Name: indirect immunofluorescence on frozen sections of mouse mammary gland
Uploaded: 2015-12-01T14:00:00.000+00:00
Duration: 11 min 13 s
Description: Watch this Scientific Journal Video about Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland at JoVE.com

作者感谢INRA MIMA2成像核心设施（INRA，UMR1198，茹伊-EN-Josas），并于IERP单位的工作人员（UE 0907，INRA，茹伊-EN-Josas）动物保健和设施。我们还要感谢IH马瑟，MC内维尔和S. Tooze为我们提供了非常有用的antibodie。

CD1小鼠饲养在INRA（UE0907 IERP，茹伊-EN-Josas，法国）。动物护理的所有道德方面符合由农业部法国文化部制定的有关准则和许可要求。所使用的程序都经当地伦理委员会（协议12/097从Comethea的Jouy-ZH-Josas / AgroParisTech）。 1.乳腺样品制备<ol><li>小鼠乳腺解剖<ol><li>颈椎脱位安乐死小鼠在哺乳期的第10天，其腹部朝上脚的动物了。 </li><li>用乙醇湿腹侧区和用纸巾擦干。 </li><li>使用镊子，拉起两条后腿之间的腹部皮肤，并用锋利的剪刀长约1厘米切口（只通过皮肤）。从这个第一切口开始，然后用剪刀剪开皮肤至鼠标的脖子。从腹膜和PI拉扯皮肤走ñ下来的皮肤在同一时间一面，伸展它教。 </li><li>收集腹部和腹股沟乳腺通过推动它们远离皮肤用棉签，最后拉动或切割他们远离腹膜。 注意:在这个步骤胭脂红染色可以以整个腺体43内可视化的乳腺上皮细胞中进行。这种方法可能是有用的，以分析在各种条件（生理发育阶段，疾病，在体内治疗）下的乳腺的全局形貌。 </li><li>除去位于腹部的连接点与腹股沟淋巴结44的淋巴结。 </li></ol></li><li>乳腺组织固定<ol><li>切割乳腺组织成3mm 3个片段用解剖刀和立即冲洗在磷酸盐缓冲盐水这些片段（PBS）的溶液，pH 7.4中，以除去尽可能多的牛奶越好。 </li><li>快速干燥的片段上的纸毛巾，并把它们在含有4％多聚甲醛（PFA，甲醛，32％甲醛溶液，小心）为10到15分钟，在冰上冷PBS溶液。 注意:这是足够的时间，以允许对乳腺组织切片随后的分析通过IIF36和/或原位杂交45。然而，由于醛固定剂渗透相当缓慢的组织片段（〜每小时1-3毫米），该时间可能会延长，以确保组织样品的最佳固定。或者，通过灌注麻醉动物用固定液（在本研究中未详述）固定在体内组织中。 </li></ol></li><li>蔗糖输液<ol><li>快速冲洗乳腺片段在冷PBS浸泡它们下温和摇动含40％蔗糖（D-蔗糖，C12H22O11议员，342.3克/摩尔）为16至48小时的冷PBS溶液在4℃下。 </li></ol></li><li>组织包埋 注意:在这一步，乳腺片段可重新切割，以便使更小的片段（2-3毫米3），或者，调整它们的形状。 <ol><li>正确标注的塑料模具和填充第三模具OCT化合物中，保持在室温的体积。放置每个模具乳腺组织中的一个片段（2-3毫米3）中，用OCT化合物中覆盖。 </li><li>将模具在液氮（以铝或使用金属筛的片材）的表面，并允许产品冻结。 注意:它必须成为沉浸在模具液氮前，固体和白色。 </li></ol></li><li>储存冷冻样品在-80℃，直到组织切片进行。 </li></ol> 2.冰冻组织切片注意:低温恒温器，它实际上是一个冰柜内的切片机，需要使冰冻组织切片。较低的温度通常需要脂肪或脂质丰富的组织如处女乳腺。 <ol><li>调节低温恒温器的温度升高到-26℃，并等待，直到它具有STABIlized。保持冷冻的组织块，在-26℃的整个切片过程。绝对避免在操作过程中解冻组织在任何时间。 </li><li>通过将它们放置在低温恒温器，至少10分钟，冷却刀片，切割支持，防滚装置和刷到-26℃。还放置低温恒温器内的滑动箱，以便能够存储载玻片作为部分制成。 </li><li>正确标注载玻片将用于收集组织切片和维护它们在RT;否则组织切片不会遵守。从低温恒温器内的模具中取出的样品。 注意:使用带正电荷的玻片将新鲜冰冻组织切片的附着力大大有利于由于较高的静电吸引。 </li><li>覆盖的金属组织光盘OCT化合物中（保持在RT）的表面和冷冻样本推到它。