这项工作得到了德意志 Forschungsgemeinschaft SFB688 (对司法部) 的支持。

小鼠育种得到了 Regierung Unterfranken 的批准, 所有的分析都严格按照德国和欧洲联盟关于实验室动物护理和使用的法律和法规进行。
1. 尿外植体培养的α4β1整合素孔板包衣
<ol><li>重组冻干小鼠α4β1整合素在200µg/毫升无菌磷酸盐缓冲盐水 (PBS)。在 5ml (37 ° c) PBS 中稀释至最终浓度为10µg/毫升。吸管20µL/稀释α4β1整合素溶液到所需数量的孔板 (1 尿/井) 的井。 注: 在孔板上涂布井时避免气泡形成。</li>
	<li>在37° c 的孔板上孵育60分钟, 将它们放置在湿润的组织培养孵化器中。</li>
	<li>用吸管小心地吸上清液。使用温和的吸力和倾斜的板, 以避免触及井底与吸管尖端。通过添加50µL 5ml (37 ° c) 培养基, 如 Dulbecco 氏改良鹰培养基 (DMEM) 冲洗两次, 并通过轻柔的吸力除去洗涤介质。</li>
	<li>关键步骤: 通过将孔板放置在湿化组织培养箱中, 在37° c 中加入50µL/0.1% (w/v) 牛血清白蛋白 (BSA)/DMEM 并孵育为≥30分钟, 以阻止自由结合点。 注: 尿外植体可附着在血清存在的各种表面。因此, 必须阻止非特异性结合位点, 在用整合素对水井进行涂布后, 并在加入含血清培养基中的外植体培养的解剖尿 (见步骤 4.4)。</li>
	<li>在4.4 步之前, 将涂覆的水井保持在37° c 的堵塞溶液中。</li>
</ol>2. 在 Dpc 8 的怀孕小鼠子宫剥离
注意: 减少假阳性妊娠率的体重增加歧视15。
<ol><li>用颈椎脱位处死小鼠。用 70% (v/v) 乙醇喷洒, 对腹部的外套进行消毒。 注意: 处理小鼠时要戴上手套。无麻醉剂的宫颈脱位可防止尿的化学污染, 这可能会干扰尿的附着。</li>
	<li>用剪刀在中线上做一个小切口, 打开腹部的皮肤。用戴手套的手指按住切口上方和下方的皮肤, 将皮肤拉向鼠标的胸部和尾部。</li>
	<li>用钳子抓住腹膜。另一方面, 用剪刀从前向后方做 V 形切口, 并将组织移开以暴露腹腔。用镊子将肠道推到一侧, 以获得子宫。</li>
	<li>找到两个子宫角。用钳子钳住宫颈 (尾段), 用细剪刀切开韧带。然后用剪刀割断输卵管的两个子宫角。</li>
	<li>用镊子将子宫转移到无菌、室温 (RT) PBS 的10厘米的无菌皿中。用细钳和/或剪刀除去子宫角周围的脂肪组织、血管和神经 (图 1A)。</li>
</ol>3. 胚胎解剖
注意: 在装有8:1 缩放范围的显微镜下执行以下步骤。
<ol><li>用剪刀在植入部位之间切割, 分离胚胎。用镊子抓住子宫芽肌的子宫内衬, 并将其拉开, 露出蜕膜 (图 1B)。</li>
	<li>使用镊子, 将子宫芽转移到一个新的10厘米的碟含有无菌, RT PBS。</li>
	<li>用钳子钳把胚胎周围的蜕膜固定下来。</li>
	<li>用细钳 (图 1C) 在蜕膜的 anti-mesometrial 一侧做切口, 并用钳子剥开蜕膜以露出胚胎 (图 1D)。 注: 蜕膜的 mesometrial 极具有极强的血管化作用, 在其基底上可比 anti-mesometrial 部分更宽, 其中包含胚胎并呈苍白。</li>
	<li>用镊子解剖胚胎 (图 1E, F)。</li>
	<li>使用微勺, 将胚胎移植到一滴无菌的 PBS 中, 包含在3.5 厘米的无菌皿中。</li>
	<li>使用微勺将胚胎移植到同一 3.5 cm 盘中的 PBS 中, 用镊子将胚胎从卵黄囊中解剖 (图 1G, H)。</li>
