对小鼠乳腺冰冻切片间接免疫荧光
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Dissection | Company | Catalog Number | Comments/Description |
| Pins | |||
| Ethanol | |||
| Scissors | |||
| Scalpel and adapted blades | |||
| Ice | |||
| Towel paper | |||
| Tissue sample preparation | Company | Catalog Number | Comments/Description |
| Phosphate Buffered Saline (pH7.4) | Sigma | P-3813 | |
| Paraformaldehyde (PFA, 32% EM grade, 100 ml) | Electron Microscopy Sciences | 15714-S | personnal protection equipment required WARNING: this product will expose you to Formaldehyde Gas, a chemical known to cause cancer |
| OCT compound/Tissue Tek | Sakura | 4583 | |
| Sucrose (D-saccharose) | VWR | 27480.294 | |
| Plastic molds | Dominique Dutscher | 39910 | |
| Liquid nitrogen | |||
| Cryostat/sample support | Leica | CM3050S | |
| Razor blades (SEC35) | Thermo Scientific | 152200 | |
| Slide box | |||
| Glass slides Superfrost/Superfrost Ultra Plus | Thermo Scientific | 10143560W90/1014356190 | |
| Brushes | |||
| IHC | Company | Catalog Number | Comments/Description |
| Super Pap Pen | Sigma | Z377821-1EA | |
| Permanent marker (black) | |||
| 50 mM NH4Cl in PBS | Sigma | A-0171 | |
| 0.1 M glycine in PBS | VWR | 24403.367 | |
| Antigen Retrieval solution: Tris 100 mM 5% urea pH9.6 | |||
| Heater (up to 100°C) | |||
| Bovine Serum Albumin (BSA) | Sigma | A7906-100G | |
| Vectashield (anti-fading mounting medium) without DAPI/with DAPI | Vector Laboratories | H-1000/H-1200 | |
| Glass coverslips 22x50mm (microscopy grade) | VWR | CORN2980-225 | |
| Nail polish | |||
| Primary antibodies | Company | Catalog Number | Comments/Description |
| Rabbit anti-mouse caseins (#7781; 1:50 dilution) | generously gifted by M.C. Neville (University of Colorado Health Sciences Center, USA) |
||
| Mouse anti-cytokeratin 8 (CK8, clone 1E8, 1:50 dilution) | Biolegend (Covance) | MMS-162P | |
| Mouse anti-cytokeratin 14 (CK14, cloneLL002, 1:50 dilution) | Thermo Scientific | MS-115-P0/P1 | |
| Rabbit anti-butyrophilin (1:300 dilution) | generously gifted by I.H. Mather (Department of Animal and Avian Sciences University of Maryland College Park, USA) | ||
| Rabbit anti-Stx6 (1:50 dilution) | generously gifted S. Tooze (Cancer Research UK, London Research Institute, London, UK) |
||
| Rabbit anti-VAMP4 (1:50 dilution) | Abcam | ab3348 | |
| Secondary antibodies | Company | Catalog Number | Comments/Description |
| Rhodamine-conjugated goat anti-rabbit IgG (H + L) (1:300 dilution) | Jackson ImmunoResearch Laboratories | 111-025-003 | |
| Counterstains | Company | Catalog Number | Comments/Description |
| Bodipy 493/503 | Life Technologies (Molecular Probes) | D-3922 | |
| DAPI (4-6-diamidino-2-phenylindole) | Life Technologies (Molecular Probes) | D-1306 | |
| Observation/Image capture | Company | Catalog Number | Comments/Description |
| conventional fluorescence microscope | Leica Leitz DMRB microscope |
Standard filters for FITC, Rhodamine and DAPI emissions, ×63 oil-immersion objective (NA 1.3), DP50 imaging camera (Olympus), CellˆF software (Olympus) |
|
| Laser Scanning Microscope (confocal microscopy) | Zeiss LSM 510 microscope |
Plan-Apochromat ×63 oil-immersion objective (NA 1.4), CLSM 510 software, Confocal facilities, MIMA2 Platform, INRA Jouy-en-Josas, France, http://mima2.jouy.inra.fr/mima2) | |
| Image treatment | Company | Catalog Number | Comments/Description |
| ImageJ 1.49k software | Free software |
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