Name: whole mount immunofluorescence and follicle quantification of cultured mouse ovaries
Uploaded: 2018-05-02T14:45:00.000+00:00
Duration: 8 min 15 s
Description: Watch this Scientific Journal Video about Whole Mount Immunofluorescence and Follicle Quantification of Cultured Mouse Ovaries at JoVE.com

我们感谢丽贝卡威廉斯和乔安娜德拉库拉克鲁兹从康奈尔的 Recknagel 成像和安德鲁的帮助建议和技术援助。这项工作得到了国家卫生研究所资助 S10-OD018516 (康奈尔的影像设施), T32HD057854 J.C.B. 和 R01-GM45415 J.C.S。

康奈尔大学的机构动物护理和使用委员会 (IACUC) 已经批准了这里描述的所有方法, 根据2004-0038 号议定书 JCS。
1. 文书和文化媒体的准备工作
<ol><li>用10% 漂白剂擦拭工作区域, 并允许漂白剂在工作区域表面保持至少5分钟的清洁。5分钟后, 用干净的纸巾和70% 乙醇 (乙醇) 去除多余的漂白剂。用70% 乙醇彻底清洁解剖显微镜。 注意: 重要的是, 工作区域必须至少半无菌, 以避免对器官培养的污染。</li>
	<li>包装干净的镊子和剪刀与纸巾, 70% 乙醇, 直到使用时间。 注意: 理想的解剖工具可能根据卵巢的年龄/大小而变化。最好的结果是使用一个尖锐的显微解剖剪刀, 两个细尖钳 (5 或 55) 和一个显微解剖虹膜剪刀。</li>
	<li>在锥形管, 使50毫升的卵巢培养基和温暖的37°c 水浴。<ol>
<li>要使子房区域性为中等4, 请补充1x 最小的基本培养基 (内存) 与10% 胎牛血清 (血清), 25 毫米 (4-(2 羟乙基)-1-piperazineethanesulfonic 酸 (HEPES), 100 单位/毫升青霉素, 100 µg/毫升链霉素, 0.25 µg/毫升两性霉素 B (例如, 45 毫升的记忆, 5 毫升的热灭活血清, 1.25 毫升的 HEPES, 0.5 毫升100x 的青霉素, 链霉素和两性霉素 b)。</li>
	</ol>
</li>
	<li>在卵巢培养前准备板材和插入物。<ol>
<li>在组织培养罩中, 将1毫升的卵巢培养基添加到35毫米的组织培养板中。</li>
		<li>预浸泡的插入膜与培养基。增加足够的培养基, 通常大约0.55 毫升每井, 到24井载体组织培养板, 并且安置细胞文化插入在井中。确保插入的膜接触介质。</li>
		<li>在实验中, 根据预期的卵巢数量, 准备35毫米板和水井的细胞培养插入物。将包含1毫升培养基的35毫米板和包含培养基的24井载体板和插入到37°c、5% CO2和大气 O2孵化器中。</li>
	</ol>
</li>
	<li>在解剖范围旁边安排一个热板, 将温度设置为37摄氏度。保持37°c, 5% CO2和大气 O2孵化器靠近解剖室。</li>
</ol>2. 卵巢解剖和器官培养
注意: 只要暴露在非理想温度下, 卵巢就可以在室温下进行解剖。
<ol><li>弄死的雌性老鼠在产后5天, 通过斩首, 一次一次。 注: 必须根据正在进行研究的设施中的 IACUC 准则进行安乐死。</li>
	<li>用70% 乙醇喷洒动物。将动物放在干纸巾顶部的解剖显微镜下。放置一个35毫米的组织培养皿, 包含卵巢培养培养基接近解剖显微镜。</li>
	<li>用镊子或显微解剖虹膜剪刀, 通过皮肤的动物和进入腹腔切开切口, 用谨慎不要切开入肠道。把肠子从腹腔推出来, 找到卵巢。提取卵巢并将其放入含有培养基的35毫米组织培养皿中。</li>
	<li>一旦卵巢已经从动物解剖, 增加了解剖显微镜的放大倍数, 以便更好地可视化卵巢和任何附加组织。用干净的细尖钳, 去除所有非卵巢组织, 如囊和脂肪组织。在去除附着的非卵巢组织时, 要注意保持卵巢完好无损。</li>
	<li>一旦卵巢被隔离, 将一对进入预浸泡细胞培养的载体24孔板 (指步骤1.4.2 和 1.4.3) 和调整培养基的体积, 以确保它足以保持器官湿润 (没有完全淹没它)。 注意: 在转移卵巢时, 注意不要在细胞培养插入物的细胞膜上戳一个洞。</li>
	<li>放置说明器官在37°c, 5% CO2和大气 O2孵化器。</li>
	<li>重复步骤 2.1-2.6, 直到每个动物的卵巢被解剖, 并放置在细胞培养插入的24井板包含卵巢培养基。</li>
	<li>每2天改变卵巢培养培养基, 使用温暖的卵巢介质整除数从37°c 水浴, 并在所需的实验时间点进行协议的3部分。</li>
</ol>3. 组织固定
<ol><li>在15毫升锥形管中, 在1x 磷酸盐缓冲盐水 (pbs) 中准备4% 多聚甲醛 (粉煤灰)(例如, 将2毫升16% 的粉煤灰电子显微镜分级为6毫升 1x PBS)。 注意: 粉煤灰是一种危险物质, 应在化学罩下处理。</li>
	<li>修复培养的卵巢, 而不删除的组织从文化插入, 通过覆盖组织与新鲜准备的4% 粉煤灰溶液。用塑料粘合剂封住盘子的边缘 (以避免蒸发), 并将样品在一夜之间放置在4摄氏度。 注意: 为了便于处理和组织完整性, 避免在整个过程中将卵巢从细胞培养中插入。在插入物表面附着的培养卵巢有利于处理, 而不损害或失去器官。</li>