	<li>(可选), 如果需要 (例如, 通过聚合酶链反应进行基因分型), 将卵黄囊转移到无菌管中, 将其与 ~ 5 µL PBS 吸进一个大孔200µL 吸管尖端。贮存在-20 ° c。 注意: 母体组织 (即, 包括赖克特膜和 ectoplacental 锥在内的顶叶囊) 可能会污染卵黄囊的制备, 导致基因分型结果错误。切除这些组织是最容易的前胚胎解剖从蛋黄囊。</li>
</ol>4. 固定化α4β1整合素的尿解剖和前子宫培养
<ol><li>使用细钳 (图 1H) 将尿从其在胚胎后端的基底插入部位切开。</li>
	<li>(可选), 如果需要发展分期, 节对胚胎的视觉检查使用的显微镜, 配有相衬光学和5×和10x 目标。 注意: 胚胎可以用镊子延长, 以方便节计数。囊附着和融合发生在 e 8.0 和 e 9.0 在胚胎与≥6节对。转动开始在胚胎与6-8 节对。</li>
	<li>吸管40µL/井的 DMEM 补充 10% (v/v) 胎牛血清, l-谷氨酰胺, 和抗生素在所需数量的预孔板井 (见第 1)。</li>
	<li>收集漂浮的尿通过吸它与〜5µL PBS 到一个大孔200µL 吸管尖端。转1尿/井。</li>
	<li>通过将孔板放置在湿润的组织培养孵化器 (37 ° c, 5% CO2) 中, 用于例如、6、12和 24 h, allantoides 孵化。为了防止干扰与尿附件, 不要移动孔板材在这些时间点之间。</li>
	<li>在每个时间点, 评分尿附件 (例如, 没有附件/浮动尿; 部分或完全连接的尿; 完全传播尿) 使用具有相衬度或差分干涉对比度光学的显微镜和5×或10x 目标 (图 2)。 注意: 新隔离和完全连接的 allantoides 的典型示例如图 2所示。前子宫, 从野生 C57BL/6J 小鼠中分离出的 allantoides 远端的末端附着在4-6 小时内 (这被称为尿的部分附着), 整个尿的完全附着是在 12 h 的培养基中实现的。18-24 h, 外植体已扁平, 并假设一个圆形 (称为完全传播尿) 与一个 CD31-positive 血管丛9,16。</li>
</ol>5. 说明尿对内皮细胞标记 CD31 的验证染色
<ol><li>通过添加40µL/4% (w/v) 甲醛/PBS, 来吸取培养基中的完全传播外植体和固定细胞。 注: 甲醛是有毒的。戴上手套和安全护目镜, 避免皮肤和眼睛接触。</li>
	<li>在 RT 处孵育10分钟, 然后抽出上清液。洗涤三次, 加入40µL/PBS, 并通过温和的吸力去除洗涤介质。</li>
	<li>Permeabilize 细胞和阻断非特异性结合位点, 添加40µL/井 0.1% (v/v) 海卫十-100/0.1% (w/v) BSA/10% (v/v) 正常山羊血清在 PBS 为30分钟 RT. 通过添加40µL 或 PBS 的好, 洗两次, 并通过温和的吸力去除洗涤介质。 注意: 在这一点上, 协议可以在一夜之间暂停。</li>
	<li>染色细胞添加20µL/良好的主要 anti-CD31 抗体稀释1:50 在 0.1% (w/v) BSA/PBS 过夜在4° c。洗涤三次, 加入40µL/PBS, 并通过温和的吸力去除洗涤介质。</li>
	<li>通过在 RT 中添加20µL/荧光标记的二级山羊鼠抗体稀释1:200 在 0.1% (w/v) BSA/PBS 为1小时, 以检测束缚的主要抗体. 保护孔板免受光通过铝箔包装或放置在一个黑暗的房间。洗涤三次, 加入40µL/PBS, 并通过温和的吸力去除洗涤介质。</li>
	<li>通过添加20µL/好的安装介质来嵌入单元格。在 RT 中孵育1小时直到安装介质凝固。在 RT 或4° c 处储存孔板, 免受光线的保护。</li>
	<li>图像的尿外植体的血管丛的荧光显微镜下配备了5×或10x 目标和合适的荧光过滤器。 注: 在图 3中显示了完全展开尿的 CD31-positive 血管丛的代表性图像。其他用于血管丛的标记, 如针对 Flk-1、外语、Tie-1 或 Tie-2 的抗体, 也可用于16。</li>
</ol>

