Name: analysis of protein-protein interactions and co-localization between components of gap, tight, and adherens junctions in murine mammary glands
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Description: Watch this Scientific Journal Video about Analysis of Protein-protein Interactions and Co-localization Between Components of Gap, Tight, and Adherens Junctions in Murine Mammary Glands at JoVE.com

知识产权由加拿大自然科学与工程研究理事会资助（NSERC＃418233-2012）资助;魁北克省魁北克省圣诞老人（FRQS），魁北克省乳腺癌基金会职业生涯奖，以及加拿大创新基金会授予的领导者资助。 ED获得了Fondation大学生Armand-Frappier奖学金。

本研究中使用的所有动物方案均由大学动物护理委员会（INRS-Institut Armand-Frappier，Laval，Canada）批准。 识别蛋白质共定位<ol><li> 从组织到显微镜片 注意:组织和部分应在干冰上处理。 <ol><li>从动物上清除乳腺（有关此程序的完整描述，请参阅Plante 等人 ）。 </li><li>将切除的组织嵌入冷冻/安装介质中的干冰上。添加足够的培养基覆盖腺体。当培养基固化时，将组织转移到-80℃的冷冻箱中供以后使用9 。 </li><li>使用设置在-35°C的冷冻切片机，将组织切成7-10μm厚的切片并将其放置在显微镜载玻片上。 注意:尽可能在每张幻灯片上放置两个部分;左侧部分将被用作特异性对照以验证抗体的特异性和组织的自发荧光，而右侧将用抗体标记。 </li><li>将部分保持在-80°C以备后用。 </li></ol></li><li> Co-IF染色 <ol><li>从冷冻箱中取出适当的微观载玻片，并在室温（RT）下将其浸入4％甲醛中15分钟立即固定切片。 </li><li>然后在室温下将载玻片浸入磷酸盐缓冲盐水（PBS）中。将幻灯片放在PBS中，直到进入下一步。 </li><li>使用市售的疏水屏障或防水实验笔（参见"材料表" ）圈住滑块的每个部分。小心不要接触组织。立即向组织中加入一滴PBS，并将载玻片置于潮湿的组织学室中，以进行剩余的操作。 注意:组织切片必须保持保湿。替代请使用带盖的盒子，并将湿纸巾放在底部。 </li><li>使用100-200μL3％牛血清白蛋白（BSA）-Tris缓冲盐水（TBS）-0.1％聚山梨醇酯20（参见材料表 ）将每个组织切片封闭 30分钟。当样品阻塞时，通过在TBS-0.1％聚山梨醇酯20中稀释抗体来制备一抗和二次抗体溶液。 注意:抗体所需的浓度由制造商提供;参见材料表和图1和图2 ，以及Dianati 等人 9 。尽管在使用大多数荧光团缀合的抗体时不需要在黑暗中工作，但避免将抗体溶液或染色的组织暴露于强烈的明亮的光线下。 </li><li>通过抽吸去除封闭溶液，并在室温下将100-200μL稀释的一抗孵育切片60分钟。 Alternatively，在4℃下与第一抗体孵育过夜。 </li><li>通过抽吸去除一级抗体溶液，并用250-500μLTBS-0.1％聚山梨醇酯20洗涤切片5分钟。通过抽吸去除洗涤液，重复洗涤两次。 </li><li>通过抽吸去除洗涤溶液，并在室温下用100-200μL适当的荧光团缀合的二抗孵育切片60分钟。 </li><li>通过抽吸去除第二抗体溶液，并用250-500μLTBS-0.1％聚山梨醇酯20洗涤切片5分钟。取出洗液并重复两次。 </li><li>重复步骤2.5-2.8，使用适当的初级和次级抗体组合，用于感兴趣的后续蛋白质。 </li><li>通过抽吸去除洗涤溶液并通过将部分与100-200μL的1mg / mL 4&#39;，6-二脒基-2-苯基吲哚（DAPI）在TBS-0.1％聚山梨醇酯20中孵育5分钟进行核染色，并进行细胞核染色RT。 </li><li>通过抽吸去除DAPI溶液，并使用水溶性，非荧光的安装介质（参见材料表 ）和盖玻片安装载玻片。一次进行一张幻灯片。 注意:孵育核染色超过5分钟不会改变染色强度。或者，移除所有载玻片上的DAPI溶液，并在安装载玻片的同时孵育PBS中的组织。 </li><li>将幻灯片平放在4°C冰箱中至少8小时。继续进行荧光显微镜成像（参见图1和图2 ）。 </li></ol></li><li> 显微成像 <ol><li>使用配备有在其特定波长处激发荧光团所需的各种激光器的共聚焦显微镜来观察荧光团缀合的二次抗体。 注意:为了能够可视化管道和肺泡，建议使用数值孔径为0.95的40X物镜。例如e的具体设置如图1所示 。 </li><li>通过在当时扫描一幅波长的图像来单独验证每种蛋白质的定位。 注意:在这个阶段，重要的是严格分析连接蛋白的定位。为了能够形成连接核酸，这些蛋白质必须位于质膜上。 </li><li>通过合并在不同波长的激光扫描的图像来确定蛋白质的共定位。 注意:蛋白质共定位可以通过在相同位置发射两个或更多个荧光团而产生的颜色变化来显现，并且可以使用适当的软件测量（ 图1和2 ;也参见参考文献9）。 </li></ol></li></ol> 2.学习PPI 注意:腹部乳腺应用于研究PPI，因为胸腺与胸部紧密相关肌肉。消毒乳腺（有关此程序的完整说明，请参阅Plante 等人 ） 14并将其保持在-80°C以备后用。 <ol><li> 裂解液制备 <ol><li>将称重纸和2 mL微量离心管放在干冰上预先冷却，然后再进行下一步骤。 </li><li>从-80°C取乳腺组织，并将其保存在干冰上。 </li><li>称重预冷计量纸上的组织，然后将组织转移到2 mL微量离心管（干冰处理）上。每个样品使用50至100 mg的组织。将组织保持在干冰上，直到步骤2.1.5。 </li><li>使用下列公式，制备所需量的补充有NaF，NaVO 3和蛋白酶/磷酸酶抑制剂的三重洗涤剂溶解缓冲液，如材料表所示。小鼠:所需缓冲液（μL）=小鼠组织重量（mg）×3;老鼠:需要缓冲液（μL）=大鼠组织重量（mg）×5。 </li><li>将所需量的冰冷裂解缓冲液（在步骤2.1.4中计算）加入到含有组织的2mL管中。 注意:在步骤2.1.5-2.1.6中，一次进行一根单管。 </li><li>使用组织研磨机上的连续匀浆将组织匀化30-40秒;始终将管保持在冰上。将组织匀浆器调整至中等速度，并轻轻地将研磨机上下移动到管内。 </li><li>与其他管重复步骤1.6和1.7。 </li><li>将溶胞产物在冰上孵育10-30分钟。 </li><li>在4℃下以170×g离心管10分钟。 </li><li>同时，为每个样品确定6-10个微量离心管（0.6 mL），并将其保存在冰上。 </li><li>离心完成后，检查管子。确保它们含有顶层脂肪，透明，黄色至粉红色的裂解物（取决于显影阶段）和颗粒。 </li><li>在脂质层中创建一个孔使用200μL移液管吸头进入液相。改变提示并收集裂解物，而不会干扰沉淀或吸入脂质层。将裂解物在冰上预先标记的管中等分（步骤2.1.11），并将其储存在-80℃。 </li><li>使用等分试样使用适当的市售试剂盒来定量蛋白质浓度（见材料表 ）。 </li></ol></li><li> 间接免疫沉淀 <ol><li>在冰上，解冻之前制备的全部乳腺裂解物的两个等分试样。 注意:一个等分试样将用于目标蛋白质的IP，而另一个将用作阴性对照。 </li><li>收集500-1,000μg裂解物，并在PBS中稀释至每个1.5 mL管中达到最终体积200μL。 注意:使用的裂解物的量取决于感兴趣的蛋白质的丰度和抗体的效率（参见图3的例如，以及材料表 ）。为了优化每个靶，应使用不同量的裂解物（ 即 500,750和1,000μg）和抗体（ 即 5,10和20μg）。继续执行以下步骤（2.2.3-2.3.7.4）。 </li><li>将针对感兴趣的抗原的抗体添加到第一个裂解物管中并保持在冰上。 注意:所需金额通常由每家公司提供的说明书中提出（见材料表 ）。 </li><li>在第二管中，通过加入与步骤2.2.3中使用的抗体相同的同种型IgG对照浓度来制备阴性对照。 </li><li>在管式辊式混合器中以4℃低速孵育管过夜。 </li><li>第二天，将50μL磁珠加入新的1.5 mL管中进行预洗。 <ol><li>基于对抗体的相对亲和力，选择蛋白A或蛋白G磁珠。 </l我&gt; <li>避免使用聚集的珠子是很重要的;轻轻混合珠悬浮液，直至将其均匀地重新悬浮，然后再加入管中。 </li></ol></li><li>将含有珠粒的管放在磁性支架上，使珠子迁移到磁体上。使用200μL移液管从珠中取出储存缓冲液。 </li><li>通过加入500μLPBS-0.1％聚山梨醇酯20洗涤珠子，并将管子剧烈旋转10秒。 </li><li>将管子放回到磁性支架上，让珠子移动到磁铁上。 </li><li>用200μL移液管移液多余的洗涤缓冲液。 </li><li>将步骤2.2.5中的反应络合物（裂解物抗体）加入到珠中，并在辊式混合器中在室温下孵育90分钟。 </li><li>将管放在磁性支架上，使珠子迁移到磁铁上。使用200μL移液管，吸出并丢弃裂解物，并将管置于冰上。 </li><li>清洗珠子通过加入500μLPBS，将管置于磁性架上，并用200μL移液管除去液体。重复这个洗涤步骤。在洗涤步骤中，避免涡旋并将样品保持在冰上。 </li><li>用PBS-0.1％聚山梨醇酯20洗涤珠子一次，不涡旋，并使用200μL移液管吸头丢弃最后的洗涤缓冲液。 </li><li>为了洗脱，向管中加入20μL的0.2M酸性甘氨酸（pH = 2.5），并在辊式混合机上摇动7分钟。 </li><li>以高速离心几秒钟（快速旋转），并将上清液收集在新鲜的冰冷管中。 </li><li>对每个管重复步骤2.2.14和2.2.15。 注意:最终体积为40μL。 </li><li>将10μL4x Laemmli缓冲液加入到步骤2.2.16的40μL洗脱样品中。 注意:由于酸性pH，颜色会变黄。 </li><li>立即将1 M Tris（pH = 8）一次一滴加入从步骤2.2.18洗脱的样品至其颜色变蓝继续下一个管。 </li><li>将样品从步骤2.2.18在70-90℃下煮沸10分钟。立即进行凝胶电泳。或者，将样品转移至-80°C的冷冻箱中直至加载。 </li></ol></li><li> 下游应用:凝胶电泳，然后进行Western印迹 <ol><li>按照标准程序准备分离和堆叠SDS-PAGE丙烯酰胺凝胶（1.5 mm厚） 15 。 注意:凝胶的选择（8-15％丙烯酰胺，梯度:参见材料表 ）应根据要沉淀的蛋白质的分子大小和待分析的潜在的结合配偶体来确定。这些蛋白质必须彼此分离，以便进行适当的免疫检测。 </li><li>在冰上解冻免疫沉淀（IP） - 样品（步骤2.2.20）。 </li><li>从相同的样品制备蛋白质裂解物（用于上述IP程序）。使用50的总裂解物并加入4X Laemmli样品缓冲液。将样品在70-90℃下煮沸5分钟，置于冰上直至加载。 注意:这些样品将在洗脱的IP样品旁边加载，以证明在总裂解物中存在沉淀的蛋白质。 </li><li>将来自步骤2.3.3的制备的裂解物和来自步骤2.2.20的沉淀的样品并排放在丙烯酰胺凝胶中，并将其运行在运行缓冲液（10×运行缓冲液:30.3g Tris，144.1g甘氨酸和10在1L蒸馏水中的SDS）在100V下进行近似95分钟，或者直到迁移蛋白的边缘到达凝胶的底部。 </li><li>使用标准方案9,15将凝胶转移到硝酸纤维素或PVDF膜上。 </li><li>在5％干牛奶-TBSBS（20mM Tris，500mM NaCl，和0.05％聚山梨醇酯20）中，在低速摇瓶上将膜封闭1小时。 </li><li>确定降水是否成功cessful。 <ol><li>探测使用针对沉淀蛋白的第一抗体的膜，以5％干奶牛TTBS以制造商推荐的浓度稀释，在4℃下在缓慢搅动的摇摆平台上过夜。 注:有关建议，请参见材料表 。 </li><li>第二天，用高速搅拌的摇摆平台上的TTBS洗涤膜3次，每次5分钟。 </li><li>在慢慢搅拌的摇摆平台上，将RTT上缀合有辣根过氧化物酶（HRP）的适当的二抗体在TTBS上孵育1小时。 注意:或者，如果检测到信号的适当装置可用，则可以使用与荧光染料缀合的二抗。 </li><li>在高搅拌的摇摆平台上进行3至6次洗涤，每次5分钟，用TTBS洗涤。通过将膜与商业化孵育来分析二抗的信号可用的鲁米诺溶液（见材料表 ），并遵循制造商说明。使用化学发光成像系统检测信号（参见材料表 ）。 注意:有关Western印迹分析的详细方案，请参见参考文献16。 </li></ol></li><li>要确定相互作用的蛋白质，请执行步骤2.3.7.1-2.3.7.4在相同印迹上使用适当的抗体。 注意:如果蛋白质相互作用，结合配偶体将与靶蛋白共同免疫沉淀，因此可通过Western印迹法检测。可以用更多的抗体重复步骤2.3.8，以确定其他蛋白质是否存在于相同的蛋白质复合物中，只要蛋白质的分子量不同就足以在凝胶和膜上分离得很好。 </li><li>要确认所识别的绑定伙伴不是工件，应执行互惠IP。 注意:这是通过重复执行的步骤2.2.1-2.2.20，其具有相同的裂解物，但沉淀步骤3.8中鉴定的结合伴侣之一。然后，使用针对目的蛋白的第一抗体重复步骤3.1-3.8。 </li></ol></li></ol>

