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>

<ol>
	<li>O'Brien, M. J., Pendola, J. K., Eppig, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Revised+Protocol+for+In+Vitro+Development+of+Mouse+Oocytes+from+Primordial+Follicles+Dramatically+Improves+Their+Developmental+Competence.">A Revised Protocol for In Vitro Development of Mouse Oocytes from Primordial Follicles Dramatically Improves Their Developmental Competence.</a> Biology of Reproduction. 68 (5), 1682-1686 (2003).</li><li>Morgan, S., Campbell, L., Allison, V., Murray, A., Spears, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+and+Co-Culture+of+Mouse+Ovaries+and+Ovarian+Follicles.">Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles.</a> Journal of Visualized Experiments. (97), (2015).</li><li>Livera, G., Rouiller-Fabre, V., Valla, J., Habert, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+retinoids+on+the+meiosis+in+the+fetal+rat+ovary+in+culture.">Effects of retinoids on the meiosis in the fetal rat ovary in culture.</a> Molecular and Cellular Endocrinology. 165 (1), 225-231 (2000).</li><li>Livera, G., Petre-Lazar, B., Guerquin, M. -. J., Trautmann, E., Coffigny, H., Habert, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p63+null+mutation+protects+mouse+oocytes+from+radio-induced+apoptosis.">p63 null mutation protects mouse oocytes from radio-induced apoptosis.</a> Reproduction. 135 (1), 3-12 (2008).</li><li>Tilly, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+follicle+counts+-+not+as+simple+as+1,+2,+3.">Ovarian follicle counts - not as simple as 1, 2, 3.</a> Reproductive Biology and Endocrinology. 1, 11 (2003).</li><li>Bucci, T. J., Bolon, B., Warbritton, A. R., Chen, J. J., Heindel, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+sampling+on+the+reproducibility+of+ovarian+follicle+counts+in+mouse+toxicity+studies.">Influence of sampling on the reproducibility of ovarian follicle counts in mouse toxicity studies.</a> Reproductive Toxicology. 11 (5), 689-696 (1997).</li><li>Skodras, A., Marcelli, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computer-Generated+Ovaries+to+Assist+Follicle+Counting+Experiments.">Computer-Generated Ovaries to Assist Follicle Counting Experiments.</a> PLOS ONE. 10 (3), e0120242 (2015).</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> Biology of Reproduction. 93 (5), (2015).</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> Scientific Reports. 7, (2017).</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=ScaleS:+an+optical+clearing+palette+for+biological+imaging.">ScaleS: an optical clearing palette for biological imaging.</a> Nature Neuroscience. 18 (10), 1518-1529 (2015).</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> Nature Neuroscience. 16 (8), 1154-1161 (2013).</li><li>Kuwajima, T., Sitko, A. A., Bhansali, P., Jurgens, C., Guido, W., Mason, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ClearT:+a+detergent-+and+solvent-free+clearing+method+for+neuronal+and+non-neuronal+tissue.">ClearT: a detergent- and solvent-free clearing method for neuronal and non-neuronal tissue.</a> Development. 140 (6), 1364-1368 (2013).</li><li>Rinaldi, V. D., Hsieh, K., Munroe, R., Bolcun-Filas, E. M., Schimenti, J. 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问题1,4进行调整。在三天和十天 (未显示数据) 的卵巢培养方面取得了可比结果, 但十天以上的动物的卵巢很大, 文化可能不那么有效, 因此突出了议定书的局限性。
如果不需要 "体外" 子房的培养, 则可以用所描述的协议修复和染色较旧的卵巢, 尽管需要修改以适应较大的组织大小 916。被描述的染色协议取决于抗体的被动扩散入器官, 表明它可能是可能的染色更大的卵巢以更长的抗体孵化步骤或划分组织成少量小片断可以在成像后重建。尿素和山梨醇在鳞片 (0) 清除剂中的使用证明对新生儿培养卵巢是有效的, 因为它需要比用尿素和甘油 (ScaleA2)8,10的清除剂所报告的潜伏期短。.此外, 使用低浓度的硼氢化钠 (NaBH4) 在通透溶液中降低了背景噪声, 并与主要和二级抗体的样品的较长孵化, 促进在组织内深染色。此外, 在通透和洗涤解决方案中使用 PVA 可以防止杂散粒子粘附到组织17,18。
在本议定书中, 培养健康的卵巢会附着在插入膜上, 而不健康的卵巢可能在固定和处理时与之分离。用镊子处理松散的组织会损害卵巢和可能的折衷成像。或者, 松散组织可以嵌入5% 琼脂糖插头或处理转移吸管, 只要尖端开口是足够宽, 不损害它。对于染色, 除本议定书所用的主要抗体可能会有不同的表现, 可能需要在通透和孵化步骤中进行优化。
最后, 该议定书描述了一种从幼卵巢中定量卵泡的替代方法, 与传统的石蜡嵌入序列切片或其他以前描述的从整个装载图像中卵泡的容积量化相比较。5,9,16. 根据研究者的要求, 该协议中描述的卵母细胞定量程序也可以用于卵泡发育的不同阶段19。该软件产生的粒子面积可用于确定细胞直径, 从而推断卵泡阶段。