<ol>
	<li>Rossant, J., Cross, 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=Placental+development:+lessons+from+mouse+mutants.">Placental development: lessons from mouse mutants.</a> Nat Rev Genet. 2 (7), 538-548 (2001).</li><li>Segerer, G., 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=An+essential+developmental+function+for+murine+phosphoglycolate+phosphatase+in+safeguarding+cell+proliferation.">An essential developmental function for murine phosphoglycolate phosphatase in safeguarding cell proliferation.</a> Sci Rep. 6, 35160 (2016).</li><li>Kane, S. V., Acquah, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Placental+transport+of+immunoglobulins:+a+clinical+review+for+gastroenterologists+who+prescribe+therapeutic+monoclonal+antibodies+to+women+during+conception+and+pregnancy.">Placental transport of immunoglobulins: a clinical review for gastroenterologists who prescribe therapeutic monoclonal antibodies to women during conception and pregnancy.</a> Am J Gastroenterol. 104 (1), 228-233 (2009).</li><li>Xu, X., Vugmeyster, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+and+opportunities+in+absorption,+distribution,+metabolism,+and+excretion+studies+of+therapeutic+biologics.">Challenges and opportunities in absorption, distribution, metabolism, and excretion studies of therapeutic biologics.</a> AAPS J. 14 (4), 781-791 (2012).</li><li>Wesolowski, S. R., Kasmi, K. C., Jonscher, K. R., Friedman, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+origins+of+NAFLD:+a+womb+with+a+clue.">Developmental origins of NAFLD: a womb with a clue.</a> Nat Rev Gastroenterol Hepatol. 14 (2), 81-96 (2017).</li><li>Racicot, K., Mor, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Risks+associated+with+viral+infections+during+pregnancy.">Risks associated with viral infections during pregnancy.</a> J Clin Invest. 127 (5), 1591-1599 (2017).</li><li>Grandjean, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life-Long+Implications+of+Developmental+Exposure+to+Environmental+Stressors:+New+Perspectives.">Life-Long Implications of Developmental Exposure to Environmental Stressors: New Perspectives.</a> Endocrinology. 156 (10), 3408-3415 (2015).</li><li>Inman, K. E., Downs, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+murine+allantois:+emerging+paradigms+in+development+of+the+mammalian+umbilical+cord+and+its+relation+to+the+fetus.">The murine allantois: emerging paradigms in development of the mammalian umbilical cord and its relation to the fetus.</a> Genesis. 45 (5), 237-258 (2007).</li><li>Arora, R., Papaioannou, V. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+murine+allantois:+a+model+system+for+the+study+of+blood+vessel+formation.">The murine allantois: a model system for the study of blood vessel formation.</a> Blood. 120 (13), 2562-2572 (2012).</li><li>Watson, E. D., Cross, 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=Development+of+structures+and+transport+functions+in+the+mouse+placenta.">Development of structures and transport functions in the mouse placenta.</a> Physiology (Bethesda). 20, 180-193 (2005).</li><li>Yang, J. T., Rayburn, H., Hynes, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+adhesion+events+mediated+by+alpha+4+integrins+are+essential+in+placental+and+cardiac+development.">Cell adhesion events mediated by alpha 4 integrins are essential in placental and cardiac development.</a> Development. 121 (2), 549-560 (1995).</li><li>Gurtner, G. C., 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=Targeted+disruption+of+the+murine+VCAM1+gene:+essential+role+of+VCAM-1+in+chorioallantoic+fusion+and+placentation.">Targeted disruption of the murine VCAM1 gene: essential role of VCAM-1 in chorioallantoic fusion and placentation.</a> Genes &amp; development. 9 (1), 1-14 (1995).</li><li>Kwee, L., 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=Defective+development+of+the+embryonic+and+extraembryonic+circulatory+systems+in+vascular+cell+adhesion+molecule+(VCAM-1)+deficient+mice.">Defective development of the embryonic and extraembryonic circulatory systems in vascular cell adhesion molecule (VCAM-1) deficient mice.</a> Development. 121 (2), 489-503 (1995).</li><li>Downs, K. M., Gardner, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+investigation+into+early+placental+ontogeny:+allantoic+attachment+to+the+chorion+is+selective+and+developmentally+regulated.">An investigation into early placental ontogeny: allantoic attachment to the chorion is selective and developmentally regulated.</a> Development. 121 (2), 407-416 (1995).</li><li>Heyne, G. W., 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+simple+and+reliable+method+for+early+pregnancy+detection+in+inbred+mice.">A simple and reliable method for early pregnancy detection in inbred mice.</a> J Am Assoc Lab Anim Sci. 54 (4), 368-371 (2015).</li><li>Downs, K. M., Temkin, R., Gifford, S., McHugh, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+of+the+murine+allantois+by+allantoic+explants.">Study of the murine allantois by allantoic explants.</a> Dev Biol. 233 (2), 347-364 (2001).</li><li>Hou, W., Sarikaya, D. P., Jerome-Majewska, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+culture+of+pre-placental+tissues+reveals+that+the+allantois+is+required+for+maintained+expression+of+Gcm1+and+Tpbpalpha.">Ex vivo culture of pre-placental tissues reveals that the allantois is required for maintained expression of Gcm1 and Tpbpalpha.</a> Placenta. 47, 12-23 (2016).</li><li>Zeigler, B. 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=The+allantois+and+chorion,+when+isolated+before+circulation+or+chorio-allantoic+fusion,+have+hematopoietic+potential.">The allantois and chorion, when isolated before circulation or chorio-allantoic fusion, have hematopoietic potential.</a> Development. 133 (21), 4183-4192 (2006).</li><li>Zaidel-Bar, R., Itzkovitz, S., Ma'ayan, A., Iyengar, R., Geiger, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+atlas+of+the+integrin+adhesome.">Functional atlas of the integrin adhesome.</a> Nature cell biology. 9 (8), 858-867 (2007).</li></ol>