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C., Berkelman, T., Yadav, G., Hammond, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+defined+methodology+for+reliable+quantification+of+Western+blot+data.">A defined methodology for reliable quantification of Western blot data.</a> Mol Biotechnol. 55 (3), 217-226 (2013).</li><li>Oxford, E. 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作者没有任何声明。

通过连接的细胞 - 细胞相互作用是许多器官（如乳腺）的正常功能和发育所必需的。研究表明，连接蛋白可以调节彼此的功能和稳定性，并通过在细胞膜10处彼此束缚来激活信号转导。目前手稿中提出的方案提供了有关连接蛋白差异表达，定位和正常鼠腺发育相互作用的有趣发现9 。鉴于连接蛋白定位对于蛋白质的功能至关重要，并且由于已知其与支架蛋白和许...

乳腺生长发育主要发生于出生后。这种器官在整个哺乳动物的生殖生活中不断地改造自己1 。成年乳腺上皮由腔内上皮细胞的内层和由基底膜2包围的主要由肌上皮细胞组成的基底细胞外层组成。为了对乳腺结构和发育的良好综述，读者可以参考Sternlicht 1 。通过间隙（GJ），紧密（TJ）和粘附（AJ）结的细胞 - 细胞相互作用对于腺体1,3,4,5,6的正常发育和功能是必需的。这些小鼠乳腺结节的主要成分是Cx26，Cx30，Cx32和Cx43（GJ）;紧密连接-1，-3，-4和-7和ZO-1（TJ）;和E-钙粘蛋白，P-钙粘蛋白和β-连环蛋白（AJ） 7,8 。这些不同连接蛋白的表达水平在乳腺发育过程中以阶段依赖的方式变化，这表明差异细胞 - 细胞相互作用的要求9 。 GJ，TJ和AJ在结构和功能上相互连接并将其他结构或调节蛋白连接到相邻细胞的相邻位点，从而产生连接性连接10 。连接关系的组成可以影响与下面的细胞骨架的桥接，以及连接通透性和稳定性，因此可以影响腺体8,9,10,11的功能。细胞间连接的组成部分存在于连接核酸中，或者彼此相互作用最近使用共免疫荧光（co-IF）和共免疫沉淀（co-IP） 9分析了乳腺发育的不同发育阶段。虽然其他技术允许评估蛋白质之间的功能关联，但这些方法在本手稿中没有提出。 由于蛋白质仅仅单独起作用，研究蛋白质 - 蛋白质相互作用（PPI），如信号转导和生物化学级联，对许多研究人员来说是必不可少的，可以提供关于蛋白质功能的重要信息。 Co-IF和微观分析有助于评估一些共享相同亚细胞空间的蛋白质。然而，目标的数量受到必须在不同动物中提高的抗体以及通过使用配备有不同波长激光器的共聚焦显微镜和用于复用的光谱检测器的抗体的限制。 Co-IP确认或揭示高亲和力的物理相互作用een两个或更多的蛋白质位于蛋白质复合物内。尽管开发了诸如荧光共振能量转移（FRET） 12和邻近连接测定（PLA） 13等新技术 ，其可以同时检测蛋白质的定位和相互作用，但共有IP仍然是一种适当和可负担的技术来研究内源蛋白。 本手册中描述的逐步方法将有助于蛋白质定位和PPI的研究，并指出在研究乳腺中的内源PPI时应避免的缺陷。该方法从对每种技术所需组织的不同保存程序的介绍开始。第1部分介绍了如何在三个步骤中研究蛋白质共定位:i）乳腺切片，ii）使用co-IF技术对不同蛋白质的双重或三重标记，以及iii）成像蛋白质定位。第2部分显示如何沉淀内源性蛋白质并以三个步骤鉴定其相互作用的蛋白质:i）裂解物制备，ii）间接蛋白质免疫沉淀，以及iii）通过SDS-PAGE和Western印迹结合配偶体鉴定。该方案的每一步都针对啮齿动物乳腺组织进行了优化，并产生了高质量，特异性和可重复的结果。该方案也可用作其他组织或细胞系中PPI研究的起点。