	<li>用70% 乙醇冲洗组织 3x, 然后进行步骤4.1 或将其存储在充满70% 乙醇的闪烁瓶中, 4 °c, 直到进一步加工。 注: 如果使用组织内在表达荧光蛋白, 储存样品与70% 乙醇可能会大大减少信号。或者, 组织可以漂洗和存储在 PBS 与0.2% 叠氮化钠 (南3)。然而, 南3是剧毒的, 并造成严重的吸入危险。在油烟机中制作库存解决方案, 并佩戴适当的个人防护设备, 如实验室外套和丁腈橡胶手套。</li>
</ol>4. 全芒免疫荧光
<ol><li>在通透步骤4.3 之前, 将固定组织放在 RT 1x PBS 中至少4小时。</li>
	<li>准备通透解决方案和堵塞解决方案。<ol>
<li>如所述, 准备通透解决方案。对 1x PBS, 添加0.2% 聚乙烯醇 (PVA), 0.1% 硼氢化钠 (NaBH4) 溶液 (从12重量 .% 在14米氢氧化钠 (氢氧化钠) 溶液) 和1.5% 聚乙二醇叔对辛醚 (非离子表面活性剂洗涤剂)。在50毫升圆锥管, 添加5毫升 10x PBS, 0.1 克聚乙烯醇, 50 µL 的 NaBH4和750µL 的非离子表面活性剂洗涤剂, 然后添加超纯水高达50毫升和搅拌良好的室温, 直到混合物完全解决。</li>
		<li>按照说明准备阻塞解决方案。到 1x PBS, 添加0.1% 非离子表面活性剂洗涤剂, 0.15% 的2.5M 甘氨酸 pH 7.4, 10% 正常山羊血清, 3% 牛血清白蛋白 (BSA), 0.2% 南3, 100 单位/毫升青霉素, 100 µg/毫升链霉素, 0.25 µg/毫升两性霉素 b. 准备一个股票解决方案2.5 M 甘氨酸将93.8 克甘氨酸溶解于超纯水中500毫升的最终容积 (pH 7.4) 和10% 南3的库存溶液, 溶解5毫升的超纯水;然后, 在50毫升锥形管中加入5毫升 10x PBS, 50 µL 非离子表面活性剂洗涤剂, 750 µL 甘氨酸溶液, 5 毫升山羊血清, 1.5 克 BSA, 1 毫升的南3库存溶液, 准备堵塞溶液。和0.5 毫升的青霉素、链霉素和两性霉素 B 的100x 库存。添加超纯水多达50毫升和注射器过滤器最终的解决方案。</li>
	</ol>
</li>
	<li>从瓶子中取出 1x PBS, 然后添加足够的通透溶液完全淹没卵巢。将样品放在通透溶液中的轨道振动筛 (例如, nutator) 室温下4小时。 注: 潜伏期可能根据器官厚度而变化。</li>
	<li>用足够的阻断溶液取代通透溶液, 完全淹没卵巢。用塑料粘合剂封住瓶子, 在一个轨道振动筛的室温下把组织孵化至少12小时。</li>
	<li>根据制造商的数据表, 准备阻止溶液中的主抗体稀释。 注意: 注意并非所有抗体都与清除剂兼容。每个样本所需的平均体积约为750µL。<ol>
<li>对于卵母细胞量化, 使用 TAp63 (核卵母细胞标记) 和 MVH (细胞质生殖细胞标记)。 注: 免疫荧光图像中使用的抗体为小鼠 anti-p63 和兔抗 MVH)。主抗体溶液可重复使用2x 或更多, 如果储存在4摄氏度之间的染色协议和妥善处理, 以避免细菌生长。</li>
	</ol>
</li>
	<li>将含有组织的插入物放置在24井板中, 并添加大约750µL 的原抗体溶液。确保卵巢完全淹没。用塑料粘合剂封住盘子, 以尽量减少蒸发, 并在眼眶上的室温下让组织孵化4天。</li>
	<li>准备洗涤液。<ol>
<li>如有说明, 准备洗涤液。制作 1x PBS, 加入 0.2% PVA 和0.15% 非离子表面活性剂洗涤剂。在50毫升圆锥管中, 加入5毫升 10x PBS, 0.1 克聚乙烯醇, 75 µL 非离子表面活性剂洗涤剂和超纯水, 最终体积为50毫升。</li>
	</ol>
</li>
	<li>去除原抗体稀释, 并添加大量的洗涤液。一定要把卵巢淹没。允许卵巢浸泡在溶液中过夜, 在室温下的轨道振动筛。</li>
	<li>更换洗涤液, 在轨道振动筛上孵育2小时以上。</li>
	<li>重复步骤4.9 再一次。</li>
	<li>在阻断溶液中准备二次抗体稀释。 注: 所用的二级抗体为山羊抗鼠488荧光染料, 山羊中饲养的抗兔594荧光染料。请参见步骤4.5。每个样本所需的平均体积。</li>
	<li>从卵巢中取出洗涤液并添加二次抗体溶液。保护样品免受光 (用铝箔包装板) 和密封板用塑料粘合剂, 以减少蒸发。在室温下在2天的轨道振动筛上孵化样品。 注: 在后续的所有孵化步骤中, 使用铝箔继续保护样品免受光线的侵害。</li>
	<li>从样品中取出二次抗体溶液并添加洗涤液。在8小时的轨道振动筛上孵化。 注: 如果需要 46-Diamidino-2-苯基吲哚 (DAPI) 染色, 添加 50 ng/毫升的 DAPI 到第一洗涤步骤 (~ 8 小时孵化)。</li>
	<li>更换洗涤液进行隔夜清洗。</li>
	<li>在准备结算解决方案时 (见第5节), 用新鲜的溶液代替隔夜洗涤液, 并将样品保存在室温下的轨道振动筛上。</li>
</ol>5. 卵巢清除和成像
<ol><li>准备刻度 (0) 清除解决方案。<ol>
<li>准备刻度 (0) 通过在给定比例中添加试剂来清除解决方案10 : 40% d-(-) 山梨醇 (w/v), 10% 甘油, 4.3 米尿素和20% 二甲基亚砜 (亚砜), pH 8.1 (例如在50毫升圆锥管, 增加2.5 毫升甘油, 10 克山梨醇, 5 毫升亚砜, 12 毫升9米尿素, 总容积为25毫升。混合解决方案通过反转在50°c 30 分钟和德加之前使用。</li>
	</ol>
</li>
	<li>卸下洗涤液并将样品浸入清除液中。为更好的结果保持在轨道振动筛的样品。</li>
	<li>每天更换清除解决方案 2x, 直到组织变得透明 (大约2天)。</li>