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 border="0" cellpadding="0" cellspacing="0" width="882">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Micro dissecting scissor 4.5&#34;, straight, sharp points</td>
			<td style="width:200px;">Roboz</td>
			<td style="width:117px;">RS-5912</td>
			<td style="width:224px;">Micro dissecting scissor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Jewelers style forceps 4-3/8&#34;, style 5</td>
			<td>Integra Miltex</td>
			<td>17-305X</td>
			<td>Fine tip forceps</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro Iris Scissors 4&#34;, 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 height="21">
			<td height="21" style="height:21px;">1X Minimal Essential Media (MEM)</td>
			<td>ThermoFisher Scientific</td>
			<td>11090-081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serium</td>
			<td>Corning</td>
			<td>35011CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES (1M)</td>
			<td>Gibco</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Antibiotic-Antimycotic (100X)</td>
			<td>Gibco</td>
			<td>15240062</td>
			<td>Used in Step 1.3.1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">35mm Tissue Culture Treated Dish</td>
			<td>Corning</td>
			<td>430165</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Nunc&#8482; 24-Well Carrier Plate for Cell Culture Inserts</td>
			<td>ThermoFisher Scientific</td>
			<td>141006</td>
			<td>Pore size: 8&#956;m</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Paraformaldehyde (PFA)</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td>16% solution</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">1X Phosphate-Buffered Saline (PBS)</td>
			<td>Gibco</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nutator mixer GyroTwister&#8482;</td>
			<td>Labnet</td>
			<td>S1000-A-B</td>
			<td>three dimensional shaker</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Normal Goat Serum</td>
			<td>VWR</td>
			<td>103219-578</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Bovine Serum Albumen (BSA)</td>
			<td>VWR</td>
			<td>97061-416</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Sodium Azide (NaN3)</td>
			<td>Sigma-Aldrich</td>
			<td>S2002-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium borohydride solution (NaBH4)</td>
			<td>Sigma-Aldrich</td>
			<td>452904-25ML</td>
			<td>Use the solution, rather than the tablet or powder form&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polyvinyl Alcohol (PVA)</td>
			<td>Sigma-Aldrich</td>
			<td>P8136-250G</td>
			<td>Cold water soluble</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton&#8482; X-100</td>
			<td>Sigma-Aldrich</td>
			<td>93443-100ML</td>
			<td>polyethylene glycol tert-octylphenyl ether</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syringe filters</td>
			<td>ThermoFisher Scientific</td>
			<td>725-2520</td>
			<td>25mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10mL Syringes</td>
			<td>BD</td>
			<td>309695</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse anti-p63 antibody (4A4)</td>
			<td>Biocare Medical</td>
			<td>CM 163A</td>
			<td>Dilution 1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rabbit anti-MVH antibody</td>
			<td>Abcam</td>
			<td>ab13840</td>