作者没有什么可透露的。

在血清的存在, 这是一个丰富的来源, pro-adhesive 细胞外基质分子, 如纤维连接蛋白, 尿植体将欣然附加到各种塑料, 玻璃, 或过滤表面9,16。因此, 如果进行的试验的目的是专门研究基因修饰或任何其他治疗对固定化α4β1尿附着的影响, 就必须阻止非特异性结合位点 (例如, 用牛血清白蛋白) 涂层后的表面与整合素和前孵化与含血清的媒体。
<p class="jo...

胎盘在子宫环境中的胚胎生长发育是必不可少的。它构成了胎儿和母体循环之间的氧气、代谢物和养分交换的界面, 也是内分泌和免疫器官的功能。任何内生或外源性侮辱, 损害胎盘发育可能导致宫内发育迟缓, 胎儿的损失, 或怀孕并发症, 在小鼠和人类的1。虽然导致胎盘生理异常的靶向小鼠突变的数量继续增加2, 但目前在小鼠基因组信息学 (凡陀) 网站上列出的219种基因型是 (http://www.informatics.jax.org/vocab/mp_ontology/MP:0010038), 许多这些表型从机械的角度看不清楚。除了基因改变, 小分子药物, 单克隆抗体, 毒素, 病原体, 过量代谢物, 或各种环境因子可能影响胎盘发育和功能3,4,5,6,7. 然而, 简单的体外分析了胎盘发育中的模型关键步骤是稀缺的。
小鼠胎盘发育在早期妊娠开始, 当尿 (脐带的发育前体) 从胚的后端出现, 作为一个芽, 生长向绒毛膜的8。Chorioadhesive 细胞在囊芽的外层斡旋在绒毛膜 (囊 "融合") 的表面上的附着和传播尿。绒毛膜随后折叠成绒毛进入其中尿的血管生长形成迷宫, 内部胎盘层在那里营养和氧气被交换在严密并列的母体血液窦和胚胎血管之间9 ,10。
尿对绒毛膜的附着是迷宫形成的初始和关键步骤, 而这一过程中的缺陷是妊娠1中最常见的胚胎致死原因之一。虽然已经描述了许多鼠标突变体, 但囊附件无法出现在10, 并且凡陀数据库当前列出了108个以异常囊融合为特征的鼠标突变体 (http://www.informatics.jax.org/vocab/mp_ontology/MP:0002824), 血管细胞黏附分子之间的相互作用 molecule-1 (VCAM-1, 表达囊胚层) 和α4β1整合素 (表达在绒毛膜皮, 这是派生自外胚层) 似乎是必不可少的囊附件和融合11,12,13。全芒野型胚的免疫组织化学染色表明, VCAM-1 在囊茎远端2/3 处以大约胚胎日 (E) 7.513 (1-4 节阶段) 表达, 其表达仍然高在囊附件和-融合11,12期间。囊附着于绒毛膜的失败通常通过子宫芽的组织学分析来检测。然而, 尿大小是生理上高度可变的在之间, 甚至在同一发育阶段的凋落物14, 和尿只能附加到绒毛膜当它已经达到足够的大小, 使身体接触。反射这自然可变性, 囊附件发生在 ~ e 8.0 和 e 9.0在子宫内, 并且一个统计地可靠评估这个过程由组织学是依赖于分析许多conceptuses 在适当的发育阶段获得足够的标本。
在这里, 我们描述一种方法来评估囊附件前子宫, 不太依赖于尿大小。我们展示了小鼠胚胎的解剖和他们的 allantoides 从怀孕的老鼠的子宫〜8天后 coitum (dpc; dpc 0.5 指定阴道插头的检测), 并随后培养尿外植体的固定α4β1整合.该方法能够对多尿外植体在不同的连续时间点上的附着和扩展行为进行快速、实用的评价。该协议可用于筛选目标突变、药物或各种环境因素对尿附着体的影响ex 体内。

