp+Med+Biol%22%5BJournal%5D)">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/pubmed?term=((Ultramicroscopy%3A+three-dimensional+visualization+of+neuronal+networks+in+the+whole+mouse+brain%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">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/pubmed?term=((Scale%3A+a+chemical+approach+for+fluorescence+imaging+and+reconstruction+of+transparent+mouse+brain%5BTitle%5D)+AND+%22Nat+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Three-dimensional+imaging+of+solvent-cleared+organs+using+3DISCO%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">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/pubmed?term=((Clear%28T%29%3A+a+detergent-+and+solvent-free+clearing+method+for+neuronal+and+non-neuronal+tissue%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">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.nature.com/neuro/journal/v16/n8/full/nn.3447.html">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/pubmed?term=((Rationalisation+and+validation+of+an+acrylamide-free+procedure+in+three-dimensional+histological+imaging%5BTitle%5D)+AND+%22PLOS+ONE%22%5BJournal%5D)">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/pubmed?term=((Whole-brain+imaging+with+single-cell+resolution+using+chemical+cocktails+and+computational+analysis%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">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/pubmed?term=((Whole-body+imaging+with+single-cell+resolution+by+tissue+decolorization%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">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="https://www.ncbi.nlm.nih.gov/pubmed/27627784">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/pubmed?term=((Fast+free-of-acrylamide+clearing+tissue+%28FACT%29-an+optimized+new+protocol+for+rapid%2C+high-resolution+imaging+of+three-dimensional+brain+tissue%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">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/pubmed?term=((In+vivo+imaging+reveals+an+essential+role+of+vasoconstriction+in+rupture+of+the+ovarian+follicle+at+ovulation%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">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/pubmed?term=((A+whole-mount+approach+for+accurate+quantitative+and+spatial+assessment+of+fetal+oocyte+dynamics+in+mice%5BTitle%5D)+AND+%22Biol+Reprod%22%5BJournal%5D)">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...

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><tbody><tr><td>Acrylamide</td><td>Vetec</td><td>v900845</td><td>http://www.sigmaaldrich.com/catalog/product/vetec/v900845</td></tr><tr><td>Alexa Flour 488 (Dilution 1:50)&amp;nbsp;</td><td>Life Technologies</td><td>A11039</td><td>https://www.thermofisher.com/antibody/product/Goat-anti-Chicken-IgY-H-L-Secondary-Antibody-Polyclonal/A-11039</td></tr><tr><td>Alexa Flour 594 (Dilution 1:50)</td><td>Life Technologies</td><td>A11012</td><td>https://www.thermofisher.com/antibody/product/Goat-anti-Rabbit-IgG-H-L-Cross-Adsorbed-Secondary-Antibody-Polyclonal/A-11012</td></tr><tr><td>Bisacrylamide</td><td>Amresco</td><td>172</td><td>http://www.amresco-inc.com/BIS-ACRYLAMIDE-0172.cmsx</td></tr><tr><td>Black wall glass bottom dish (Willco-Dish)</td><td>Ted Pella</td><td>14032</td><td>http://www.tedpella.com/section_html/706dish.htm#black_wall</td></tr><tr><td>Boric acid</td><td>Sinopharm Chemical Reagent</td><td>10004818</td><td>http://en.reagent.com.cn/enshowproduct.jsp?id=10004818</td></tr><tr><td>Disodium hydrogen phosphate dodecahydrate (Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; 12H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Sinopharm Chemical Reagent</td><td>10020318</td><td>http://en.reagent.com.cn/enshowproduct.jsp?id=10020318</td></tr><tr><td>FocusClear</td><td>Celexplorer</td><td>FC-102</td><td>http://www.celexplorer.com/product_list.asp?MainType=107&amp;amp;BRDarea=1</td></tr><tr><td>Parafilm</td><td>Bemis</td><td>PM996</td><td>http://www.parafilm.com/products</td></tr><tr><td>Paraformaldehyde</td><td>Sinopharm Chemical Reagent</td><td>80096618</td><td>http://en.reagent.com.cn/enshowproduct.jsp?id=80096618</td></tr><tr><td>PECAM1/CD31, platelet-endothelial cell adhesion molecule 1 (Dilution 1:10)</td><td>Abcam</td><td>ab28364</td><td>http://www.abcam.com/cd31-antibody-ab28364.html</td></tr><tr><td>Photoinitiator VA044</td><td>Wako</td><td>va-044/225-02111</td><td>http://www.wako-chem.co.jp/specialty/waterazo/VA-044.htm</td></tr><tr><td>Sodium azide</td><td>Sigma</td><td>S2002</td><td>http://www.sigmaaldrich.com/catalog/product/sial/s2002?lang=en&amp;amp;region=US</td></tr><tr><td>Sodium chloride (NaCl)</td><td>Sinopharm Chemical Reagent</td><td>10019318</td><td>http://en.reagent.com.cn/enshowproduct.jsp?id=10019318</td></tr><tr><td>Sodium dihydrogen phosphate dihydrate (NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; 2H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Sinopharm Chemical Reagent</td><td>20040718</td><td>http://en.reagent.com.cn/enshowproduct.jsp?id=20040718</td></tr><tr><td>Sodium dodecyl sulfate</td><td>Sinopharm Chemical Reagent</td><td>30166428</td><td>http://en.reagent.com.cn/enshowproduct.jsp?id=30166428</td></tr><tr><td>Sodium hydroxide (NaOH)</td><td>Sinopharm Chemical Reagent</td><td>10019718</td><td>http://en.reagent.com.cn/enshowproduct.jsp?id=10019718</td></tr><tr><td>Triton X-100</td><td>Sinopharm Chemical Reagent</td><td>30188928</td><td>http://en.reagent.com.cn/enshowproduct.jsp?id=30188928</td></tr><tr><td>Tyrosine hydroxylase (TH, Dilution 1:50)</td><td>Abcam</td><td>ab76442</td><td>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)。
<p class="jove_content" fo:keep-toget...