			<td>Dilution 1:600</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor&#174; goat anti-mouse 594</td>
			<td>ThermoFisher Scientific</td>
			<td>A-11032</td>
			<td>Dilution 1:1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor&#174; goat anti-rabbit 488</td>
			<td>ThermoFisher Scientific</td>
			<td>A-11034</td>
			<td>Dilution 1:1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4,6-Diamidino-2-phenylindole (DAPI)&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>62248</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-(-)sorbitol&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>240850-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G9012-100ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Urea</td>
			<td>Sigma-Aldrich</td>
			<td>U5378-500G</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D2650-5X10ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FIJI-ImageJ</td>
			<td></td>
			<td></td>
			<td>Image processing software</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">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) 和1小时后 (右) 向子房添加清除解决方案。卵巢将开始变得半透明在被淹没在清除解决方案的几分钟内。一旦清除, 像在图 2A中映像的小卵巢变得难以处理而不造成损害。因此, 使用培养的说明卵巢是有利的, 因为它们会附着在其培养的插入膜的多孔表面。将卵巢附着在插入膜上使实验者能够处理膜插入而不是子房本身 (图 2B 和图 3)。此外, 近距离培养的卵巢将保险丝和图 2B显示附加的融合卵巢在(左)和清除(右)之后, 苏木素染色样品。
在无清除的情况下执行培养卵巢的光学切片是可能的;然而, 在组织内部的细胞有一个信号, 这是很难区分的背景和缺乏明确的信号妨碍适当的卵母细胞量化 (图 3A)。与图 3A相比,图 3B是一个清除样本的代表性图像, 在该示例中, 可以在整个器官中对卵母细胞进行量化。图 3B演示了经过清除的培养卵巢的整个器官成像可以进行, 而不会在组织中有明显的信号丢失, 如显示的图像 (图 3B) 可以很容易地量化。在样本中对卵母细胞进行量化的一种方法是将 Z 堆栈系列的光学剖面转换为最大强度投影, 然后用图像处理包进一步处理该文件。图 4和补充图 1突出显示了用于在图 3B中使用斐济-ImageJ 来量化卵母细胞数量的步骤。补充表 1包括斐济-ImageJ 的粒子 (卵母细胞) 量化。使用这种方法对粒子进行量化也可以根据卵母细胞大小对不同卵泡进行分析, 因为该软件计算了粒子数量和每个粒子对应面积的信息, 并提供给用户。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57593/57593fig1.jpg"> 图 1: 整个协议的示意图表示形式.从卵巢解剖 (1) 开始的协议的视觉总结, 其次是卵巢培养 (2), 组织固定与 4%PFA (3), 染色使用特异抗体 (4), 清除组织为深成像 (5), 获得光学部分使用共聚焦显微镜 (6) 和结束与卵母细胞定量 (7)。<a href="/files/ftp_upload/57593/57593fig1large.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/57593/57593fig2.jpg"> 图 2: 未清除和清除的卵巢之间组织不透明度的差异.(A)子房从产后一天五小狗没有清除 (左) 和在温和的清除以后大约 1 h (正确)。(B)在近距离培养七天的多个卵巢。显示了每个样本的两个不同放大。最左边的图像是没有清除, 最右边的图像是与清除。卵巢将附加到文化插入膜, 并可以更好地处理, 如果保持附加在整个议定书。为了提高白光成像组织的对比度, 固定后卵巢被浸入苏木素中5分钟。<a href="/files/ftp_upload/57593/57593fig2large.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/57593/57593fig3.jpg"> 图 3: 未清除和清除卵巢的免疫荧光成像.从产后5只幼鼠的卵巢培养7天, 并根据整个芒特免疫荧光染色协议染色。以红色显示的细胞标记与老鼠的同源 (MVH), 以确定生殖细胞和在绿色是细胞标记与核卵卵子特异标记, p63。蓝色的 DAPI 被用来标记所有细胞核。(A)卵巢无清除的免疫荧光图像。(B)卵巢带清除的免疫荧光图像。<a href="/files/ftp_upload/57593/57593fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57593/57593fig4.jpg"> 图 4: 有关如何使用斐济-ImageJ 软件量化卵母细胞的可视工作流.为了便于数据分析, 从共焦平面获得的图像可以减少到最大强度投影而不是3D 图像。利用最大强度投影, 用户可以定义图像阈值参数, 使目标粒子变得明显。一旦获得了简化的图像, 该软件用于计数卵母细胞。在设置参数时严格检查图像是至关重要的。此图显示了如何设置粒子/卵母细胞阈值的示例。每个图像上方的文本突出显示了用于在斐济-ImageJ 中获取该图像的参数。软件生成一个带有粒子面积测量的表 (请参见补充表 1)。<a href="/files/ftp_upload/57593/57593fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
补充图 1: 有关如何使用斐济-ImageJ 软件 (更低的放大倍数) 量化卵母细胞的可视工作流.该图显示了近距离两个卵巢的卵母细胞定量。执行的步骤与图 4中相同。2436微粒或卵泡由软件计数。从软件获得的量化数据可以在补充表 1中找到。<a href="/files/ftp_upload/57593/Supplemental_Figure1_fix.pdf">请单击此处下载此文件.</a>
补充表 1: 由斐济 ImageJ 计算的卵泡定量.此表包含计数卵泡的列表以及补充图 1中从图像生成的相应区域。用于量化的粒子大小由10µm2-无穷大设置。<a href="/files/ftp_upload/57593/Supplementary_table_1.xlsx">请单击此处下载此文件.</a>