<table><tbody><tr><td>C57BL/6J wildtype mice</td><td>The Jackson Laboratory</td><td>Stock No:&amp;nbsp;000664</td><td></td></tr><tr><td>70 % (v/v) Ethanol</td><td>VWR International</td><td>930031006</td><td></td></tr><tr><td>Standard pattern forceps</td><td>Fine Science Tools</td><td>11000-12</td><td>Forceps used to open the abdominal cavity.</td></tr><tr><td>Micro dissecting forceps</td><td>Fine Science Tools</td><td>11254-20</td><td>Dumont # 5 medical biology forcepts with fine, sharp tip.&amp;nbsp;</td></tr><tr><td>Fine scissors&amp;nbsp;</td><td>Fine Science Tools</td><td>14094-11</td><td>Straight blades.</td></tr><tr><td>Spring scissors&amp;nbsp;</td><td>Fine Science Tools</td><td>15012-12</td><td>Noyes spring scissors with 14 mm blades for the dissection of uterus buds.</td></tr><tr><td>Microspoon</td><td>Carl Roth</td><td>AT18.1</td><td>5 mm Spoon diameter.</td></tr><tr><td>Microtiter plates</td><td>ibidi</td><td>81501</td><td>We use uncoated, sterile angiogenesis slides (internal volume 50 &amp;micro;L) with a hydrophobic surface that is not tissue-culture treated to reduce non-&amp;#945;4&amp;#946;1-mediated binding to plastic.</td></tr><tr><td>10 cm dish</td><td>Nunc</td><td>150350</td><td>Sterile tissue culture dishes.</td></tr><tr><td>3.5 cm dish</td><td>Nunc</td><td>150288</td><td>Sterile tissue culture dishes.</td></tr><tr><td>Large orifice pipette tips&amp;nbsp;</td><td>Biozym</td><td>VT0140X</td><td>Low binding pipette tips, 200 &amp;micro;L.</td></tr><tr><td>&amp;#945;4&amp;#946;1 integrin</td><td>R&amp;amp;D Systems</td><td>6054-A4</td><td>Murine &amp;#945;4&amp;#946;1 integrin recombinantly expressed and purified from a Chinese hamster ovary cell line.</td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline (PBS)</td><td>Sigma Aldrich</td><td>D8537</td><td>Without Ca&lt;sup&gt;2+&lt;/sup&gt; and Mg&lt;sup&gt;2+&lt;/sup&gt;.</td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle Medium (DMEM)</td><td>PAN Biotech</td><td>P04-03600</td><td>The formulation contains 4.5 g/L glucose, 110 mg/L sodium pyruvate and 3.7 g/L NaHCO&lt;sub&gt;3&lt;/sub&gt; but no L-glutamine, which is added separately.</td></tr><tr><td>Penicillin/Streptomycin</td><td>Gibco (ThermoFisher Scientific)</td><td>15140-122</td><td>100 x (10,000 U/mL Penicillin and 10,000 &amp;micro;g/mL streptomycin) stock solution.&amp;nbsp;Prepare and store aliquots at -20 &amp;deg;C to avoid freeze/thaw.</td></tr><tr><td>L-Glutamine</td><td>PAN Biotech</td><td>P04-80100</td><td>100 x (200 mM) Stock solution. Prepare and store aliquots at -20 &amp;deg;C to avoid freeze/thaw.</td></tr><tr><td>Fetal bovine serum&amp;nbsp;</td><td>Biochrom</td><td>S 0115</td><td>We use heat-inactivated FBS (heated to 56 &amp;deg;C for 30 min with mixing to inactivate complement).</td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma Aldrich</td><td>A7030</td><td>We use protease- and fatty acid-free albumin. Prepare a 0.1 % (w/v) solution in DMEM. Sterile filter the solution through a disposable cell culture filter with a 0.22 &amp;micro;m pore size and low protein-binding membrane, and store aliquots at -20 &amp;deg;C.</td></tr><tr><td>&lt;em&gt;para&lt;/em&gt;-Formaldehyde</td><td>Carl Roth</td><td>0335.2</td><td>To make a formaldehyde solution in PBS, weigh &lt;em&gt;para&lt;/em&gt;-formaldehyde powder in a ventilated hood, and add it to PBS preheated to ca. 60 &amp;deg;C in a beaker on a stir plate. Add 1 N NaOH dropwise until solution clears. Let solution cool to room temperature, and filter through 0.22 mm filter syringe. Adjust pH with diluted HCl to ca. 6.9, and adjust to final volume with PBS. We aliquot and freeze the solution, but it can also be stored at 4 &amp;deg;C for approximately four weeks.&amp;nbsp;</td></tr><tr><td>Triton X-100</td><td>Sigma Aldrich</td><td>X100</td><td>Use wide-orifice pipette tips to handle undiluted Triton X-100.</td></tr><tr><td>Normal goat serum</td><td>Sigma Aldrich</td><td>G9023</td><td>To block non-specific binding of antibodies in immunofluorescence analyses.</td></tr><tr><td>&amp;#945;-CD31 Antibodies</td><td>BD Biosciences</td><td>550274</td><td>Purified rat anti-mouse monoclonal antibodies, clone MEC 13.3.</td></tr><tr><td>Secondary goat anti-rat antibodies</td><td>Thermo Fisher</td><td>A-11006</td><td>Goat anti-rat IgG (heavy and light chains), cross-adsorbed, fluorescently labeled with Alexa Fluor 488. Other fluorescent labels are also possible.</td></tr><tr><td>Mowiol 4-88</td><td>Sigma Aldrich</td><td>81381</td><td>Mounting medium for cytochemistry. MW ~31,000</td></tr><tr><td>Labovert&amp;nbsp;</td><td>Leitz</td><td>Labovert&amp;nbsp;</td><td>Inverted phase contrast microscope equipped with a 4 x and 10 x objective for somite pair counting. Other phase contrast microscopes (such as those that are routinely used in tissue culture) are also suitable.&amp;nbsp;&amp;nbsp;</td></tr><tr><td>M80</td><td>Leica Microsystems</td><td>M80</td><td>Stereomicroscope with 8 : 1 zoom range (yielding a magnification between 7.5 x and 60 x) for embryo and allantois dissection.</td></tr><tr><td>DM4000B</td><td>Leica Microsystems</td><td>DM4000B</td><td>Microscope system for histology with LED illumination. We use HCX PL FLUOTAR 5 x/0.15 and HC PL FLUOTAR 10 x/0.30 objectives. Other microscopes equipped phase contrast and fluorescence optics can be used to score allantois attachment.&amp;nbsp;</td></tr><tr><td>Digital camera</td><td>JVC</td><td>KY-F75U</td><td>3-CCD digital capture camera attached to the DM4000B LED microscope. We use Diskus software (Hilgers) to operate both the microscope and the camera.</td></tr><tr><td>Confocal microscope</td><td>Leica Microsystems</td><td>SP5</td><td>Confocal microscope for higher-resolution imaging of endothelia in the vascular plexus of allantois explants. Immunofluorescence images were taken with a 40 x objective.</td></tr></tbody></table>