	<li>一旦组织被清除, 准备标本进行成像, 仔细删除含有培养卵巢的插入膜与一个精细的尖端手术刀, 并将样品放在玻璃滑梯上。</li>
	<li>在能够进行光学切片的显微镜上对样品进行成像。</li>
</ol>6. 卵母细胞量化
注意: 有许多不同的计算机程序可以量化细胞。下面描述的是使用非商业图像处理包 (斐济-ImageJ14) 进行卵母细胞量化的协议。
<ol><li>对于感兴趣的特定蜂窝标记, 使用Z堆栈图像系列的扁平最大强度投影 (没有 DAPI 通道), 请将图像转换为图像下拉列表Type下的黑白8位图像。菜单。</li>
	<li>在图像下拉菜单下, 突出显示调整选项卡, 然后选择阈值。</li>
	<li>手动将阈值调整为最佳标识每个卵母细胞的级别。确定级别后, 单击应用生成黑白图像。完成后, 通过使用椭圆或手绘图标对示例进行量化。 注: 斐济-ImageJ 有不同的选择, 以微调的图像, 将用于粒子量化。例如, 在进程 |二进制选项卡中, 有不同的选项可用于改进对要计算的内容的检测。在图 4中, 用于更好地个性化卵泡的选项是侵蚀, 后跟填充孔, 最后是分水岭。</li>
	<li>在分析下拉菜单下, 选择分析粒子。根据所需的分辨率设置参数。</li>
</ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+Inhibition+of+the+DNA+Damage+Checkpoint+Prevents+Radiation-Induced+Oocyte+Death.">Pharmacological Inhibition of the DNA Damage Checkpoint Prevents Radiation-Induced Oocyte Death.</a> Genetics. , (2017).</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> Nature Methods. 9 (7), 676-682 (2012).</li><li>Obata, Y., Kono, T., Hatada, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oogenesis:+Maturation+of+mouse+fetal+germ+cells+in+vitro.">Oogenesis: Maturation of mouse fetal germ cells in vitro.</a> Nature. 418 (6897), 497 (2002).</li><li>Faire, 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=Follicle+dynamics+and+global+organization+in+the+intact+mouse+ovary.">Follicle dynamics and global organization in the intact mouse ovary.</a> Developmental Biology. 403 (1), 69-79 (2015).</li><li>Byrne, C., Hardman, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-Mount+Assays+for+Gene+Induction+and+Barrier+Formation+in+the+Developing+Epidermis.">Whole-Mount Assays for Gene Induction and Barrier Formation in the Developing Epidermis.</a> Epidermal Cells. , 127-136 (2005).</li><li>Lauter, G., S&#246;ll, I., Hauptmann, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicolor+fluorescent+in+situ+hybridization+to+define+abutting+and+overlapping+gene+expression+in+the+embryonic+zebrafish+brain.">Multicolor fluorescent in situ hybridization to define abutting and overlapping gene expression in the embryonic zebrafish brain.</a> Neural Development. 6, 10 (2011).</li><li>Laronda, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+bioprosthetic+ovary+created+using+3D+printed+microporous+scaffolds+restores+ovarian+function+in+sterilized+mice.">A bioprosthetic ovary created using 3D printed microporous scaffolds restores ovarian function in sterilized mice.</a> Nature Communications. 8, (2017).</li></ol>