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

Nanyang Technological University

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 ...

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 ...

Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques

Due to its formidable tools for molecular genetic studies, Arabidopsis thaliana is one of the most prominent model species in plant biology and, especially, in plant reproductive biology. However, plant morphological, anatomical, and ultrastructural analyses traditionally involve time-consuming embedding and sectioning procedures for bright field, scanning, and electron microscopy. Recent progress in confocal fluorescence microscopy, state-of-the-art 3-D computer-aided microscopic analyses, and the continuous refinement of molecular techniques to be used on minimally processed whole-mount specimens, has led to an increased demand for developing efficient and minimal sample processing techniques. In this protocol, we describe techniques for properly dissecting Arabidopsis flowers and siliques, basic clearing techniques, and some staining procedures for whole-mount observations of reproductive structures.

Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques

In this protocol, we describe techniques for the proper dissection of Arabidopsis flowers and siliques, some basic clearing techniques, and selected staining procedures for whole-mount observations of reproductive structures.

拟南芥花器官和角果的全装清和染色

Due to its formidable tools for molecular genetic studies, Arabidopsis thaliana is one of the most prominent model species in plant biology and, ...

Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development and organogenesis are two areas of research that have benefitted greatly from these advances, due to the high quality of data that can be obtained directly from imaging pre-implantation embryos or ex vivo organs. While pre-implantation embryos have yielded data with especially high spatial resolution, later stages have been less amenable to three-dimensional reconstruction. Obtaining high-quality 3D or volumetric data for known embryonic structures in combination with fate mapping or genetic lineage tracing will allow for a more comprehensive analysis of the morphogenetic events taking place during embryogenesis.
This protocol describes a whole-mount immunofluorescence approach that allows for the labeling, visualization, and quantification of progenitor cell populations within the developing cardiac crescent, a key structure formed during heart development. The approach is designed in such a way that both cell- and tissue-level information can be obtained. Using confocal microscopy and image processing, this protocol allows for three-dimensional spatial reconstruction of the cardiac crescent, thereby providing the ability to analyze the localization and organization of specific progenitor populations during this critical phase of heart development. Importantly, the use of reference antibodies allows for successive masking of the cardiac crescent and subsequent quantitative measurements of areas within the crescent. This protocol will not only enable a detailed examination of early heart development, but with adaptations should be applicable to most organ systems in the gastrula to early somite stage mouse embryo.

Here, we describe a protocol for whole mount immunofluorescence and image-based quantitative volumetric analysis of early stage mouse embryos. We present this technique as a powerful approach to qualitatively and quantitatively assess cardiac structures during development, and propose that it may be widely adaptable to other organ systems.

小鼠胚胎心肌内祖群的定量全贴免疫荧光分析

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development ...