dissection and explant culture of murine allantois for the in vitro analysis of allantoic attachment

胎盘对哺乳动物胚胎的生长发育至关重要。由于这个原因, 许多基因的改变和环境的侮辱, 扰乱胎盘的发展或功能可能导致早孕损失的小鼠和人类。然而, 简单的体外检测对胎盘形成的潜在影响是缺乏的。在这里, 我们的重点是建模的第一步和关键步骤的胎盘形成, 其中包括附着的尿的绒毛膜。我们描述了一种快速评估囊外植体附着在固定化α4β1整合素上的方法, 它是一种 chorio 模拟的基底。这种体外方法可以对不同连续时间点的多个尿的附着和扩散行为进行定性评估。该协议可用于调查有针对性的小鼠突变, 药物, 或各种环境因素的影响, 已与妊娠并发症或胎儿损失的尿附着在前体内。

本协议描述了一种方法来隔离和外植体小鼠 allantoides前体, 并详细的定性评估的尿附着α4β1整合素, 一个关键的过程中, 在体内囊融合。在图 1A-H中显示的代表性结果演示了从怀孕子宫开始的尿隔离的连续步骤。围绕子宫角的母体组织, 如脂肪组织和血管 (图 1A) 被移除, 并在打开肌肉子宫层?...

Watch this Scientific Journal Video about Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment at JoVE.com

Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment

我们描述了一个体外测定模型囊附着, 第一步胎盘形成。该协议展示了小鼠 allantoides 在固定化α4β1整合素上的解剖和外植体培养。尿附件是在预定的时间点显微镜下评估的。

小鼠尿的解剖和外植体培养对囊附着的作用分析

The placenta is essential for the growth and development of mammalian embryos. For this reason, numerous genetic alterations and likely also environmental ...

dissection-explant-culture-murine-allantois-for-vitro-analysis

Research

JoVE Journal

Medicine

Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment

7.8K 次观看.  University of Würzburg. 我们描述了一个体外测定模型囊附着, 第一步胎盘形成。该协议展示了小鼠 allantoides 在固定化α4β1整合素上的解剖和外植体培养。尿附件是在预定的时间点显微镜下评估的。

视频: Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic lamellae and smooth muscle cells (SMCs), and an outer layer of fibroblasts and extracellular matrix. In contrast to the widespread study of pathological models (e.g., atherosclerosis) in the adult aorta, much less is known about the embryonic and perinatal aorta. Here, we focus on SMCs and provide protocols for the analysis of the morphogenesis and pathogenesis of embryonic and perinatal aortic SMCs in normal development and disease. Specifically, the four protocols included are: i) in vivo embryonic fate mapping and clonal analysis; ii) explant embryonic aorta culture; iii) SMC isolation from the perinatal aorta; and iv) subcutaneous osmotic mini-pump placement in pregnant (or non-pregnant) mice. Thus, these approaches facilitate the investigation of the origin(s), fate, and clonal architecture of SMCs in the aorta in vivo. They allow for modulating embryonic aorta morphogenesis in utero by continuous exposure to pharmacological agents. In addition, isolated aortic tissue explants or aortic SMCs can be used to gain insights into the role of specific gene targets during fundamental processes such as muscularization, proliferation, and migration. These hypothesis-generating experiments on isolated SMCs and the explanted aorta can then be assessed in the in vivo context through pharmacological and genetic approaches.

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

Protocols for studying the embryonic and perinatal murine aorta using in vivo clonal analysis and fate mapping, aortic explants, and isolated smooth muscle cells are detailed here. These diverse approaches facilitate the investigation of the morphogenesis of the embryonic and perinatal aorta in normal development and the pathogenesis in disease.

使用体内和组织和细胞外植体方法研究胚胎和围产期主动脉的形态和发病机制

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic ...