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

Research

JoVE Journal

Biology

8.7K 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

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all gynecologic malignancies among US women 1,2,3. Serous tumors are the most widespread forms of ovarian cancer and 4,5 the Tg-MISIIR-TAg transgenic represents the only mouse model that spontaneously develops this type of tumors. Tg-MISIIR-TAg mice express SV40 transforming region under control of the Mullerian Inhibitory Substance type II Receptor (MISIIR) gene promoter 6. Additional transgenic lines have been identified that express the SV40 TAg transgene, but do not develop ovarian tumors. Non-tumor prone mice exhibit typical lifespan for C57Bl/6 mice and are fertile. These mice can be used as syngeneic allograft recipients for tumor cells isolated from Tg-MISIIR-TAg-DR26 mice.
Objective: Although tumor imaging is possible 7, early detection of deep tumors is challenging in small living animals. To enable preclinical studies in an immunologically intact animal model for serous ovarian cancer, we describe a syngeneic mouse model for this type of ovarian cancer that permits in vivo imaging, studies of the tumor microenvironment and tumor immune responses.
Methods: We first derived a TAg+ mouse cancer cell line (MOV1) from a spontaneous ovarian tumor harvested in a 26 week-old DR26 Tg-MISIIR-TAg female. Then, we stably transduced MOV1 cells with TurboFP635 Lentivirus mammalian vector that encodes Katushka, a far-red mutant of the red fluorescent protein from sea anemone Entacmaea quadricolor with excitation/emission maxima at 588/635 nm 8,9,10. We orthotopically implanted MOV1Kat in the ovary 11,12,13,14 of non-tumor prone Tg-MISIIR-TAg female mice. Tumor progression was followed by in vivo optical imaging and tumor microenvironment was analyzed by immunohistochemistry.
Results: Orthotopically implanted MOV1Kat cells developed serous ovarian tumors. MOV1Kat tumors could be visualized by in vivo imaging up to three weeks after implantation (fig. 1) and were infiltrated with leukocytes, as observed in human ovarian cancers 15 (fig. 2).
Conclusions: We describe an orthotopic model of ovarian cancer suitable for in vivo imaging of early tumors due to the high pH-stability and photostability of Katushka in deep tissues. We propose the use of this novel syngeneic model of serous ovarian cancer for in vivo imaging studies and monitoring of tumor immune responses and immunotherapies.

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

To study in vivo tumor growth and tumor microenvironment, we used a syngeneic and orthotopic mouse model of ovarian cancer in immunocompetent animals. We transduced a mouse tumor cell line (MOV1) with Katushka fluorescent protein (MOV1KAT) and here we show its orthotopic implantation in ovary and in vivo imaging.

浆液性卵巢癌原位模型在免疫小鼠，为在体内肿瘤显像和肿瘤免疫反应的监测

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all ...

科研

免疫与感染

Whole Mount Immunofluorescence and Follicle Quantification of Cultured Mouse Ovaries

Research in the field of mammalian reproductive biology often involves evaluating the overall health of ovaries and testes. Specifically, in females, ovarian fitness is often assessed by visualizing and quantifying follicles and oocytes. Because the ovary is an opaque three-dimensional tissue, traditional approaches require laboriously slicing the tissue into numerous serial sections in order to visualize cells throughout the entire organ. Furthermore, because quantification by this method typically entails scoring only a subset of the sections separated by the approximate diameter of an oocyte, it is prone to inaccuracy. Here, a protocol is described that instead utilizes whole organ tissue clearing and immunofluorescence staining of mouse ovaries to visualize follicles and oocytes. Compared to more traditional approaches, this protocol is advantageous for visualizing cells within the ovary for numerous reasons: 1) the ovary remains intact throughout sample preparation and processing; 2) small ovaries, which are difficult to section, can be examined with ease; 3) cellular quantification is more readily and accurately achieved; and 4) the whole organ imaged.