放置在低温恒温器内部的湿片，让它合作OL至少15分钟。 </li><li>将湿安装在低温恒温器的盘夹持器。调整切割厚度为5-6微米，如果可能的话，使用新的锋利的刀片，或者至少改变区用来切割每个样品，因为一些组织会迅速沉闷它的刀片。 </li><li>通过使封固剂的切口，直到切片形成均匀且正确地调整在剃刀刀片的防滚装置的位置。理想的是，防滚装置将跳过的刀片由约1mm。 </li><li>一旦设置正确，转动轮连续匀速运动进行组织切片。除非温度是理想的，组织部分将在本质上，尽量蜷缩。 <ol><li>用刷子抓住和操纵在舞台上一节，以便根据需要在载玻片上放置。用刷子清理冰冻组织块和/或刀片上可能存在的遗迹。 </li></ol></li><li>拉组织切片朝向用户和避免按压到低温恒温器阶段。避免按组织切片上低温恒温器阶段，因为它可能导致在舞台上的组织切片，因此无法与载玻片恢复它的粘附性。 </li><li>通过拾取起来在一个玻璃载片的表面上，通过保持它上面的部分和垂钓下来触摸组织切片获取组织切片一个接一个。 注:组织切片快坚持以热烈的玻璃由于静电吸引力。如果几个组织切片放在同一张幻灯片上，注意不要重叠，他们和他们的空间足够，能够单独将它们放在一个疏水圈（参见3.1.1节）。 </li></ol> 3.间接免疫荧光<ol><li>定位部分<ol><li>使用的疏水性屏障用笔画出载玻片上的组织周围的疏水性循环。让圈子干在室温约1分钟。周围绘制的T线用细黑永久性标记问题的部分，以及，但在玻璃载片相对的之一，组织切片是侧。 注意:此圆是拒水和丙酮和醇不溶性的。因此，它提供了一个屏障，IHC过程期间所使用的水溶液，并减少所需的试剂的体积。 </li><li>通过覆盖它们用一滴〜250微升PBS在RT几分钟再水合的组织切片。通过覆盖它们与〜250微升在PBS中新鲜制备的3％PFA溶液10至15分钟，固定的组织切片。 注意:任选在这种情况下使用的醛淬火液（50mM氯化铵（NH 4氯，先生53.5克/摩尔）在PBS或0.1M甘氨酸（C 2 H 5 NO 2，先生75.07克/摩尔）在PBS中）停止结合反应。简单和丰富的PBS洗涤一般足以除去未反应的醛。 </li></ol></li><li>抗原修复（可选） <br/&gt;注:虽然大多数抗原可无抗原检索（AR）进行检测，其他人将被只有在执行这一步观察。在某些情况下，AR也允许观察到的信号的增强。 <ol><li>放置在一个烧杯中的AR溶液（100毫摩尔Tris（C 4 H 11 NO 3议员，121.14）5％尿素（NH 2 CONH 2，先生60.06），PH值9.6）。 AR的溶液的体积必须足以完全覆盖玻片置于玻璃支架上。 </li><li>预热AR溶液至95℃，通过用温度计监测温度，然后将在合适的机架载玻片，浸没机架在热缓冲器，罩以限制蒸发并孵育10分钟，在95℃。 </li><li>除去从水浴的烧杯中，并离开该玻片在缓冲区中的另一10分钟。 </li></ol></li><li>免疫检测<ol><li>冲洗组织切片用PBS（〜250微升/部分），并与溶液饱和他们的3％牛血清白蛋白（BSA，〜250微升/部分）在PBS中至少30分钟，在RT。 </li><li>放30-50微升稀释在含有2％BSA的每个组织切片的PBS第一抗体。 注意:此卷是足以形成一滴完全覆盖组织切片。 </li><li>放置稀释剂（在PBS中的2％的BSA）的相同体积独自上的组织切片来执行无第一抗体的阴性对照。 <ol><li>系统包括该阴性对照中的每个IHC实验和用于估计实验的背景的每个第二抗体（非特异性标记由于第二抗体和/或所述组织自发荧光）执行。其他类型的阳性或阴性对照的，也可以进行，以确保标签的特异性（见讨论）。 </li></ol></li><li>将玻片在加湿箱O / N在4℃下。 注意:使用初级抗体是小鼠单克隆抗细胞角蛋白8（CK8，1:50稀释），鼠单克隆抗细胞角蛋白14（CK14，1:50稀释），兔多克隆抗小鼠酪蛋白（＃7781，1:50稀释，慷慨MC内维尔，科罗拉多州卫生大学提供科学中心，CO，USA），兔多克隆抗BTN1（1:300稀释，用IH马瑟，动物和鸟类科学系，美国马里兰大学，学院公园，MD，USA），兔多克隆抗Stx6（慷慨提供1:50的稀释，由S. Tooze，英国癌症研究所，伦敦研究所，伦敦，英国）和兔多克隆抗VAMP4（1:50稀释）慷慨地提供。 </li><li>彻底冲洗组织切片用PBS在室温10分钟的至少四倍。 </li><li>稀释适当的二级抗体（罗丹明缀合的山羊抗兔IgG（H + L），1:300稀释度）在含有2％BSA的PBS，放置30-50微升上的所有组织切片的该溶液，并孵育1.5小时在RT。 <ol><li>由于荧光对光敏感的分子，不揭露组织切片光，直到他们的分析。对于IIF上的组​​织切片，有利于耦合到红色荧光团的二抗自细胞膜倾向于产生一个绿色自发荧光可与低标签干扰。此外，选择红色荧光团偶联的二级抗体允许中性脂质（见下文）的伴随标记。 </li></ol></li><li>彻底PBS洗涤组织切片至少四次在室温10分钟。 </li></ol></li><li>后固定（可选） <ol><li>对于一些实验中，通过将用2％PFA样品在PBS中稀释，在室温10分钟，在以稳定的抗原/抗体支架进行后固定。但是，这一步骤可以与在大多数情况下，分配。 </li></ol></li><li>中性脂类和DNA复染<ol><li>以通过将组织切片形象化限流二极管和MFGS，颜色中性脂质在30-50微升含有BODIPY 493 3微克/ ml的PBS溶液/ 503在室温10分钟。迅速冲洗组织切片两次用PBS。 </li><li>染液用30-50微升含有3微米的DAPI（4-6二脒基-2-苯基吲哚，5毫克/ ml储备溶液）在室温10分钟的PBS溶液核DNA。安装的幻灯片观察之前用PBS洗两次组织切片。 </li></ol></li><li>部分安装<ol><li>除去PBS并放置一滴每个组织切片安装介质。 </li><li>将盖玻片的一侧，在对幻灯片上的角度，使得与液滴外缘接触，然后放下顶盖慢慢地，避免气泡。让液体在载玻片和盖玻片之间流传了几分钟，然后去除多余的安装介质纸巾的。 </li><li>密封盖玻片与指甲油和储存组织切片的载玻片在4℃，以防止其暴露在光线下，直至观察。 </li></ol></li></ol>4.荧光观察和图像采集注:配有由图像采集软件控制的摄像头必须遵守的IHC结果的荧光显微镜。 <ol><li>获取图像之前，检查标记的强度和通过查看阴性对照评估实验的背景。获得的每一个荧光标记（颜色通道）单独的照片。 </li><li>获得的所有照片，包括那些对应的控制，在用于每个颜色通道的相同条件（曝光和常规设置）。 </li><li>传统的显微镜<ol><li>用装有标准滤波器异硫氰酸荧光素（FITC，绿色），若丹明（红色）和DAPI（蓝色）的排放量的显微镜进行荧光显微镜，×20至×63（油浸，NA 1.3）的目标和一个DP50成像照相机。 </li></ol></li><li>共聚焦显微镜</li><li>与MICROS执行共聚焦显微镜型箱搭载用ZEN软件，用×20至×63（油浸，NA 1.4）的目标和激光的488-和568纳米的激发波长。 </li></ol> 5.图像处理注意:所有图像后处理使用ImageJ的自由软件（http://imagej.nih.gov/ij/）执行。 <ol><li>叠加图像（合并） <ol><li>打开，将被组合（文件/打开）各信道获得的图像。如果与8位灰度图像时，属性人工颜色使用查找表（图像/查找表）的每个信道。 </li><li>通过使用命令"合并信道"（图像/色彩/合并通道），然后归因一种颜色给每个信道生成的灰度或彩色图像的复合图像。 </li><li>执行图像栈叠加以同样的方式通过打开栈中，将被组合（文件/打开）各信道获取和使用命令"合并通道与＃8221; （图片/颜色/合并通道），以属性的颜色给每个通道。复合堆保存为图像序列或电影（参见5.4节）。 </li></ol></li><li>图像堆栈ž投影<ol><li>使用Z投影函数（图像/堆叠/ Zproject，最多强度）通过投影它们沿轴线垂直于图像平面（z轴），以提供一种图像栈的所有图片的一个二维视图。 "最大强度"选项创建，其中每个像素包含在堆栈中的所有图像的最大值的图像。这将生成一个单一的图像，允许所有通过整个图像堆栈针对特定通道或多个通道的叠加后观察到的染色的可视化。 </li></ol></li><li>图像堆栈3D投影<ol><li>使用3D投影命令（图像/堆叠/ 3D项目，亮的点，y轴）产生旋转体积的突起的一个序列上的平面。苏的可视化呈现rfaces和内部结构取决于两个投影方法（最接近点，亮的点（这里使用的），或平均值）和所选择的可视化参数。动画序列的每个帧是从不同的视角突出的结果。 </li><li>绕每个所述三个正交轴（y轴这里被选择）的创建的3D图像。除制作成一个图像或电影的序列。 </li></ol></li><li>图像堆栈电影转换<ol><li>打开图像堆栈（文件/打开），并将其保存为一个电影.AVI格式，使用命令"AVI"（文件/另存为/ AVI）。 </li></ol></li></ol>