作者没有什么可透露的。

研究哺乳动物的繁殖需要使用和量化的专门细胞, 不定期服从细胞培养。但是, "体外" 区域性系统在维护子房和卵泡生存能力 1,15中是有效的。在卵巢培养过程中, 组织需要更大的表面积来通过扩散来交换养分。因此, 5 天大鼠卵巢是理想的大小和形状的器官培养。该协议是为七天的文化中保持的卵巢进行了优化, 但卵巢培养长度可以根据具体的科学...

为了研究哺乳动物卵巢的细胞组成和形态学特征, 科学家们经常依赖于体内实验, 其次是石蜡嵌入卵巢的免疫组织化学染色。最近, 虽然整个卵巢器官培养已被证明是研究卵巢功能的有效替代方案1,2,3,4 , 因为该技术可以结合更好的可视化和量化工具。传统上, 卵巢形态学分析依靠石蜡嵌入序列切片重建三维卵巢结构, 但除了费力和耗时, 连续切片石蜡嵌入组织不保证器官的适当的重建, 并且区段经常丢失或错误地命令在过程中。
除了与串行切片相关的技术难题外, 通常用于量化每个子房的卵泡数的方法也有变化5,6。目前使用的方法变异在研究中损害了卵巢储备的 meta 分析5,7。例如, 不同研究文章中的卵泡数在特定应变6中的相似发育年龄之间可能会有10倍或更多的变化。这些在报告的卵泡数量上的巨大差异会导致混淆, 并阻碍了交叉研究的比较。实验中, 从序列切片中进行卵泡定量的传统方法是通过计数一个预先定义的节数 (例如,每第五、第十或其他部分) 的卵泡来完成的。使用这种方法的卵泡计数的变异性不仅来自于计算节数的周期性, 而且还产生于截面厚度的变化, 以及生成串行部分5、6的技术经验。除了变异外, 传统组织切片的另一个缺点是, 幼兽的小卵巢的切片特别具有挑战性, 并且高度依赖于组织方向8。
下面的协议描述了常规使用的卵巢培养技术1 , 但大大改善了传统的卵泡定量替代物理切片与组织清除和光学切片使用共焦显微镜8 ,9。用组织浸泡 (不需要经颅灌注或电泳) 在尿素和山梨醇基溶液中 (例如,刻度 (0)10) 的清除被证明与染色兼容, 并允许减少清除时间而不损害成像深度。其他报告的方法 (例如、ScaleA28、10、SeeDB11、ClearT12和 ClearT212) 要么更耗时, 要么不允许对示例进行深入的光学解析。光学切片是有利的, 因为它是少劳动密集型和维护器官的三维体系结构7,8。这种方法的另一个好处是, 样品的制备不需要昂贵的试剂来清除组织, 而且可以相对容易地进行。
具体而言, 所描述的协议已被优化为培养的小鼠卵巢在产后五天, 但已经进行了卵巢范围从产后一天 0-10。该方法利用卵巢培养系统, 组织自然附着在其培养的膜上, 促进器官的处理和操作。所描述的培养系统可用于维持说明卵巢长达10天, 并评估不同实验条件对卵母细胞存活率的影响13。所描述的量化程序是使用非商业图像处理包 (斐济-ImageJ14 ) 进行的, 可以在大多数个人计算机上进行。此外, 用于量化的图像可以广泛地提供给科学界, 从而允许将来进行 meta 分析。

<table><tbody><tr><td>Micro dissecting scissor 4.5&quot;, straight, sharp points</td><td>Roboz</td><td>RS-5912</td><td>Micro dissecting scissor</td></tr><tr><td>Jewelers style forceps 4-3/8&quot;, style 5</td><td>Integra Miltex</td><td>17-305X</td><td>Fine tip forceps</td></tr><tr><td>Micro Iris Scissors 4&quot;, straight, sharp points</td><td>Integra Miltex</td><td>18-1618</td><td>Micro dissecting iris scissor or micro dissecting spring scissor</td></tr><tr><td>1X Minimal Essential Media (MEM)</td><td>ThermoFisher Scientific</td><td>11090-081</td><td></td></tr><tr><td>Fetal Bovine Serium</td><td>Corning</td><td>35011CV</td><td></td></tr><tr><td>HEPES (1M)</td><td>Gibco</td><td>15630080</td><td></td></tr><tr><td>Antibiotic-Antimycotic (100X)</td><td>Gibco</td><td>15240062</td><td>Used in Step 1.3.1</td></tr><tr><td>35mm Tissue Culture Treated Dish</td><td>Corning</td><td>430165</td><td></td></tr><tr><td>Nunc&amp;trade; 24-Well Carrier Plate for Cell Culture Inserts</td><td>ThermoFisher Scientific</td><td>141006</td><td>Pore