<table><tbody><tr><td>Mice strain and stage</td><td>St. Constant, Quebec, Canada</td><td></td><td>C57BL/6 Femals; pregnancy day 18 (P18) and lactation day 14 (L14), Charles River Canada&amp;nbsp;</td></tr><tr><td>PBS 10x (stock)</td><td></td><td></td><td>1) Dissolve 80 g NaCl (F.W.: 58.44), 2 g KCl (F.W. 74.55), 26.8 g Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;#183;7H&lt;sub&gt;2&lt;/sub&gt;O (F.W. 268.07) and 2.4 g KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; (F.W.:136.09) in 800 mL distilled water;&lt;br/&gt; 2) Adjust the PH to 7.4;&lt;br/&gt; 3) Add water to reach to the 1 L final volume.</td></tr><tr><td>TBS 10x (stock)</td><td></td><td></td><td>1) Dissolve 60.5 g TRIS, 87.6 g NaCl in 800 mL distilled water;&lt;br/&gt;2) Adjust the pH to 7.5;&lt;br /&gt;3) Add water to reach to the 1 L final volume.</td></tr><tr><td colspan="4">&lt;strong&gt;Part 1: Immunofluorescence&lt;/strong&gt;</td></tr><tr><td>Freezing media</td><td>VWR International, Ville Mont-Royal, QC, Canada</td><td>95067-840</td><td>VMR frozen sections compound&amp;nbsp;</td></tr><tr><td>Microtome</td><td>Mississauga, ON, Canada</td><td>956640</td><td>Microm HM525, Thermo fisher scientific HM525 NX Cryostat 115 V 60 Hz</td></tr><tr><td>Blades</td><td>C.L. Sturkey, Inc. Les Produits&amp;nbsp;Scientifiques&amp;nbsp;ESBE&amp;nbsp;St-Laurent, QC, Canada</td><td>BLM1001C</td><td>High profile gold coated blades</td></tr><tr><td>Pap pen</td><td>Cedarlane, Burlington, ON, Canada</td><td>8899</td><td>Super PAP Pen, Thermo fisher scientific</td></tr><tr><td>Microscopic slides</td><td>Fisher Scientific, Burlington, ON, Canada</td><td>12-550-15</td><td>Fisherbrand&amp;nbsp;Superfrost Plus Microscope Slides</td></tr><tr><td>Formaldehyde</td><td>BioShop Canada Inc, Burlington, ON, Canada</td><td>FOR201.1</td><td>Forlmadehyde</td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Santa Cruz Biotechnology, Inc, California, USA</td><td></td><td></td></tr><tr><td>Blocking solution</td><td></td><td></td><td>3% BSA in TBS</td></tr><tr><td>Wash solution</td><td></td><td></td><td>TBS-Tween 20 0.1%</td></tr><tr><td>Polysorbate 20</td><td>Oakville, ON, Canada</td><td>P 9416</td><td>Tween 20, Sigma-Aldrich</td></tr><tr><td>Mounting media</td><td>Cedarlane, Burlington, ON</td><td>17984-25(EM)</td><td>Fluoromount-G</td></tr><tr><td>First &amp;amp; secondary antibodies</td><td>Cell Signaling, Beverly, MA, USA</td><td>See Comments</td><td>E-Cadherin (4A2) Mouse mAb (#14472s) 1/50 (Cell Signaling) with anti-mouse IgG Fab2 Alexa Fluor 555 (#4409s), Cell Signaling&amp;nbsp;</td></tr><tr><td>First &amp;amp; secondary antibodies&amp;nbsp;</td><td>Life technologies, Waltham, MA, USA &amp;amp; Cell Signaling, Beverly, MA, USA</td><td>See Comments</td><td>Claudin-7 (#34-9100) 1/100 (Life Technologies) with anti-rabbit IgG Fab2 Alexa Fluor 488 (#4412s) (Cell Signaling)&amp;nbsp;</td></tr><tr><td>First &amp;amp; secondary antibodies&amp;nbsp;</td><td>Santa Cruz Biotechnology, Inc, California, USA; Fischer Scientific, Burlington, ON, Canada&amp;nbsp;</td><td>See Comments</td><td>&amp;beta;-Catenin Antibody (C-18): sc-1496 (SANTA CRUZ) with anti-Goat IgG (H+L) Alexa Fluor 568 (#A11057), Molecular Probe (Fisher Scientific)</td></tr><tr><td>First &amp;amp; secondary antibodies&amp;nbsp;</td><td>Life technologies, Waltham, MA, USA &amp;amp; Cell Signaling, Beverly, MA, USA</td><td>See Comments</td><td>Connexin26&amp;nbsp; (#33-5800) 1/75 (Life Technologies) with anti-mouse IgG Fab2 Alexa Fluor 647 (#4410s)&amp;nbsp;</td></tr><tr><td>Nuclei stain</td><td>Fisher Scientific, Burlington, ON, Canada</td><td>D1306</td><td>DAPI (4&#039;,6-Diamidino-2-Phenylindole, Dihydrochloride) 1/1,000 in PBS</td></tr><tr><td>Fluorescent microscope</td><td>Nikon Canada, Mississauga, On, Canada</td><td></td><td>Nikon A1R+ confocal microscopic laser equipped with a spectral detector&amp;nbsp;</td></tr><tr><td>Software of IF images analysis</td><td>Nikon Canada, Mississauga, On, Canada</td><td></td><td>NIS-elements software (version 4)</td></tr><tr><td colspan="4">&lt;strong&gt;Part 2: Immunoprecipitation&lt;/strong&gt;</td></tr><tr><td>Triple-detergent Lysis buffer (100&amp;nbsp;mL) pH=8.0</td><td></td><td></td><td>1) Mix 50 mM TRIS (F.W.: 121.14), 150&amp;nbsp;mM NaCl (F.W.: 58.44), 0.02% Sodium Azide, 0.1% SDS, 1% NONIdET P40, 0.5% Sodium Deoxycholate in 80 mL distilled H&lt;sub&gt;2&lt;/sub&gt;O.&lt;br/&gt; 2) Adjust the pH to 8.0 with HCl 6&amp;nbsp;N (~0.5&amp;nbsp;mL).&lt;br/&gt; 3) Adjust the volume to 100&amp;nbsp;mL. Keep it in fridge.