Here, we present a protocol to quantify follicles in cultured ovaries of young mice without serial sectioning. Using whole organ immunofluorescence and tissue clearing, physical sectioning is replaced with optical sectioning. This method of sample preparation and visualization maintains organ integrity and facilitates automated quantification of specific cells.

培养小鼠卵巢的全贴装免疫荧光和卵泡定量研究

Research in the field of mammalian reproductive biology often involves evaluating the overall health of ovaries and testes. Specifically, in females, ...

发育生物学

Ovarian Tissue Culture to Visualize Phenomena in Mouse Ovary

Mammalian females periodically ovulate an almost constant number of oocytes during each estrus cycle. To sustain such regularity and periodicity, regulation occurs at the hypothalamic-pituitary-gonadal axis level and on developing follicles in the ovary. Despite active studies, follicle development mechanisms are not clear because of the several steps involved from the dormant primordial follicle activation to ovulation, and because of the regulation complexity that differs at each follicular stage. To investigate the mechanisms of follicle development, and the dynamics of follicles throughout the estrus cycle, we developed a mouse ovarian tissue culture model that can be used to observe follicle development using a microscope. Systematic follicle development, periodical ovulation, and follicle atresia can all be reproduced in the cultured ovary model, and the culture conditions can be experimentally modulated. Here, we demonstrate the usefulness of this method in the study of the regulatory mechanisms of follicle development and other ovarian phenomena.

Ovarian tissue cultures can be used as models of follicle development, ovulation, and follicle atresia and indicate regulatory mechanisms of dynamic ovarian processes.

卵巢组织培养对小鼠卵巢现象的可视化

Mammalian females periodically ovulate an almost constant number of oocytes during each estrus cycle. To sustain such regularity and periodicity, ...

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, ...

生物学

Accurate Follicle Enumeration in Adult Mouse Ovaries

Sexually reproducing female mammals are born with their entire lifetime supply of oocytes. Immature, quiescent oocytes are found within primordial follicles, the storage unit of the female germline. They are non-renewable, thus their number at birth and subsequent rate of loss largely dictates the female fertile lifespan. Accurate quantification of primordial follicle numbers in women and animals is essential for determining the impact of medicines and toxicants on the ovarian reserve. It is also necessary for evaluating the need for, and success of, existing and emerging fertility preservation techniques. Currently, no methods exist to accurately measure the number of primordial follicles comprising the ovarian reserve in women. Furthermore, obtaining ovarian tissue from large animals or endangered species for experimentation is often not feasible. Thus, mice have become an essential model for such studies, and the ability to evaluate primordial follicle numbers in whole mouse ovaries is a critical tool. However, reports of absolute follicle numbers in mouse ovaries in the literature are highly variable, making it difficult to compare and/or replicate data. This is due to a number of factors including strain, age, treatment groups, as well as technical differences in the methods of counting employed. In this article, we provide a step-by-step instructional guide for preparing histological sections and counting primordial follicles in mouse ovaries using two different methods: [1] stereology, which relies on the fractionator/optical dissector technique; and [2] the direct count technique. Some of the key advantages and drawbacks of each method will be highlighted so that reproducibility can be improved in the field and to enable researchers to select the most appropriate method for their studies.

Here, we describe, compare, and contrast two different techniques for accurate follicle counting of fixed mouse ovarian tissues.

成人小鼠卵巢中的精确卵泡图解

Sexually reproducing female mammals are born with their entire lifetime supply of oocytes. Immature, quiescent oocytes are found within primordial ...