size: 8&amp;mu;m</td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Electron Microscopy Sciences</td><td>15710</td><td>16% solution</td></tr><tr><td>1X Phosphate-Buffered Saline (PBS)</td><td>Gibco</td><td>10010023</td><td></td></tr><tr><td>Nutator mixer GyroTwister&amp;trade;</td><td>Labnet</td><td>S1000-A-B</td><td>three dimensional shaker</td></tr><tr><td>Normal Goat Serum</td><td>VWR</td><td>103219-578</td><td></td></tr><tr><td>Bovine Serum Albumen (BSA)</td><td>VWR</td><td>97061-416</td><td></td></tr><tr><td>Sodium Azide (NaN3)</td><td>Sigma-Aldrich</td><td>S2002-25G</td><td></td></tr><tr><td>Sodium borohydride solution (NaBH&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>452904-25ML</td><td>Use the solution, rather than the tablet or powder form&amp;nbsp;</td></tr><tr><td>Polyvinyl Alcohol (PVA)</td><td>Sigma-Aldrich</td><td>P8136-250G</td><td>Cold water soluble</td></tr><tr><td>Triton&amp;trade; X-100</td><td>Sigma-Aldrich</td><td>93443-100ML</td><td>polyethylene glycol tert-octylphenyl ether</td></tr><tr><td>Syringe filters</td><td>ThermoFisher Scientific</td><td>725-2520</td><td>25mm</td></tr><tr><td>10mL Syringes</td><td>BD</td><td>309695</td><td></td></tr><tr><td>Mouse anti-p63 antibody (4A4)</td><td>Biocare Medical</td><td>CM 163A</td><td>Dilution 1:500</td></tr><tr><td>Rabbit anti-MVH antibody</td><td>Abcam</td><td>ab13840</td><td>Dilution 1:600</td></tr><tr><td>Alexa Fluor&amp;reg; goat anti-mouse 594</td><td>ThermoFisher Scientific</td><td>A-11032</td><td>Dilution 1:1000</td></tr><tr><td>Alexa Fluor&amp;reg; goat anti-rabbit 488</td><td>ThermoFisher Scientific</td><td>A-11034</td><td>Dilution 1:1000</td></tr><tr><td>4,6-Diamidino-2-phenylindole (DAPI)&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>62248</td><td></td></tr><tr><td>D-(-)sorbitol&amp;nbsp;</td><td>Sigma-Aldrich</td><td>240850-100G</td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G9012-100ML</td><td></td></tr><tr><td>Urea</td><td>Sigma-Aldrich</td><td>U5378-500G</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>D2650-5X10ML</td><td></td></tr><tr><td>FIJI-ImageJ</td><td></td><td></td><td>Image processing software</td></tr><tr><td>Disposable plastic transfer pipettes</td><td>VWR</td><td>414044-034</td><td></td></tr></tbody></table>