Dissection and Staining of Drosophila Pupal Ovaries

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a pupal case. The challenge of dissecting pupal ovaries also lies in their physical location within the pupa: the ovaries are surrounded by fat body cells inside the pupal abdomen, and these fat cells must be removed to allow for proper antibody staining. To overcome these challenges, this protocol utilizes customized Pasteur pipets to extract fat body cells from the pupal abdomen. Moreover, a chambered coverglass is used in place of a microcentrifuge tube during the staining process to improve visibility of the pupae. However, despite these and other advantages of the tools used in this protocol, successful execution of these techniques may still involve several days of practice due to the small size of pupal ovaries. The techniques outlined in this protocol could be applied to time course experiments in which ovaries are analyzed at various stages of pupal development.

Dissection and Staining of Drosophila Pupal Ovaries

The Drosophila ovary is an excellent model system for studying stem cell niche development. Though methods for dissecting larval and adult ovaries have been published, pupal ovary dissections require different techniques that have not been published in detail. Here we outline a protocol for dissecting, staining, and mounting pupal ovaries.

果蝇蛹卵巢的解剖与染色

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

用扫描电镜和全贴免疫荧光技术可视化小鼠胚胎中的节点和外型板

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

Analysis of Cardiac Chamber Development During Mouse Embryogenesis Using Whole Mount Epifluorescence

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during heart development using ventricular specific fluorescent reporter knock-in mice (MLC-2v-tdTomato mice). Heart development involves a linear heart tube formation, the heart tube looping, and four chamber septation. These complex processes are highly conserved in all vertebrates. The mouse embryonic heart has been widely used for heart developmental studies. However, due to their extremely small size, dissecting mouse embryonic hearts is technically challenging. In addition, visualization of cardiac chamber formation often needs in situ hybridization, beta-galactosidase staining using LacZ reporter mice, or immunostaining of sectioned embryonic hearts. Here, we describe how to dissect mouse embryonic hearts and directly visualize ventricular chamber formation of MLC-2v-tdTomato mice using whole mount epifluorescent microscopy. With this method, it is possible to directly examine heart tube formation and looping, and four chamber formation without further experimental manipulation of mouse embryos. Although the MLC-2v-tdTomato reporter knock-in mouse line is used in this protocol as an example, this protocol can be applied to other heart-specific fluorescent reporter transgenic mouse lines.

We present the protocols to examine mouse heart development using whole mount epifluorescent microscopy on mouse embryos dissected from ventricular specific MLC-2v-tdTomato reporter knock-in mice. This method allows us to directly visualize each stage of the ventricular formation during mouse heart development without labor-intensive histochemical methods.

全山荧光在小鼠胚胎发生过程中心脏室发育的分析

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during ...

Laser Capture Microdissection of Mouse Embryonic Cartilage and Bone for Gene Expression Analysis

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and molecular complexity of skeletal elements increases with development. Tissue heterogeneity, such as at the interface of cartilaginous and osseous elements with each other or with surrounding tissues, is one obstacle to the study of developing cartilage and bone. Our protocol provides a rapid method of tissue processing and isolation of cartilage and bone that yields high quality RNA for gene expression analysis. Fresh frozen tissues of mouse embryos are sectioned and brief cresyl violet staining is used to visualize cartilage and bone with colors distinct from surrounding tissues. Slides are then rapidly dehydrated, and cartilage and bone are isolated subsequently by LCM. The minimization of exposure to aqueous solutions during this process maintains RNA integrity. Mouse Meckel&rsquo;s cartilage and mandibular bone at E16.5 were successfully collected and gene expression analysis showed differential expression of marker genes for osteoblasts, osteocytes, osteoclasts, and chondrocytes. High quality RNA was also isolated from a range of tissues and embryonic ages. This protocol details sample preparation for LCM including cryoembedding, sectioning, staining and dehydrating fresh frozen tissues, and precise isolation of cartilage and bone by LCM resulting in high quality RNA for transcriptomic analysis.

This protocol describes laser capture microdissection for the isolation of cartilage and bone from fresh frozen sections of the mouse embryo. Cartilage and bone can be rapidly visualized by cresyl violet staining and collected precisely to yield high quality RNA for transcriptomic analysis.

用于基因表达分析的小鼠胚胎软骨和骨骼的激光捕获微分

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and ...