Whole Ovary Immunofluorescence, Clearing, and Multiphoton Microscopy for Quantitative 3D Analysis of the Developing Ovarian Reserve in Mouse

Female fertility and reproductive lifespan depend on the quality and quantity of the ovarian oocyte reserve. An estimated 80% of female germ cells entering meiotic prophase I are eliminated during Fetal Oocyte Attrition (FOA) and the first week of postnatal life. Three major mechanisms regulate the number of oocytes that survive during development and establish the ovarian reserve in females entering puberty. In the first wave of oocyte loss, 30-50% of the oocytes are eliminated during early FOA, a phenomenon that is attributed to high Long interspersed nuclear element-1 (LINE-1) expression. The second wave of oocyte loss is the elimination of oocytes with meiotic defects by a meiotic quality checkpoint. The third wave of oocyte loss occurs perinatally during primordial follicle formation when some oocytes fail to form follicles. It remains unclear what regulates each of these three waves of oocyte loss and how they shape the ovarian reserve in either mice or humans.
Immunofluorescence and 3D visualization have opened a new avenue to image and analyze oocyte development in the context of the whole ovary rather than in less informative 2D sections. This article provides a comprehensive protocol for whole ovary immunostaining and optical clearing, yielding preparations for imaging using multiphoton microscopy and 3D modeling using commercially available software. It shows how this method can be used to show the dynamics of oocyte attrition during ovarian development in C57BL/6J mice and quantify oocyte loss during the three waves of oocyte elimination. This protocol can be applied to prenatal and early postnatal ovaries for oocyte visualization and quantification, as well as other quantitative approaches. Importantly, the protocol was strategically developed to accommodate high-throughput, reliable, and repeatable processing that can meet the needs in toxicology, clinical diagnostics, and genomic assays of ovarian function.

Here, we present an optimized protocol for imaging entire ovaries for quantitative and qualitative analyses using whole-mount immunostaining, multiphoton microscopy, and 3D visualization and analysis. This protocol accommodates high-throughput, reliable, and repeatable processing that is applicable for toxicology, clinical diagnostics, and genomic assays of ovarian function.

全卵巢免疫荧光、清除和多光子显微镜用于小鼠发育中卵巢储备的定量3D分析

Female fertility and reproductive lifespan depend on the quality and quantity of the ovarian oocyte reserve. An estimated 80% of female germ cells ...

Microdissection and Dissociation of the Murine Oviduct: Individual Segment Identification and Single Cell Isolation

Mouse model systems are unmatched for the analysis of disease processes because of their genetic manipulability and the low cost of experimental treatments. However, because of their small body size, some structures, such as the oviduct with a diameter of 200-400 &#956;m, have proven to be relatively difficult to study except by immunohistochemistry. Recently, immunohistochemical studies have uncovered more complex differences in oviduct segments than were previously recognized; thus, the oviduct is divided into four functional segments with different ratios of seven distinct epithelial cell types. The different embryological origins and ratios of the epithelial cell types likely make the four functional regions differentially susceptible to disease. For example, precursor lesions to serous intraepithelial carcinomas arise from the infundibulum in mouse models and from the corresponding fimbrial region in the human fallopian tube. The protocol described here details a method for microdissection to subdivide the oviduct in such a way to yield a sufficient amount and purity of RNA necessary for downstream analysis such as reverse transcription-quantitative PCR (RT-qPCR) and RNA sequencing (RNAseq). Also described is a mostly non-enzymatic tissue dissociation method appropriate for flow cytometry or single cell RNAseq analysis of fully differentiated oviductal cells. The methods described will facilitate further research utilizing the murine oviduct in the field of reproduction, fertility, cancer, and immunology.

A method for microdissection of the mouse oviduct that allows collection of the individual segments while maintaining RNA integrity is presented. In addition, non-enzymatic oviductal cell dissociation procedure is described. The methods are appropriate for subsequent gene and protein analysis of the functionally different oviductal segments and dissociated oviductal cells.

小鼠输卵管的显微解剖和解离:个体片段鉴定和单细胞分离

Mouse model systems are unmatched for the analysis of disease processes because of their genetic manipulability and the low cost of experimental ...

<meta charset="utf8"></head><body>使用组织透明化和荧光标记进行视网膜周细胞可视化

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.

<meta charset="utf8"></head><body>通过免疫染色对小鼠视网膜血管周细胞进行 3D 可视化

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.

通过免疫染色对小鼠视网膜血管周细胞进行 3D 可视化 

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

无源清清除小鼠卵巢血管构筑的三维重建

摘要

探索更多视频

浆液性卵巢癌原位模型在免疫小鼠，为在体内肿瘤显像和肿瘤免疫反应的监测

培养小鼠卵巢的全贴装免疫荧光和卵泡定量研究

卵巢组织培养对小鼠卵巢现象的可视化

嗨 - C：一个方法研究基因组的三维架构。

成人小鼠卵巢中的精确卵泡图解

全卵巢免疫荧光、清除和多光子显微镜用于小鼠发育中卵巢储备的定量3D分析

小鼠输卵管的显微解剖和解离:个体片段鉴定和单细胞分离

通过免疫染色对小鼠视网膜血管周细胞进行 3D 可视化

通过免疫染色对小鼠视网膜血管周细胞进行 3D 可视化

通过免疫染色对小鼠视网膜血管周细胞进行 3D 可视化