whole mount immunofluorescence and follicle quantification of cultured mouse ovaries

在哺乳动物生殖生物学领域的研究通常涉及评估卵巢和睾丸的整体健康。具体来说, 在女性中, 卵巢健康通常通过形象化和定量卵泡和卵母细胞来评估。由于卵巢是一个不透明的三维组织, 传统的方法需要费力地切片的组织成许多序列切片, 以便可视化整个器官细胞。此外, 由于这种方法的量化通常只需要评分的一部分, 由大约卵母细胞的直径分开, 它容易不准确。在这里, 一个协议被描述, 而不是利用整个器官组织清除和免疫荧光染色小鼠卵巢, 以可视化卵泡和卵母细胞。与传统方法相比, 该协议有利于在卵巢内可视化细胞, 原因有很多: 1) 卵巢在样品制备和加工过程中保持完好;2) 小卵巢不易切片, 可轻松检查;3) 细胞量化更容易、更准确地实现;和 4) 整个器官成像。

该协议包括在卵巢解剖后的6个主要步骤, 如图 1所述。图 2, 图 3, 图 4突出显示了该协议最新颖的功能, 包括使用斐济 ImageJ 优化卵巢组织清除和全组织卵母细胞定量. 图 2A显示了落后5天产后固定子房的图像 (左)...

Watch this Scientific Journal Video about Whole Mount Immunofluorescence and Follicle Quantification of Cultured Mouse Ovaries at JoVE.com

Whole Mount Immunofluorescence and Follicle Quantification of Cultured Mouse Ovaries

在这里, 我们提出一项协议, 以量化卵泡在培养卵巢小鼠没有连续切片。用全器官免疫荧光和组织清除, 用光学切片代替物理切片。这种样品制备和可视化方法保持了器官的完整性, 促进了特定细胞的自动定量。

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, ...

whole-mount-immunofluorescence-follicle-quantification-cultured-mouse

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

10.6K 次观看.  Cornell University. 在这里, 我们提出一项协议, 以量化卵泡在培养卵巢小鼠没有连续切片。用全器官免疫荧光和组织清除, 用光学切片代替物理切片。这种样品制备和可视化方法保持了器官的完整性, 促进了特定细胞的自动定量。

视频: Whole Mount Immunofluorescence and Follicle Quantification of Cultured Mouse Ovaries

Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together within a basement membrane. A finite pool of follicles is laid down during embryonic development, when oocytes in meiotic arrest form a close association with flattened granulosa cells, forming primordial follicles. By or shortly after birth, mammalian ovaries contain their lifetime&#8217;s supply of primordial follicles, from which point onwards there is a steady release of follicles into the growing follicular pool.
The ovary is particularly amenable to development in vitro, with follicles growing in a highly physiological manner in culture. This work describes the culture of whole neonatal ovaries containing primordial follicles, and the culture of individual ovarian follicles, a method which can support the development of follicles from an immature through to the preovulatory stage, after which their oocytes are able to undergo fertilization in vitro. The work outlined here uses culture systems to determine how the ovary is affected by exposure to external compounds. We also describe a co-culture system, which allows investigation of the interactions that occur between growing follicles and the non-growing pool of primordial follicles.