作者宣称，他们有没有竞争的财务权益。

IHC是一个相对简单的和直接的实验方法来定位抗原在组织切片，这主要取决于特定的表位 - 抗体相互作用。虽然有大量的协议被用来通过IIF本地化的蛋白质，这些程序的核心是几乎总是相同的。不过，也有可强烈影响的结果，因此，必须为每个单独的IHC研究优化某些关键方面。这种方法的最具挑战性的方面是，以确定最佳的实验条件， 即 ，那些产生用于所感兴趣的抗原的强烈和特定信号?...

乳腺是非典型哺乳动物外分泌器官，其主要功能是产生乳喂养新生儿。乳腺组织的发育主要发生在出生后，其特征是一种独特的方法，其中上皮侵入周围的基质。此组织经历了许多变化（生长，分化和回归），特别是在成人生活，与之同时变化生殖状态（ 图1）。除了 ​​组织的整体形态，不同的细胞类型以及它们的乳腺内排列的比例发展1-5中显着改变。 在胚胎生命，乳腺上皮细胞从乳房乳线，这是由外胚层的轻微增厚和分层定义导出，脱颖而出和后肢之间围绕10.5天胚胎（E10.5）（图1A中线的每一侧）。上E11.5，牛奶线分解成单个placodes，它们沿在可重现的位置的乳房乳线对称地定位，并且周围的间质开始凝结。所述placodes开始下沉更深进入真皮和乳腺间质组织周围的乳腺芽（E12.5-E14.5）同心层。作为E15.5的，乳腺上皮细胞，开始增殖和细长形成初级发芽是推动通过向脂肪垫乳腺间质。主芽开发的中空内腔具有开口到皮肤上，打上了乳头鞘的形成。在E18.5，在延长管道已成长为脂肪垫，并分流到包括在脂肪垫一个小arborized导管系统。发展基本上逮捕和基本的乳腺仍然morphogenetically静态直到青春期。在男性胚胎，雄激素受体的活化导致的芽，其消失的变性由E15.5。作为E18的，乳腺发育停止，直到青春期6-9。 出生时，乳腺藏着一个基本的导管系统，拉长和分支机构缓慢（等速生长）。在青春期开始，位于所述导管的尖端球形结构称为末端芽（TEBS），被帽细胞的外层和细胞的多层内芯（体细胞）的形成。这些结构是高度增殖和浸润周围基质组织响应于激素线索。该TEBS导致乳腺导管内延伸扩散，加上分支形态。该过程导致建立一个基本上皮arborized网络从乳头（图1B，青春期）发出的。在〜出生后10-12周，当上皮侵入整个脂肪垫，其扩张停止，并且TEBS消失。导管发展然后经历动态变化，也就是说，successi根据动情周期10（ 图1B，成人）已经增殖的上皮细胞和回归。 从妊娠开始时，乳腺组织经历了重要的增长和形态变化，为哺乳做准备。乳腺上皮细胞广泛增殖和分化，导致了高度支肾小管肺泡网络。与此同时，乳腺上皮细胞（的MECs）成为偏振光，并且能够合成和分泌奶制品。的MECs组织成无数肺泡结构（腺泡）是由收缩肌上皮细胞包围，并在一个的结缔组织和脂肪组织，血管和神经末端（图1B，怀孕）组成基质并入。此外，的MECs的基部侧与基底膜（细胞外基质）紧密接触，并且在这两个实体之间的相互作用紧紧调节乳房的两个形态发生和分泌功能RY上皮11-13。 所有这些方法依赖于各种环境因素，其中最重要的是hormones14，旁分泌因子和细胞外基质的作用。例如，孕激素诱导广泛侧分支15和alveologenesis即，结合催乳激素（PRL）的16,17，促 ​​进和维持肺泡的分化。除了 ​​类固醇和PRL18，细胞因子和与发育相关的信号通路（Wnt信号和Notch信号转导通路）也参与了乳腺谱系提交和发展19-21。在怀孕的最后，管腔的MECs开始产生一个富含蛋白质的乳在肺泡的管腔被称为初乳。此外，孕酮作用于上皮通透性并且由于紧密连接仍然是开放的，初乳也见于母体血液流。 分娩后，mammarÿ上皮占据了几乎所有的乳腺体积和高度有组织的（图2，乳腺上皮细胞）。牛奶生产单元，即肺泡（图2，肺泡），由极化的乳腺上皮分泌细胞（MESCs）的单层形成，其顶端质膜界定的内腔中。肺泡安排自己成分为连接到漏奶到外环境（ 图2，叶）导管叶小叶。哺乳期时， 即 ，MESCs开始分泌大量丰富的牛奶，主要由胎盘激素的下降（主要是黄体酮）（ 图1B期，哺乳期）触发。牛奶中的蛋白质基因在规定的时间时程，从怀孕到哺乳期9,22,23激活，主要是针对在哺乳时释放的垂体泌乳素。与此同时，MESCs和细胞外基质在两者之间的接触刺激乳蛋白SYNThesis通过通过细胞整合和层粘连蛋白24,25之间的相互作用介导的，而抑制细胞凋亡MESCs 26,27信号。这些信号通路导致的乳蛋白基因启动子28通过的特异性转录激活因子29的活化。细胞 - 细胞接触也可用于分化的一些方面包括建立心尖极​​性和牛奶制品的矢量分泌重要。紧密连接泌乳和MESCs开始之后迅速接近微细编排分子的摄取从血液以及牛奶成分的合成，运输和分泌，响应于新生儿的营养需求。在哺乳时，周围的肺泡中的肌上皮细胞收缩发生在响应于催产素和通过导管并进入乳头导致牛奶的喷射。奶是一种复杂的流体，它包含蛋白质（大多酪蛋白），糖（主要是乳糖），脂质和矿物质，以及生物活性分子，例如免疫球蛋白A（IgA的），生长因子和激素。酪蛋白被合成，组装在超分子结构，即酪蛋白胶束，沿分泌途径转运，然后通过胞吐作用释放时， 即 ，融合的含酪蛋白分泌囊泡（SV）上与卓制的顶质膜（ 图2）。 细胞内的流量依赖于膜室之间的物质交换，涉及可溶性N-乙基马来酰亚胺敏感融合（NSF）附着蛋白（SNAP）受体（SNARE）30,31。的SNARE蛋白家族被细分在囊泡斯耐尔（ⅴ-斯耐尔），存在于泡膜，和目标斯耐尔（叔斯耐尔），定位于目标膜。通过他们的卷曲螺旋结构域压缩和解，v型和叔斯耐尔组装形成一个高度稳定的四螺旋束复合物，被称为第ËSNARE复合体。这种复杂的，通过逐步使它们变成接近30,32促进了两种对立的脂质双层的融合。然后，圈套络合物由NSF三磷酸腺苷酶解离和其接头蛋白的SNAP和SNARE蛋白被再循环回到原点33的其隔室。有趣的是，每SNARE蛋白主要位于不同的细胞区室和SNARE配对可能有助于胞内融合事件34的特异性。以往的研究表明，至少有突触相关蛋白23（SNAP23）和囊泡相关膜蛋白8（VAMP8），和syntaxins（STX）-7和-12起到酪蛋白的胞外分泌35,36的作用。这些蛋白也被发现与牛奶的脂质级分， 即 ，乳脂肪球（MFGS）37。目前通行的模型假设，胞浆脂滴（限流二极管）是由中立升堆积而成ipids（主要是甘油三酯和甾醇酯）和胆固醇从内质网（ER）膜38-41的两个小叶之间的母亲饮食衍生的。大限流二极管形成，至少部分地由较小的限流二极管的融合而被运送到MESCs的顶侧在那里它们被释放为MFGS（1-10微米直径）通过出芽，被包裹由卓制顶部质膜40-42。泌乳停止幼仔断奶和MESCs逐步通过凋亡死亡，导致乳腺组织回青春期状态（图1B，退化）的回归之后。 免疫荧光（IF）是在生物学的几乎所有方面中使用的共同的分析实验室方法，无论是在研究和在临床诊断。 IF技术可以对组织切片进行（免疫组化，IHC）或细胞（免疫细胞化学，ICC）的样品。这种强大的方法依赖于使用fluorescent-特异性结合（直接或间接）到感兴趣的抗原，因此允许其组织分布通过荧光显微镜可视化标记的抗体。荧光信号大多依赖于抗体的检体的质量和浓度和适当的处理。一个简单的间接免疫荧光（IIF）协议被呈现给检测奶制品（酪蛋白和MFGS）以及参与牛奶产品分泌蛋白质（嗜乳脂蛋白（BTN1），SNARE蛋白质）对小鼠乳腺组织（图3）的冷冻切片。虽然该协议提供了一个完整的IHC概述，从组织收集到的图像后处理，关键和可选步骤以及一些技术建议也被提出和讨论。