&lt;br/&gt; At the day of protein extraction, use 1/100 NaVo&lt;sub&gt;3&lt;/sub&gt;, 1/100 protease/phosphatase inhibitor and 1/25 NAF in calculated amount of Triple detergent lysis buffer:&lt;br/&gt; Sodium Fluoride (stock) solution 1.25&amp;nbsp;M (F.W.: 41.98), Sodium Orthovanadate (stock) Solution 1&amp;nbsp;M (F.W.: 183.9)</td></tr><tr><td>Protease/phosphatase inhibitor</td><td>Fisher Scientific, Burlington, ON</td><td>78441</td><td>Halt Protease and Phosphatase Inhibitor Cocktail, EDTA-free (100x)</td></tr><tr><td>Protein dosage</td><td>Thermo Scientific, Rockford, Illinois, USA</td><td>23225</td><td>Pierce BCA protein assay kit&amp;nbsp;</td></tr><tr><td>Tissue grinder</td><td>Fisher Scientific, Burlington, ON</td><td>FTH-115</td><td>Power 125, Model FTH-115</td></tr><tr><td>Magnetic beads and stand</td><td>Millipore, Etobicoke, ON, Canada</td><td></td><td>PureProteome Protein G Magnetic Bead System (LSKMAGG02)</td></tr><tr><td>Wash solution for IP</td><td></td><td></td><td>PBS or PBS-Tween20 0.1% depending to the step</td></tr><tr><td>Primary antibodies for immunoprecipitation</td><td>Cell Signaling, Beverly, MA, USA</td><td>See Comments</td><td>IgG Rabbit (rabbit (DA1E) mAb IgG Isotype control (#3900s) (Cell Signaling) 0.5 &amp;micro;L/200 &amp;micro;L</td></tr><tr><td>Primary antibodies for immunoprecipitation</td><td>Cell Signaling, Beverly, MA, USA</td><td>See Comments</td><td>IgG Mouse mouse (G3A1) mAb IgG Isotype control (#5415s) (Cell Signaling) 0.5 &amp;micro;L/200 &amp;micro;L</td></tr><tr><td>Primary antibodies for immunoprecipitation</td><td>Sigma-Aldrich, Oakville, ON, Canada</td><td>See Comments</td><td>Connexin43 (#C6219) (Sigma-Aldrich) 4&amp;nbsp;&amp;micro;L/200&amp;nbsp;&amp;micro;L</td></tr><tr><td>Primary antibodies for immunoprecipitation</td><td>Cell Signaling, Beverly, MA, USA</td><td>See Comments</td><td>E-cadherin (4A2) Mouse mAb (#14472s) (Cell Signaling) 1 &amp;micro;L/200 &amp;micro;L</td></tr><tr><td>Laemmli buffer&amp;nbsp;</td><td>BIO-RAD, Mississauga, Ontario, Canada</td><td>1610747</td><td>4x Laemmli Sample Buffer (Add &amp;beta;-mercaptoethanol following manufacturer recommendation)</td></tr><tr><td>Acidic glycine&amp;nbsp;</td><td>Fisher Scientific, Burlington, ON</td><td>PB381-5</td><td>0.2 M glycine; adjust pH=2.5 with HCl&amp;nbsp;</td></tr><tr><td>Tris&amp;nbsp;</td><td>Fisher Scientific, Burlington, ON</td><td>BP152-1</td><td>1 M (pH=8)&amp;nbsp;</td></tr><tr><td>SDS-PAGE acrylamide gels&amp;nbsp;</td><td>BIO-RAD, Mississauga, ON, Canada</td><td>1610180 -5</td><td>TGX Stain-Free FastCast Acrylamide Solutionss (7.8%, 10%, 12%)</td></tr><tr><td>Running buffer 10x&amp;nbsp;</td><td>BIO-RAD, Mississauga, ON, Canada</td><td>1704272</td><td>Tris 30.3&amp;nbsp;g/glycine 144.1&amp;nbsp;g /SDS 10&amp;nbsp;g in 1 L distilled water</td></tr><tr><td>Membranes</td><td>BIO-RAD, Mississauga, ON, Canada</td><td>1704272</td><td>PVDF membranes, Trans-Blot Turbo RTA Mini PVDF Transfer Kit</td></tr><tr><td>Transfer method</td><td>BIO-RAD, Mississauga, ON, Canada</td><td>1704155</td><td>Trans-Blot Turbo Transfer System</td></tr><tr><td>Dry Milk</td><td>Smucker Food of Canada Co, Markham, ON, Canada</td><td></td><td>Fat Free Instant Skim Milk Powder, Carnation</td></tr><tr><td>Blocking solution for blots</td><td></td><td></td><td>5% dry milk in TBS-Tween 20 0.1%</td></tr><tr><td>Washing solutions for blots</td><td></td><td></td><td>TBS-Tween 20 0.1%</td></tr><tr><td>Primary and secondary antibodies for blots (10&amp;nbsp;mL)</td><td>Sigma-Aldrich, Oakville, Ontario &amp;amp; Abcam, Toronto, ON, Canada</td><td>See Comments</td><td>Connexin43 (#C6219) (Sigma-Aldrich) 1/2,500 with HRP-conjugated Veriblot for IP secondary antibody (ab131366) 1/5,000 (Abcam, Toronto, ON, Canada)</td></tr><tr><td>Primary and secondary antibodies for blots (10&amp;nbsp;mL)</td><td>Cell Signaling, Beverly, MA, USA &amp;amp; Abcam, Toronto, ON, Canada</td><td>See Comments</td><td>E-cadherin (24E10) rabbit mAb 1/1,000 (#3195s) (Cell Signaling) 1/1,000 with HRP-conjugated Veriblot for IP secondary antibody (ab131366) 1/5,000 (Abcam, Toronto, ON, Canada)</td></tr><tr><td>Primary and secondary antibodies for blots (10&amp;nbsp;mL)</td><td>Life technologies, Waltham, MA, USA &amp;amp; Abcam, Toronto, ON, Canada</td><td>See Comments</td><td>Claudin-7 (#34-9100) (Life technologies) 1/1,000 with HRP-conjugated Veriblot for IP secondary antibody (ab131366) 1/5,000 (Abcam, Toronto, ON, Canada)</td></tr><tr><td>Primary and secondary antibodies for blots (10&amp;nbsp;mL)</td><td>Life technologies, Waltham, MA, USA &amp;amp; Abcam, Toronto, ON, Canada</td><td>See Comments</td><td>Claudin3 (#34-1700) (Life technologies) 1/1,000 with HRP-conjugated Veriblot for IP secondary antibody (ab131366) 1/5,000 (Abcam, Toronto, ON, Canada)</td></tr><tr><td>Luminol solution for signal detection on blots</td><td>BIO-RAD, Mississauga, ON, Canada</td><td>1705061</td><td>Clarity Western ECL Blotting Substrate</td></tr><tr><td>Imaging blots</td><td>BIO-RAD, Mississauga, ON, Canada</td><td>1708280</td><td>ChemiDoc MP imaging system</td></tr><tr><td>Analayzing blots</td><td>BIO-RAD, Mississauga, ON, Canada</td><td></td><td>ImageLab 5.2 software&amp;nbsp;</td></tr></tbody></table>