This protocol describes the primary culture/co-culture of mouse ovarian tissue, using ovaries from neonatal mice and individual ovarian follicles from prepubertal mice. The culture techniques support development in a highly physiological manner, allowing investigation of the effect of extrinsic agents on the ovary, and of interactions between ovarian follicles.

文化和共同培养小鼠的卵巢和卵巢卵泡

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together ...

科研

发育生物学

Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We first designed a reporter screen using MEFs from human CD34-tTA/TetO-H2BGFP (34/H2BGFP) double transgenic mice. CD34+ cells from these mice label H2B histones with GFP, and cease labeling upon addition of doxycycline (DOX). MEFS were transduced with candidate TFs and then observed for the emergence of GFP+ cells that would indicate the acquisition of a hematopoietic or endothelial cell fate. Starting with 18 candidate TFs, and through a process of combinatorial elimination, we obtained a minimal set of factors that would induce the highest percentage of GFP+ cells. We found that Gata2, Gfi1b, and cFos were necessary and the addition of Etv6 provided the optimal induction. A series of gene expression analyses done at different time points during the reprogramming process revealed that these cells appeared to go through a precursor cell that underwent an endothelial to hematopoietic transition (EHT). As such, this reprogramming process mimics developmental hematopoiesis "in a dish," allowing study of hematopoiesis in vitro and a platform to identify the mechanisms that underlie this specification. This methodology also provides a framework for translation of this work to the human system in the hopes of generating an alternative source of patient-specific hematopoietic stem cells (HSCs) for a number of applications in the treatment and study of hematologic diseases.

The protocol described here details the induction of a hemogenic program in mouse embryonic fibroblasts via overexpression of a minimal set of transcription factors. This technology may be translated to the human system to provide platforms for future study of hematopoiesis, hematologic disease, and hematopoietic stem cell transplant.

重新编程的小鼠胚胎成纤维细胞转录因子诱导Hemogenic计划

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We ...

Organ Culture and Whole Mount Immunofluorescence Staining of Mouse Wolffian Ducts

Tubal morphogenesis is a fundamental requirement for the development of most mammalian organs, including the male reproductive system. The epididymis, an integral part of the male reproductive tract, is responsible for sperm storage, maturation, and transport. The adult epididymis is a highly coiled tube that develops from a simple and straight embryonic precursor known as Wolffian duct (WD). Proper coiling of the epididymis is essential for male fertility, as sperm in the testis are unable to fertilize an oocyte. However, the mechanism responsible for epididymal development and coiling remains unclear, partially due to the lack of whole organ culture and imaging methods. In this study, we describe an in vitro culture system and whole mount immunofluorescence protocol to better visualize the process of WD coiling and development, which may also be applied to study other tubular organs.

We present a method for isolation and culture of the mouse Wolffian duct (WD). We also demonstrate a detailed procedure for whole mount immunostaining of cultured/freshly isolated WDs with fluorescently tagged antibodies. Together, these techniques enable the study of WD development, coiling, and differentiation.

器官培养和鼠标沃尔弗导管的全山免疫染色

Tubal morphogenesis is a fundamental requirement for the development of most mammalian organs, including the male reproductive system. The epididymis, an ...