<table><tbody><tr><td>Dissection</td><td></td><td></td><td></td></tr><tr><td>Pins</td><td></td><td></td><td></td></tr><tr><td>Ethanol</td><td></td><td></td><td></td></tr><tr><td>Scissors</td><td></td><td></td><td></td></tr><tr><td>Scalpel and adapted blades</td><td></td><td></td><td></td></tr><tr><td>Ice</td><td></td><td></td><td></td></tr><tr><td>Towel paper</td><td></td><td></td><td></td></tr><tr><td>Tissue sample preparation</td><td>Company</td><td>Catalog Number</td><td>Comments/Description</td></tr><tr><td>Phosphate Buffered Saline (pH7.4)</td><td>Sigma</td><td>P-3813</td><td></td></tr><tr><td>Paraformaldehyde (PFA, 32% EM grade, 100 ml)</td><td>Electron Microscopy Sciences</td><td>15714-S</td><td>personnal protection equipment required WARNING: this product will expose you to Formaldehyde Gas, a chemical known to cause cancer</td></tr><tr><td>OCT compound/Tissue Tek</td><td>Sakura</td><td>4583</td><td></td></tr><tr><td>Sucrose (D-saccharose)</td><td>VWR</td><td>27480.294</td><td></td></tr><tr><td>Plastic molds</td><td>Dominique Dutscher</td><td>39910</td><td></td></tr><tr><td>Liquid nitrogen</td><td></td><td></td><td></td></tr><tr><td>Cryostat/sample support</td><td>Leica&amp;nbsp;</td><td>CM3050S</td><td></td></tr><tr><td>Razor blades (SEC35)</td><td>Thermo Scientific</td><td>152200</td><td></td></tr><tr><td>Slide box</td><td></td><td></td><td></td></tr><tr><td>Glass slides Superfrost/Superfrost Ultra Plus</td><td>Thermo Scientific</td><td>10143560W90/1014356190</td><td></td></tr><tr><td>Brushes</td><td></td><td></td><td></td></tr><tr><td>IHC</td><td>Company</td><td>Catalog Number</td><td>Comments/Description</td></tr><tr><td>Super Pap Pen</td><td>Sigma</td><td>Z377821-1EA</td><td></td></tr><tr><td>Permanent marker (black)</td><td></td><td></td><td></td></tr><tr><td>50 mM NH&lt;sub&gt;4&lt;/sub&gt;Cl in PBS</td><td>Sigma</td><td>A-0171</td><td></td></tr><tr><td>0.1 M glycine in PBS</td><td>VWR</td><td>24403.367</td><td></td></tr><tr><td>Antigen Retrieval solution: Tris 100 mM 5% urea pH9.6</td><td></td><td></td><td></td></tr><tr><td>Heater (up to 100&amp;deg;C)</td><td></td><td></td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma</td><td>A7906-100G</td><td></td></tr><tr><td>Vectashield (anti-fading mounting medium) without DAPI/with DAPI</td><td>Vector Laboratories</td><td>H-1000/H-1200</td><td></td></tr><tr><td>Glass coverslips 22x50mm (microscopy grade)</td><td>VWR</td><td>CORN2980-225</td><td></td></tr><tr><td>Nail polish</td><td></td><td></td><td></td></tr><tr><td>Primary antibodies</td><td>Company</td><td>Catalog Number</td><td>Comments/Description</td></tr><tr><td>Rabbit anti-mouse caseins (#7781; 1:50 dilution)</td><td>generously gifted by M.C. Neville (University of Colorado Health Sciences&lt;br/&gt; Center, USA)</td><td></td><td></td></tr><tr><td>Mouse anti-cytokeratin 8 (CK8, clone 1E8, 1:50 dilution)</td><td>Biolegend (Covance)</td><td>MMS-162P</td><td></td></tr><tr><td>Mouse anti-cytokeratin 14 (CK14, cloneLL002, 1:50 dilution)</td><td>Thermo Scientific</td><td>MS-115-P0/P1</td><td></td></tr><tr><td>Rabbit anti-butyrophilin (1:300 dilution)</td><td>generously gifted by I.H. Mather (Department of Animal and Avian Sciences University of Maryland College Park, USA)</td><td></td><td></td></tr><tr><td>Rabbit anti-Stx6 (1:50 dilution)</td><td>generously gifted S. Tooze&lt;br/&gt; (Cancer Research UK, London Research Institute, London, UK)</td><td></td><td></td></tr><tr><td>Rabbit anti-VAMP4 (1:50 dilution)</td><td>Abcam</td><td>ab3348</td><td></td></tr><tr><td>Secondary antibodies</td><td>Company</td><td>Catalog Number</td><td>Comments/Description</td></tr><tr><td>Rhodamine-conjugated goat anti-rabbit IgG (H + L) (1:300 dilution)</td><td>Jackson ImmunoResearch Laboratories</td><td>111-025-003</td><td></td></tr><tr><td>Counterstains</td><td>Company</td><td>Catalog Number</td><td>Comments/Description</td></tr><tr><td>Bodipy 493/503</td><td>Life Technologies (Molecular Probes)</td><td>D-3922</td><td></td></tr><tr><td>DAPI (4-6-diamidino-2-phenylindole)</td><td>Life Technologies (Molecular Probes)</td><td>D-1306</td><td></td></tr><tr><td>Observation/Image capture</td><td>&lt;span style=&quot;line-height: 20.8px;&quot;&gt;Company&lt;/span&gt;</td><td>Catalog Number</td><td>Comments/Description</td></tr><tr><td>conventional fluorescence microscope</td><td>Leica Leitz DMRB&lt;br/&gt; microscope</td><td></td><td>Standard filters for FITC, Rhodamine&lt;br/&gt; and DAPI emissions,&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; &amp;times;63 oil-immersion objective (NA 1.3), DP50 imaging camera (Olympus), Cell&amp;circ;F software (Olympus)</td></tr><tr><td>Laser Scanning Microscope (confocal microscopy)</td><td>Zeiss LSM&lt;br/&gt; 510 microscope</td><td></td><td>Plan-Apochromat &amp;times;63 oil-immersion objective (NA 1.4),&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; CLSM 510 software,&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Confocal facilities, MIMA2 Platform, INRA Jouy-en-Josas, France, http://mima2.jouy.inra.fr/mima2)</td></tr><tr><td>Image treatment</td><td>Company</td><td>Catalog Number</td><td>Comments/Description</td></tr><tr><td>ImageJ 1.49k software</td><td></td><td></td><td>Free software</td></tr></tbody></table>