analysis of protein-protein interactions and co-localization between components of gap, tight, and adherens junctions in murine mammary glands

细胞间的相互作用在保持组织完整性和乳腺不同隔室之间的屏障方面发挥关键作用。这些相互作用由在相邻细胞之间形成核酸的连接蛋白提供。功能蛋白质错位和与其结合配偶体的物理缔合减少可导致功能丧失，从而导致器官功能障碍。因此，在正常和疾病相关组织中鉴定蛋白质定位和蛋白质 - 蛋白质相互作用（PPI）对于找到导致疾病发展或发育状态改变的新证据和机制是至关重要的。该手稿提供了两步法评估鼠乳腺中的PPI。在方案1中，描述了使用针对感兴趣的蛋白质产生的抗体，随后用荧光染料标记的二抗进行共免疫荧光（co-IF）的方法。虽然共同所有为了证明蛋白质的接近程度，它确实有可能研究其物理相互作用。因此，在协议第2节中提供了共免疫沉淀（co-IP）的详细方案。该方法用于确定蛋白质之间的物理相互作用，而不确认这些相互作用是直接还是间接的。在过去几年中，共同中心和共同IP技术已经证明，细胞间连接的某些组成部分共同定位并相互作用，从而在乳腺发育过程中产生不同阶段依赖的连接性核酸。

为了确定GJ，AJ和TJ组分是否可以在乳腺中相互作用，首先进行共IF测定。分别用特异性抗体探测Cx26，GJ蛋白和β-Catenin，一种AJ蛋白，分别用荧光团缀合的小鼠-647（绿色，假色）和山羊-568（红色）抗体（ 图1B和C ） 。数据显示，在哺乳期第7天（L7），它们在小鼠乳腺的上皮细胞的细胞膜上共同定位，如黄色所反映的（ 图1D ）。其次，?...

Watch this Scientific Journal Video about Analysis of Protein-protein Interactions and Co-localization Between Components of Gap, Tight, and Adherens Junctions in Murine Mammary Glands at JoVE.com

Analysis of Protein-protein Interactions and Co-localization Between Components of Gap, Tight, and Adherens Junctions in Murine Mammary Glands

细胞间连接是乳腺阶段特异性功能和发育的必需条件。该手稿提供了一个详细的方法，用于研究蛋白质 - 蛋白质相互作用（PPI）和使用鼠乳腺的共定位。这些技术允许调查不同发育阶段的细胞间连接之间的物理联系的动力学。

蛋白质 - 蛋白质相互作用的分析和鼠乳腺中间隙，紧密和粘附连接部位之间的共定位

Cell-cell interactions play a pivotal role in preserving tissue integrity and the barrier between the different compartments of the mammary gland. These ...

analysis-protein-protein-interactions-co-localization-between

Research

JoVE Journal

Developmental Biology

10.0K 次观看.  INRS, Institut Armand-Frappier. 细胞间连接是乳腺阶段特异性功能和发育的必需条件。该手稿提供了一个详细的方法，用于研究蛋白质 - 蛋白质相互作用（PPI）和使用鼠乳腺的共定位。这些技术允许调查不同发育阶段的细胞间连接之间的物理联系的动力学。 

视频: Analysis of Protein-protein Interactions and Co-localization Between Components of Gap, Tight, and Adherens Junctions in Murine Mammary Glands

Differentiation of Mouse Breast Epithelial HC11 and EpH4 Cells

Cadherins play an important role in the regulation of cell differentiation as well as neoplasia. Here we describe the origins and methods of the induction of differentiation of two mouse breast epithelial cell lines, HC11 and EpH4, and their use to study complementary stages of mammary gland development and neoplastic transformation.
The HC11 mouse breast epithelial cell line originated from the mammary gland of a pregnant Balb/c mouse. It differentiates when grown to confluence attached to a plastic Petri dish surface in medium containing fetal calf serum and Hydrocortisone, Insulin and Prolactin (HIP medium). Under these conditions, HC11 cells produce the milk proteins &#946;-casein and whey acidic protein (WAP), similar to lactating mammary epithelial cells, and form rudimentary mammary gland-like structures termed &#34;domes&#34;.
The EpH4 cell line was derived from spontaneously immortalized mouse mammary gland epithelial cells isolated from a pregnant Balb/c mouse. Unlike HC11, EpH4 cells can fully differentiate into spheroids (also called mammospheres) when cultured under three-dimensional (3D) growth conditions in HIP medium. Cells are trypsinized, suspended in a 20% matrix consisting of a mixture of extracellular matrix proteins produced by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, plated on top of a layer of concentrated matrix coating a plastic Petri dish or multiwell plate, and covered with a layer of 10% matrix-containing HIP medium. Under these conditions, EpH4 cells form hollow spheroids that exhibit apical-basal polarity, a hollow lumen, and produce &#946;-casein and WAP.
Using these techniques, our results demonstrated that the intensity of the cadherin/Rac signal is critical for the differentiation of HC11 cells. While Rac1 is necessary for differentiation and low levels of activated RacV12 increase differentiation, high RacV12 levels block differentiation while inducing neoplasia. In contrast, EpH4 cells represent an earlier stage in mammary epithelial differentiation, which is inhibited by even low levels of RacV12.