科研

发育生物学

Application of Aorta-gonad-mesonephros Explant Culture System in Developmental Hematopoiesis

The limitation of using mouse embryos for hematopoiesis studies is the added inconvenience in operations, which is largely due to the intrauterine development of the embryo. Although genetic data from knockout (KO) mice are convincing, it is not realistic to generate KO mice for all genes as needed. In addition, performing in vivo rescue experiments to consolidate the data obtained from KO mice is not convenient. To overcome these limitations, the Aorta-Gonad-Mesonephros (AGM) explant culture was developed as an appropriate system to study hematopoietic stem cell (HSC) development. Especially for rescue experiments, it can be used to recover the impaired hematopoiesis in KO mice. By adding the appropriate chemicals into the medium, the impaired signaling can be reactivated or up-regulated pathways can be inhibited. With the use of this method, many experiments can be performed to identify the critical regulators of HSC development, including HSC related gene expression at mRNA and protein levels, colony formation ability, and reconstitution capacity. This series of experiments would be helpful in defining the underlying mechanisms essential for HSC development in mammals.

This protocol describes using cultured Aorta-Gonad-Mesonephros for expression analyses, colony-forming units in the culture and spleen, and long-term reconstitution to determine the effect of regulatory factors and signaling pathways on hematopoietic stem cell development. This has been demonstrated as an effective system for studying hematopoietic stem cell biology and function.

主动脉-性腺-肾外植体培养体系在发育性造血中的应用

The limitation of using mouse embryos for hematopoiesis studies is the added inconvenience in operations, which is largely due to the intrauterine ...

Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing fetus. This tolerance is initiated during coitus, established during fecundation and implantation, and maintained throughout pregnancy. Active cellular and molecular mediators of maternal-fetal tolerance are enriched at the site of contact between fetal and maternal tissues, known as the maternal-fetal interface, which includes the placenta and the uterine and decidual tissues. This interface is comprised of stromal cells and infiltrating leukocytes, and their abundance and phenotypic characteristics change over the course of pregnancy. Infiltrating leukocytes at the maternal-fetal interface include neutrophils, macrophages, dendritic cells, mast cells, T cells, B cells, NK cells, and NKT cells that together create the local micro-environment that sustains pregnancy. An imbalance among these cells or any inappropriate alteration in their phenotypes is considered a mechanism of disease in pregnancy. Therefore, the study of leukocytes that infiltrate the maternal-fetal interface is essential in order to elucidate the immune mechanisms that lead to pregnancy-related complications. Described herein is a protocol that uses a combination of gentle mechanical dissociation followed by a robust enzymatic disaggregation with a proteolytic and collagenolytic enzymatic cocktail to isolate the infiltrating leukocytes from the murine tissues at the maternal-fetal interface. This protocol allows for the isolation of high numbers of viable leukocytes (&#62;70%) with sufficiently conserved antigenic and functional properties. Isolated leukocytes can then be analyzed by several techniques, including immunophenotyping, cell sorting, imaging, immunoblotting, mRNA expression, cell culture, and in vitro functional assays such as mixed leukocyte reactions, proliferation, or cytotoxicity assays.

Described herein is a protocol to isolate and analyze the infiltrating leukocytes of tissues at the maternal-fetal interface (uterus, decidua, and placenta) of mice. This protocol maintains the integrity of most cell surface markers and yields enough viable cells for downstream applications including flow cytometry analysis.

从小鼠组织在母胎界面分离白细胞

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing ...

免疫与感染

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the complexities of the organoid growth characteristics carry significant caveats for the investigator. Specifically, the growth patterns of each individual organoid are highly variable and create a heterogeneous population of epithelial cells in culture. With such caveats, common tissue culture practices cannot be simply applied to the organoid system due to the complexity of the cellular structure. Counting and plating based solely on cell number, which is common for individually separated cells, such as cell lines, is not a reliable method for organoids unless some normalization technique is applied. Normalizing to total protein content is made complex due to the resident protein matrix. These characteristics in terms of cell number, shape and cell type should be taken into consideration when evaluating secreted contents from the organoid mass. This protocol has been generated to outline a simple procedure to culture and treat small intestinal organoids with microbial pathogens and pathogen associated molecular patterns (PAMPs). It also emphasizes the normalization techniques that should be applied when protein analysis are conducted after such a challenge.

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Here, a protocol to harvest, maintain, and treat mouse small intestinal organoids with pathogen associated molecular patterns (PAMPs) and Listeria monocytogenes is described, as well as emphasis on gene expression and proper normalization techniques for protein.

该<em&gt;离体</em&gt;文化与模式识别小鼠肠道组织体受体刺激

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the ...

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

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

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

人胎盘及蜕膜器官培养研究在母胎界面感染

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

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 ...