indirect immunofluorescence on frozen sections of mouse mammary gland

间接免疫荧光来检测和定位感兴趣的蛋白质中的组织。这里介绍的协议描述了用于免疫检测的蛋白质的完整和简单的方法，鼠标哺乳期乳腺被作为例子。一种协议，它的组织样本的准备，特别是关于小鼠乳腺，组织固定和冷冻组织切片的剥离，有详细。一个标准的协议进行间接免疫荧光法，包括可选的抗原修复步骤，还赠送。也指出了标记的组织切片的观察以及图像获取和后处理。此过程提供了一个完整的概述，从动物组织收集到的蛋白质的细胞定位。虽然这种通常的方法可以适用于其他组织样品，应当适应每个组织/一级抗体耦合影响。

乳腺是位于沿两个胸部和在啮齿类动物的腹部的腹部结构的皮下腺。五对妊娠期间的小鼠的腺体的位置示于图4。乳腺的形态其发展过程中极大地改变，反映到全泌乳（图1B）制备所需的功能修改。在处女或动物未产妇，乳腺由一个人烟稀少分支导管上皮内嵌入一个薄的脂肪基质可能很难看到。从发病妊娠，乳腺上皮细胞的激增和膨胀，导致更大的乳...

Watch this Scientific Journal Video about Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland at JoVE.com

Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland

在这篇文章中所描述的间接免疫荧光协议允许的检测和蛋白在小鼠乳腺定位。一个完整的方法是考虑到制备乳腺样品，利用荧光显微镜进行免疫组织化学，将图像的组织切片，并重建图像。

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 ...

indirect-immunofluorescence-on-frozen-sections-of-mouse-mammary-gland

Research

JoVE Journal

Biology

36.8K 次观看.  INRA. 在这篇文章中所描述的间接免疫荧光协议允许的检测和蛋白在小鼠乳腺定位。一个完整的方法是考虑到制备乳腺样品，利用荧光显微镜进行免疫组织化学，将图像的组织切片，并重建图像。 

视频: Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland

Sectioning Mammary Gland Whole Mounts for Lesion Identification

Normal mammary gland development may be altered by exposure to environmental toxicants and pharmaceutical products, excessive exposure to hormones, and genetic alterations. Mammary gland whole mounts are an inexpensive method to capture the progression of morphological changes that may arise after exposure. However, in later life, when abnormalities are more prone to develop, sole reliance on this one method may not always provide enough information to make a proper diagnosis of the abnormality. Historically, in chemical test guideline studies, a single mammary gland is removed at necropsy and prepared as a hematoxylin and eosin (H&#38;E)-stained section. The incorporation of contralateral mammary whole-mount collection and analysis decreases the likelihood of a false-negative assessment. Evaluation of the whole mount is limited by the presence of one or two entire mammary glands on a slide, and in some cases, the abnormalities observed in the whole mount are not uniformly represented in the H&#38;E section.&#160; The goal of this study was to develop a protocol for converting coverslipped mammary whole mounts to H&#38;E-stained sections so that lesions that would otherwise have been missed or that are difficult to diagnose can be identified. Here, we detail a method to produce a high-quality, paraffin-embedded H&#38;E section from a mammary gland that was initially prepared as a whole mount. In comparison to a tissue that was intentionally prepared for H&#38;E sectioning, the whole mount requires additional preparation for tissue removal and processing. However, this method is considered inexpensive, as it requires common lab reagents and little additional time. As a result, this method can provide invaluable information on how chemical and environmental exposures alter normal mammary development, as well as display changes that occur because of genetic modifications.