We describe techniques for differentiation induction of two breast epithelial lines, HC11 and EpH4. While both require fetal calf serum, insulin, and prolactin to produce milk proteins, EpH4 cells can fully differentiate into mammospheres in three-dimensional culture. These complementary models are useful for signal transduction studies of differentiation and neoplasia.

小鼠乳腺皮下HC11和EpH4细胞的分化

Cadherins play an important role in the regulation of cell differentiation as well as neoplasia. Here we describe the origins and methods of the induction ...

科研

癌症研究

Quantifying Branching Density in Rat Mammary Gland Whole-mounts Using the Sholl Analysis Method

An increasing number of studies are utilizing the rodent mammary gland as an endpoint for assessing the developmental toxicity of a chemical exposure. The effects these exposures have on mammary gland development are typically evaluated using either basic dimensional measurements or by scoring morphological characteristics. However, the broad range of methods for interpreting developmental changes could lead to inconsistent translations across laboratories. A common method of assessment is needed so that proper interpretations can be formed from data being compared across studies. The present study describes the application of the Sholl analysis method to quantify mammary gland branching characteristics. The Sholl method was originally developed for use in quantifying neuronal dendritic patterns. By using ImageJ, an open-source image analysis software package, and a plugin developed for this analysis, the mammary gland branching density and the complexity of a mammary gland from a peripubertal female rat were determined. The methods described here will enable the use of the Sholl analysis as an effective tool for quantifying an important characteristic of mammary gland development.

Mammary gland development in the rodent has typically been evaluated using descriptive assessments or by measuring basic physical attributes. Branching density is an indicator of mammary development that is difficult to quantify objectively. This protocol describes a reliable method for the quantitative assessment of mammary gland branching characteristics.

使用Sholl分析方法量化大鼠乳腺组织中的分支密度

An increasing number of studies are utilizing the rodent mammary gland as an endpoint for assessing the developmental toxicity of a chemical exposure. The ...

发育生物学

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

工程三维上皮组织细胞外基质中嵌入

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

生物工程

Generation of Mosaic Mammary Organoids by Differential Trypsinization

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine mammary gland is composed of two distinct epithelial cell compartments, serving different functions: the outer, contractile myoepithelial compartment and the inner, secretory luminal compartment. Here, we describe a method by which the cells comprising these compartments are isolated and then combined to investigate their individual lineage contributions to mammary gland morphogenesis and differentiation. The method is simple and efficient and does not require sophisticated separation technologies such as fluorescence activated cell sorting. Instead, we harvest and enzymatically digest the tissue, seed the epithelium on adherent tissue culture dishes, and then use differential trypsinization to separate myoepithelial from luminal cells with ~90% purity. The cells are then plated in an extracellular matrix where they organize into bilayered, three-dimensional (3D) organoids that can be differentiated to produce milk after 10 days in culture. To test the effects of genetic mutations, cells can be harvested from wild type or genetically engineered mouse models, or they can be genetically manipulated prior to 3D culture. This technique can be used to generate mosaic organoids that allow investigation of gene function specifically in the luminal or myoepithelial compartment.

The mammary gland is a bilayered structure, comprising outer myoepithelial and inner luminal epithelial cells. Presented is a protocol to prepare organoids using differential trypsinization. This efficient method allows researchers to separately manipulate these two cell types to explore questions concerning their roles in mammary gland form and function.

通过微分试穿素生成马赛克乳腺有机体

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine ...

Evaluation of Mammary Gland Development and Function in Mouse Models

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves composed of a number of terminal duct lobular units made of secretory alveoli and converging ducts1. In mice, a similar architecture is observed at pregnancy in which ducts and alveoli are interspersed within the connective tissue stroma. The mouse mammary gland epithelium is a tree like system of ducts composed of two layers of cells, an inner layer of luminal cells surrounded by an outer layer of myoepithelial cells denoted by the confines of a basement membrane2. At birth, only a rudimental ductal tree is present, composed of a primary duct and 15-20 branches. Branch elongation and amplification start at the beginning of puberty, around 4 weeks old, under the influence of hormones3,4,5. At 10 weeks, most of the stroma is invaded by a complex system of ducts that will undergo cycles of branching and regression in each estrous cycle until pregnancy2. At the onset of pregnancy, a second phase of development begins, with the proliferation and differentiation of the epithelium to form grape-shaped milk secretory structures called alveoli6,7. Following parturition and throughout lactation, milk is produced by luminal secretory cells and stored within the lumen of alveoli. Oxytocin release, stimulated by a neural reflex induced by suckling of pups, induces synchronized contractions of the myoepithelial cells around the alveoli and along the ducts, allowing milk to be transported through the ducts to the nipple where it becomes available to the pups 8. Mammary gland development, differentiation and function are tightly orchestrated and require, not only interactions between the stroma and the epithelium, but also between myoepithelial and luminal cells within the epithelium9,10,11. Thereby, mutations in many genes implicated in these interactions may impair either ductal elongation during puberty or alveoli formation during early pregnancy, differentiation during late pregnancy and secretory activation leading to lactation12,13. In this article, we describe how to dissect mouse mammary glands and assess their development using whole mounts. We also demonstrate how to evaluate myoepithelial contractions and milk ejection using an ex-vivo oxytocin-based functional assay. The effect of a gene mutation on mammary gland development and function can thus be determined in situ by performing these two techniques in mutant and wild-type control mice.

This method describes how to dissect and assess mammary gland development and function from mice. Excised mammary glands are assessed for the degree of development using whole mount while milk ejection is evaluated using an oxytocin-based myoepithelial cell contraction assay.

乳腺的发育和功能在小鼠模型的评价

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves ...

生物学

Reconstruction of 3-Dimensional Histology Volume and its Application to Study Mouse Mammary Glands

Histology volume reconstruction facilitates the study of 3D shape and volume change of an organ at the level of macrostructures made up of cells. It can also be used to investigate and validate novel techniques and algorithms in volumetric medical imaging and therapies. Creating 3D high-resolution atlases of different organs1,2,3 is another application of histology volume reconstruction. This provides a resource for investigating tissue structures and the spatial relationship between various cellular features. We present an image registration approach for histology volume reconstruction, which uses a set of optical blockface images. The reconstructed histology volume represents a reliable shape of the processed specimen with no propagated post-processing registration error. The Hematoxylin and Eosin (H&#38;E) stained sections of two mouse mammary glands were registered to their corresponding blockface images using boundary points extracted from the edges of the specimen in histology and blockface images. The accuracy of the registration was visually evaluated. The alignment of the macrostructures of the mammary glands was also visually assessed at high resolution.
This study delineates the different steps of this image registration pipeline, ranging from excision of the mammary gland through to 3D histology volume reconstruction. While 2D histology images reveal the structural differences between pairs of sections, 3D histology volume provides the ability to visualize the differences in shape and volume of the mammary glands.