An Explant Assay for Assessing Cellular Behavior of the Cranial Mesenchyme

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the neural tube in a process known as neurulation. During neurulation, morphogenesis of the mesenchyme that underlies the neural plate is believed to drive neural fold elevation. The cranial mesenchyme is comprised of the paraxial mesoderm and neural crest cells. The cells of the cranial mesenchyme form a pourous meshwork composed of stellate shaped cells and intermingling extracellular matrix (ECM) strands that support the neural folds. During neurulation, the cranial mesenchyme undergoes stereotypical rearrangements resulting in its expansion and these movements are believed to provide a driving force for neural fold elevation. However, the pathways and cellular behaviors that drive cranial mesenchyme morphogenesis remain poorly studied. Interactions between the ECM and the cells of the cranial mesenchyme underly these cell behaviors. Here we describe a simple ex vivo explant assay devised to characterize the behaviors of these cells. This assay is amendable to pharmacological manipulations to dissect the signaling pathways involved and live imaging analyses to further characterize the behavior of these cells. We present a representative experiment demonstrating the utility of this assay in characterizing the migratory properties of the cranial mesenchyme on a variety of ECM components.

The cranial mesenchyme undergoes dramatic morphogenic movements that likely provides a driving force for elevation of the neural folds1,2. Here we describe a simple ex vivo explant assay to characterize the cellular behaviors of the cranial mesenchyme during neurulation. This assay has numerous applications including being amenable to pharmacological manipulations and live imaging analyses.

为外植体含量的颅间充质细胞行为评定

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the ...

生物学

Co-Culture of Murine Small Intestine Epithelial Organoids with Innate Lymphoid Cells

Complex co-cultures of organoids with immune cells provide a versatile tool for interrogating the bi-directional interactions that underpin the delicate balance of mucosal homeostasis. These 3D, multi-cellular systems offer a reductionist model for addressing multi-factorial diseases and resolving technical difficulties that arise when studying rare cell types such as tissue-resident innate lymphoid cells (ILCs). This article describes a murine system that combines small intestine organoids and small intestine lamina propria derived helper-like type-1 ILCs (ILC1s), which can be readily extended to other ILC or immune populations. ILCs are a tissue-resident population that is particularly enriched in the mucosa, where they promote homeostasis and rapidly respond to damage or infection. Organoid co-cultures with ILCs have already begun shedding light on new epithelial-immune signaling modules in the gut, revealing how different ILC subsets impact intestinal epithelial barrier integrity and regeneration. This protocol will enable further investigations into reciprocal interactions between epithelial and immune cells, which hold the potential to provide new insights into the mechanisms of mucosal homeostasis and inflammation.

This protocol offers detailed instructions for establishing murine small intestine organoids, isolating type-1 innate lymphoid cells from the murine small intestine lamina propria, and establishing 3-dimensional (3D) co-cultures between both cell types to study bi-directional interactions between intestinal epithelial cells and type-1 innate lymphoid cells.

小鼠小肠上皮类器官与先天性淋巴细胞的共培养

Complex co-cultures of organoids with immune cells provide a versatile tool for interrogating the bi-directional interactions that underpin the delicate ...

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over developmental stages. Three-dimensional information from thin serial sections can be challenging to interpret because of the difficulty of reconstructing serial sections perfectly and maintaining proper orientation of the tissue over serial sections. Recent findings by Grosse et al., 2011 highlight the importance of three- dimensional information in understanding morphogenesis of the developing villi of the intestine1. Three-dimensional reconstruction of singly labeled intestinal cells demonstrated that the majority of the intestinal epithelial cells contact both the apical and basal surfaces. Furthermore, three-dimensional reconstruction of the actin cytoskeleton at the apical surface of the epithelium demonstrated that the intestinal lumen is continuous and that secondary lumens are an artifact of sectioning. Those two points, along with the demonstration of interkinetic nuclear migration in the intestinal epithelium, defined the developing intestinal epithelium as a pseudostratified epithelium and not stratified as previously thought1. The ability to observe the epithelium three-dimensionally was seminal to demonstrating this point and redefining epithelial morphogenesis in the fetal intestine. With the evolution of multi-photon imaging technology and three-dimensional reconstruction software, the ability to visualize intact, developing organs is rapidly improving. Two-photon excitation allows less damaging penetration deeper into tissues with high resolution. Two-photon imaging and 3D reconstruction of the whole fetal mouse intestines in Walton et al., 2012 helped to define the pattern of villus outgrowth2. Here we describe a whole organ culture system that allows ex vivo development of villi and extensions of that culture system to allow the intestines to be three-dimensionally imaged during their development.

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Improved imaging technology is allowing three-dimensional imaging of organs during development. Here we describe a whole organ culture system that allows live imaging of the developing villi in the fetal mouse intestine.

老鼠胎儿整肠文化系统<em&gt;前体内</em&gt;信号通路和绒毛开发的三维实时成像的操作

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over ...

Establishing Villous and Decidual Organ Cultures as Ex Vivo Models of Human Maternal-Fetal Interface

Source: Rizzuto, G. A., et al. <a href="https://app.jove.com/v/54237">Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface.</a> J. Vis. Exp. (2016).
This video demonstrates the establishment of primary human placental villous and decidual organ cultures in ex vivo conditions. These models are well-suited for studying pathogenesis at the human maternal-fetal interface.

建立绒毛和蜕膜器官培养物作为人类母胎界面的离体模型

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human ...