Use of Single Molecule Fluorescent In Situ Hybridization (SM-FISH) to Quantify and Localize mRNAs in Murine Oocytes

Current methods routinely used to quantify mRNA in oocytes and embryos include digital reverse-transcription polymerase chain reaction (dPCR), quantitative, real-time RT-PCR (RT-qPCR) and RNA sequencing. When these techniques are performed using a single oocyte or embryo, low-copy mRNAs are not reliably detected. To overcome this problem, oocytes or embryos can be pooled together for analysis; however, this often leads to high variability amongst samples. In this protocol, we describe the use of fluorescence in situ hybridization (FISH) using branched DNA chemistry. This technique identifies the spatial pattern of mRNAs in individual cells. When the technique is coupled with Spot Finding and Tracking&#160;computer software, the abundance of mRNAs in the cell can also be quantified. Using this technique, there is reduced variability within an experimental group and fewer oocytes and embryos are required to detect significant differences between experimental groups. Commercially available branched-DNA SM-FISH kits have been optimized to detect mRNAs in sectioned tissues or adherent cells on slides. However, oocytes do not effectively adhere to slides and some reagents in the kit were too harsh resulting in oocyte lysis. To prevent this lysis, several modifications were made to the FISH kit. Specifically, oocyte permeabilization and wash&#160;buffers designed for the immunofluorescence of oocytes and embryos replaced the proprietary buffers. The permeabilization, washes, and incubations with probes and amplifier were performed in 6-well plates and oocytes were placed on slides at the end of the protocol using mounting media. These modifications were able to overcome the limitations of the commercially available kit, in particular, the oocyte lysis. To accurately and reproducibly count the number of mRNAs in individual oocytes, computer software was used. Together, this protocol represents an alternative to PCR and sequencing to compare the expression of specific transcripts in single cells.

Use of Single Molecule Fluorescent In Situ Hybridization SM-FISH to Quantify and Localize mRNAs in Murine Oocytes

To reproducibly count the numbers of mRNAs in individual oocytes, single molecule RNA fluorescence in situ hybridization (RNA-FISH) was optimized for non-adherent cells. Oocytes were collected, hybridized with the transcript specific probes, and quantified using an image quantification software.

单分子荧光原位杂交 (SM-FISH) 在小鼠卵母细胞中 Mrna 的定量和定位

Current methods routinely used to quantify mRNA in oocytes and embryos include digital reverse-transcription polymerase chain reaction (dPCR), ...

遗传学

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We quantitatively assessed follicle survival, measured follicle and oocyte diameters, and examined ultrastructure of the oocytes produced from the system. We observed decreased follicle survival, downregulation of expressions of proliferating cell nuclear antigen and growth differentiation factor 9, as indicators for the development of granulosa cells and oocytes, respectively, and oocyte ultrastructural abnormalities under the simulated microgravity condition. The simulated microgravity experimental setup needs to be optimized to provide a model for investigation of the mechanisms involved in the oocyte/follicle in vitro development.

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

A highly promising technique to generate tissue constructs without using matrix is to culture cells in a simulated microgravity condition. Using a rotary culture system, we examined ovarian follicle growth and oocyte maturation in terms of follicle survival, morphology, growth, and oocyte function under the simulated microgravity condition.

体外模拟微重力下小鼠腔卵泡生长的研究

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We ...

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and optical transparency. In particular, the zebrafish embryo has become an important organism to study vertebrate kidney organogenesis as well as multiciliated cell (MCC) development. To visualize MCCs in the embryonic zebrafish kidney, we have developed a combined protocol of whole-mount fluorescent in situ hybridization (FISH) and whole mount immunofluorescence (IF) that enables high resolution imaging. This manuscript describes our technique for co-localizing RNA transcripts and protein as a tool to better understand the regulation of developmental programs through the expression of various lineage factors.

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

Cilia development is vital to proper organogenesis. This protocol describes an optimized method to label and visualize ciliated cells of the zebrafish.

全贴装荧光原位杂交和免疫荧光法在斑马鱼中 Multiciliated 细胞的可视化

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and ...

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, ...

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 ...

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

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 system, but the complex structural relationships and three-dimensional (3D) vasculature architectures of the ovary are not well described. In order to visualize the 3D connections and architecture of blood vessels in the intact ovary, the first important step is to make the ovary optically clear. In order to avoid tissue shrinkage, we used the hydrogel fixation-based passive CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/ Immunostaining/In situ-hybridization-compatible Tissue Hydrogel) protocol method to clear an intact ovary. Immunostaining, advanced multiphoton confocal microscopy, and 3D image-reconstructions were then used for the visualization of ovarian vessels and follicular capillaries. Using this approach, we showed a significant positive correlation (P &#60;0.01) between the length of the follicular capillaries and volume of the follicular wall.

Here we present an adaptation of the passive CLARITY and 3D reconstruction method for visualization of the ovarian vasculature and follicular capillaries in intact mouse ovaries.

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 ...