We present an image registration approach for 3-dimensional (3D) histology volume reconstruction, which facilitates the study of the changes of an organ at the level of macrostructures made up of cells . Using this approach, we studied the 3D changes between wild-type and Igfbp7-null mammary glands.

3维组织学音量和其研究小鼠乳腺腺体的应用改造

Histology volume reconstruction facilitates the study of 3D shape and volume change of an organ at the level of macrostructures made up of cells. It can ...

A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. A variety of molecules, such as peptides or oligonucleotides can be introduced into essentially 100% of the cells in a non-traumatic manner.&#160; Here, we describe how it can be used to study intercellular, gap junctional communication. Lucifer yellow penetrates into the cells when an electric pulse, applied to the conductive surface on which they are growing, causes pores to form through the cell membrane. This is electroporation. Cells growing on the nonconductive&#160;glass surface immediately adjacent to the electroporated region do not take up Lucifer yellow by electroporation but do acquire the fluorescent dye as it is passed to them via gap junctions that link them to the electroporated cells. The results of the transfer of dye from cell to cell can be observed microscopically under fluorescence illumination. This technique allows for precise quantitation of gap junctional communication. In addition, it can be used for the introduction of peptides or other non-permeant molecules, and the transfer of small electroporated peptides via gap junctions to inhibit the signal in the adjacent, non-electroporated cells is a powerful demonstration of signal inhibition.

This presentation demonstrates a method whereby electroporation of adherent, cultured cells is used for the study of intercellular, junctional communication, while the cells grow on a slide coated with conductive and transparent indium-tin oxide.

对缝隙连接检查功能测定;贴壁细胞的氧化铟锡电

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. ...

Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple method for the immune detection of proteins, the mouse lactating mammary gland being taken as an example. A protocol for the preparation of the tissue samples, especially concerning the dissection of mouse mammary gland, tissue fixation and frozen tissue sectioning, are detailed. A standard protocol to perform indirect immunofluorescence, including an optional antigen retrieval step, is also presented. The observation of the labeled tissue sections as well as image acquisition and post-treatments are also stated. This procedure gives a full overview, from the collection of animal tissue to the cellular localization of a protein. Although this general method can be applied to other tissue samples, it should be adapted to each tissue/primary antibody couple studied.

The indirect immunofluorescence protocol described in this article allows the detection and the localization of proteins in the mouse mammary gland. A complete method is given to prepare mammary gland samples, to perform immunohistochemistry, to image the tissue sections by fluorescence microscopy, and to reconstruct images.

对小鼠乳腺冰冻切片间接免疫荧光

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple ...

Isolation of Intact, Whole Mouse Mammary Glands for Analysis of Extracellular Matrix Expression and Gland Morphology

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal architecture. Here, a small pocket below the skin was created using Mayo scissors, allowing separation of the glands within the subcutaneous tissue from the underlying peritoneum. Visualization of the glands was aided by the use of 3.5x-R surgical micro loupes. The pelt was inverted and pinned back allowing identification of the intact mammary fat pads. Each of the #4 abdominal glands was bluntly dissected by sliding the scalpel blade laterally between the subcutaneous layer and the glands. Immediately post-harvest, glands were placed in 10% neutral buffered formalin for subsequent tissue processing. Excision of the entire gland is advantageous because it primarily eliminates the risk of excluding important tissue-wide interactions between ductal epithelial cells and other microenvironmental cellular populations that could be missed in a partial biopsy. One drawback of the methodology is the use of serial sections from fixed tissues which limits analyses of ductal morphogenesis and protein expression to discrete locations within the gland. As such, changes in ductal architecture and protein expression in 3 dimensions (3D) is not readily obtainable. Overall, the technique is applicable to studies requiring whole intact murine mammary glands for downstream investigations such as developmental ductal morphogenesis or breast cancer.

Here, we present a protocol for the isolation of whole, intact mouse mammary glands to investigate extracellular matrix (ECM) expression and ductal morphology. Mouse #4 abdominal glands were extracted from 8-10 week old female nulliparous mice, fixed in neutral buffered formalin, sectioned and stained using immunohistochemistry for ECM proteins.

完整、全小鼠乳腺的分离及细胞外基质表达及腺体形态学分析

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal ...

Longitudinal Intravital Microscopy Using a Mammary Imaging Window with Replaceable Lid

The branched structure of the mammary gland is highly dynamic and undergoes several phases of growth and remodeling after birth. Intravital microscopy in combination with skin flap surgery or implantation of imaging windows has been used to study the dynamics of the healthy mammary gland at different developmental stages. Most mammary imaging technologies are limited to a time frame of hours to days, whereas the majority of mammary gland remodeling processes occur in time frames of days to weeks. To study mammary gland remodeling, methods that allow optical access to the tissue of interest for extended time frames are required. Here, an improved version of the titanium mammary imaging window with a replaceable lid (R.MIW) is described that allows high-resolution imaging of the mammary gland with a cellular resolution for up to several weeks. Importantly, the R.MIW provides tissue access over the entire duration of the intravital imaging experiment and could therefore be used for local tissue manipulation, labeling, drug administration, or image-guided microdissection. Taken together, the R.MIW enables high-resolution characterization of the cellular dynamics during mammary gland development, homeostasis, and disease.

This protocol describes a novel mammary imaging window with a replaceable lid (R.MIW). Intravital microscopy after implantation of the R.MIW allows for longitudinal and multi-day imaging of the healthy and diseased mammary gland with a cellular resolution during the different developmental stages.

使用带有可更换盖子的乳腺成像窗口进行纵向活体腔内显微镜检查

The branched structure of the mammary gland is highly dynamic and undergoes several phases of growth and remodeling after birth. Intravital microscopy in ...