We developed a method to successfully remove, process, section, and stain, for histopathological evaluation, mammary tissue that had originally been fixed on slides as whole mounts. This method may promote the collection and evaluation of mammary gland whole mounts in reproductive and developmental test guideline studies.

切片乳腺全身为病变鉴别

Normal mammary gland development may be altered by exposure to environmental toxicants and pharmaceutical products, excessive exposure to hormones, and ...

科研

癌症研究

Immunohistochemistry on Paraffin Sections of Mouse Epidermis Using Fluorescent Antibodies

In the epidermis, immunohistochemistry is an efficient means of localizing specific proteins to their relative expression compartment; namely the basal, suprabasal, and stratum corneum layers. The precise localization within the epidermis of a particular protein lends clues toward its functional role within the epidermis. In this chapter, we describe a reliable method for immunolocalization within the epidermis modified for both frozen and paraffin sections that we use very routinely in our laboratory. Paraffin sections generally provide much better morphology, hence, superior results and photographs; however, not all antibodies will work with the harsh fixation and treatment involved in their processing. Therefore, the protocol for frozen sectioning is also included. Within paraffin sectioning, two fixation protocols are described (Bouin's and paraformaldehyde); the choice of fixative will be directly related to the antibody specifications and may require another fixing method.

We describe a reliable method for immunolocalization within the epidermis modified for both frozen and paraffin sections that we use very routinely in our laboratory.

使用荧光抗体对小鼠表皮石蜡切片的免疫组织化学

In the epidermis, immunohistochemistry is an efficient means of localizing specific proteins to their relative expression compartment; namely the basal, ...

生物学

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

Cell-cell interactions play a pivotal role in preserving tissue integrity and the barrier between the different compartments of the mammary gland. These interactions are provided by junctional proteins that form nexuses between adjacent cells. Junctional protein mislocalization and reduced physical associations with their binding partners can result in the loss of function and, consequently, to organ dysfunction. Thus, identifying protein localization and protein-protein interactions (PPIs) in normal and disease-related tissues is essential to finding new evidences and mechanisms leading to the progression of diseases or alterations in developmental status. This manuscript presents a two-step method to evaluate PPIs in murine mammary glands. In protocol section 1, a method to perform co-immunofluorescence (co-IF) using antibodies raised against the proteins of interest, followed by secondary antibodies labeled with fluorochromes, is described. Although co-IF allows for the demonstration of the proximity of the proteins, it does make it possible to study their physical interactions. Therefore, a detailed protocol for co-immunoprecipitation (co-IP) is provided in protocol section 2. This method is used to determine the physical interactions between proteins, without confirming whether these interactions are direct or indirect. In the last few years, co-IF and co-IP techniques have demonstrated that certain components of intercellular junctions co-localize and interact together, creating stage-dependent junctional nexuses that vary during mammary gland development.

Intercellular junctions are requisites for mammary gland stage-specific functions and development. This manuscript provides a detailed protocol for the study of protein-protein interactions (PPIs) and co-localization using murine mammary glands. These techniques allow for the investigation of the dynamics of the physical association between intercellular junctions at different developmental stages.

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 ...

发育生物学

In Vivo Gene Delivery into Mouse Mammary Epithelial Cells Through Mammary Intraductal Injection

Mouse mammary glands comprise ductal trees, which are lined by epithelial cells and have one opening at the tip of each nipple. The epithelial cells play a major role in mammary gland function and are the origin of most mammary tumors. Introducing genes of interest into mouse mammary epithelial cells is a critical step in evaluating gene function in epithelial cells and generating mouse mammary tumor models. This goal can be accomplished through the intraductal injection of a viral vector carrying the genes of interest into the mouse mammary ductal tree. The injected virus subsequently infects mammary epithelial cells, bringing in the genes of interest. The viral vector can be lentiviral, retroviral, adenoviral, or adenovirus-associated viral (AAV). This study demonstrates how a gene of interest is delivered into mammary epithelial cells through mouse mammary intraductal injection of a viral vector. A lentivirus carrying GFP is used to show stable expression of a delivered gene, and a retrovirus carrying Erbb2 (HER2/Neu) is used to demonstrate oncogene-induced atypical hyperplastic lesions and mammary tumors.

In Vivo Gene Delivery into Mouse Mammary Epithelial Cells Through Mammary Intraductal Injection

The present protocol describes intraductal injection of viral vectors via the teat to deliver genes of interest into the mammary epithelial cells.

体内 通过乳腺导管内注射将基因传递到小鼠乳腺上皮细胞

Mouse mammary glands comprise ductal trees, which are lined by epithelial cells and have one opening at the tip of each nipple. The epithelial cells play ...

Isolation, Fixation, and Immunofluorescence Imaging of Mouse Adrenal Glands

Immunofluorescence is a well-established technique for detection of antigens in tissues with the employment of fluorochrome-conjugated antibodies and has a broad spectrum of applications. Detection of antigens allows for characterization and identification of multiple cell types. Located above the kidneys and encapsulated by a layer of mesenchymal cells, the adrenal gland is an endocrine organ composed by two different tissues with different embryological origins, the mesonephric intermediate mesoderm-derived outer cortex and the neural crest-derived inner medulla. The adrenal cortex secretes steroids (i.e., mineralocorticoids, glucocorticoids, sex hormones), whereas the adrenal medulla produces catecholamines (i.e., adrenaline, noradrenaline). While conducting adrenal research, it is important to be able to distinguish unique cells with different functions. Here we provide a protocol developed in our laboratory that describes a series of sequential steps required for obtaining immunofluorescence staining to characterize the cell types of the adrenal gland. We focus first on the dissection of the mouse adrenal glands, the microscopic removal of periadrenal fat followed by the fixation, processing and paraffin embedding of the tissue. We then describe sectioning of the tissue blocks with a rotary microtome. Lastly, we detail a protocol for immunofluorescent staining of adrenal glands that we have developed to minimize both non-specific antibody binding and autofluorescence in order to achieve an optimal signal.

Here we present a method to isolate adrenal glands from mice, fix the tissues, section them, and perform immunofluorescence staining.

小鼠肾上腺的分离、固定和免疫荧光成像

Immunofluorescence is a well-established technique for detection of antigens in tissues with the employment of fluorochrome-conjugated antibodies and has ...

免疫与感染

A Rapid Method for Multispectral Fluorescence Imaging of Frozen Tissue Sections

Multispectral fluorescence imaging on formalin-fixed paraffin-embedded (FFPE) tissues enables the detection of multiple markers in a single tissue sample that can provide information about antigen coexpression and spatial distribution of the markers. However, a lack of suitable antibodies for formalin-fixed tissues may restrict the nature of markers that can be detected. In addition, the staining method is time-consuming. Here we describe a rapid method to perform multispectral fluorescence imaging on frozen tissues. The method includes the fluorophore combinations used, detailed steps for the staining of mouse and human frozen tissues, and the scanning, acquisition, and analysis procedures. For staining analysis, a commercially available semiautomated multispectral fluorescence imaging system is used. Through this method, up to six different markers were stained and detected in a single frozen tissue section. The machine learning analysis software can phenotype cells that can be used for quantitative analysis. The method described here for frozen tissues is useful for the detection of markers that cannot be detected in FFPE tissues or for which antibodies are not available for FFPE tissues.

We describe a rapid staining method to perform multispectral imaging on frozen tissues.

冷冻组织部分多光谱荧光成像的快速方法

Multispectral fluorescence imaging on formalin-fixed paraffin-embedded (FFPE) tissues enables the detection of multiple markers in a single tissue sample ...

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 ...

Intravital Subcellular Microscopy of the Mammary Gland

The mammary gland constitutes a model par excellence for investigating epithelial functions, including tissue remodeling, cell polarity, and secretory mechanisms. During pregnancy, the gland expands from a primitive ductal tree embedded in a fat pad to a highly branched alveolar network primed for the formation and secretion of colostrum and milk. Post-partum, the gland supplies all the nutrients required for neonatal survival, including membrane-coated lipid droplets (LDs), proteins, carbohydrates, ions, and water. Various milk components, including lactose, casein micelles, and skim-milk proteins, are synthesized within the alveolar cells and secreted from vesicles by exocytosis at the apical surface. LDs are transported from sites of synthesis in the rough endoplasmic reticulum to the cell apex, coated with cellular membranes, and secreted by a unique apocrine mechanism. Other preformed constituents, including antibodies and hormones, are transported from the serosal side of the epithelium into milk by transcytosis. These processes are amenable to intravital microscopy because the mammary gland is a skin gland and, therefore, directly accessible to experimental manipulation. In this paper, a facile procedure is described to investigate the kinetics of LD secretion in situ, in real-time, in live anesthetized mice. Boron-dipyrromethene (BODIPY)665/676&#160;or monodansylpentane are used to label the neutral lipid fraction of transgenic mice, which either express soluble EGFP (enhanced green fluorescent protein) in the cytoplasm, or a membrane-targeted peptide fused to either EGFP or tdTomato. The membrane-tagged fusion proteins serve as markers of cell surfaces, and the lipid dyes resolve LDs &#8805; 0.7 &#181;m. Time-lapse images can be recorded by standard laser scanning confocal microscopy down to a depth of 15-25 &#181;m or by multiphoton microscopy for imaging deeper in the tissue. The mammary gland may be bathed with pharmacological agents or fluorescent dyes throughout the surgery, providing a platform for acute experimental manipulations as required.

<meta charset="utf8"></head><body>乳腺活体亚细胞显微镜检查

The present protocol describes a facile technique for the intravital imaging of the lactating mouse mammary gland by laser scanning confocal and multiphoton microscopy.

乳腺活体亚细胞显微镜检查

The mammary gland constitutes a model par excellence for investigating epithelial functions, including tissue remodeling, cell polarity, and secretory ...

对小鼠乳腺冰冻切片间接免疫荧光

摘要

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此视频中的章节

切片乳腺全身为病变鉴别

使用荧光抗体对小鼠表皮石蜡切片的免疫组织化学

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

体内通过乳腺导管内注射将基因传递到小鼠乳腺上皮细胞

小鼠肾上腺的分离、固定和免疫荧光成像

冷冻组织部分多光谱荧光成像的快速方法

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

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

乳腺活体亚细胞显微镜检查

乳腺活体亚细胞显微镜检查

对小鼠乳腺冰冻切片间接免疫荧光

摘要

探索更多视频

此视频中的章节

切片乳腺全身为病变鉴别

使用荧光抗体对小鼠表皮石蜡切片的免疫组织化学

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

体内 通过乳腺导管内注射将基因传递到小鼠乳腺上皮细胞

小鼠肾上腺的分离、固定和免疫荧光成像

冷冻组织部分多光谱荧光成像的快速方法

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

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

乳腺活体亚细胞显微镜检查

乳腺活体亚细胞显微镜检查

体内通过乳腺导管内注射将基因传递到小鼠乳腺上皮细胞