小鼠颅面组织和未切除骨骼的组织制备及免疫染色
Summary
在这里,我们提出了一个详细的协议,以小鼠颅面组织为例,通过免疫染色来检测和量化颅面形态形成/发病机制期间的蛋白质水平。此外,我们描述了一种制备和冷冻方法,从幼鼠的未分化硬组织免疫染色。
Abstract
组织免疫染色提供对给定组织内感兴趣的蛋白质的高度特定和可靠的检测。在这里,我们描述了一个完整和简单的协议,以小鼠颅面组织为例,用于检测颅面形态形成/发病过程中的蛋白质表达。该方案包括组织制备和冷冻、间接免疫荧光、图像采集和定量。此外,还介绍了一种制备和冷冻未分化的硬组织进行免疫染色的方法,以颅面组织和长骨为例。这些方法是确定颅面形态形成/发病过程中各种组织中的蛋白质表达和形态/解剖变化的关键。它们也适用于具有适当修改的其他组织。组织学知识和各节的高质量对于从实验结果中得出科学结论至关重要。该方法的潜在局限性包括但不限于抗体的特异性和定量困难,这里还讨论了这些缺陷。
Introduction
面部是人类身份的关键部分,由几种不同的组织组成,如上皮、肌肉、骨骼、软骨、牙齿。这些组织来自所有三个生殖层、内皮和中代1、2。为了正确形成颅面组织,细胞增殖、死亡和分化需要高度协调和由特定的信号通路调节,如Wnt、Fgf、Hh和Bmp通路3、4 , 5.细胞增殖、存活或分化的缺陷会导致颅面畸形,这是最常见的先天性先天缺陷之一。转基因小鼠是研究颅面形态形成和发病机制的有用工具,其作用是1、2、3、4、5。了解颅面结构在发育和发病过程中的变化将有助于澄清关键的发育原理以及颅面畸形的机制1,2,3 ,4,5.
用特定抗体染色整个安装或切片组织是确定感兴趣的蛋白质的空间分布的宝贵技术。从形式上讲,组织免疫染色可以依赖于免疫组织化学 (IHC) 或免疫荧光 (IF)。与IHC用3,3′-Diaminobenzidine(DAB)等致色基质产生的不透明反应产物相比,IF涉及使用荧光显微镜可见的荧光结合物。因此,IF 可以清楚地区分正细胞和背景噪声,并允许通过 ImageJ 和 Adobe Photoshop7、8等软件以简单的方式对图像进行定量分析和增强。整个安装染色方法适用于小块组织(厚度小于 5 mm),可提供蛋白质/抗原位置的三维信息,而无需从第9节、第 10 节开始重建.然而,与组织部分相比,整个安装免疫染色是耗时的,需要大量的抗体溶液。并非所有抗体都与基本的整体安装方法兼容。此外,抗体的不完全渗透将导致不均匀的染色或错误的阴性染色。在这里,我们将重点介绍切片组织上蛋白质/抗原的免疫荧光检测。对于硬组织(如头部、牙齿、长骨),钙在发育/发病过程中沉积使样品难以分段,在免疫染色治疗期间容易冲洗掉11、12。目前可用的大多数协议在嵌入之前对硬组织进行标记,使切片更容易,这非常耗时,如果处理不当,可以破坏样品的形态和抗原。为了克服这些问题,我们优化了一种在不去钙化的情况下冷冻硬组织的方法,从而改进了信号蛋白的形态和分布的可视化。
此处描述的方案用于确定BMP转基因小鼠颅面组织的形态学和组织学变化。具体来说,该方案包括(1)收获和解剖头组织,(2)实验标记的截面和免疫染色(Ki67,pSmad1/5/9)以及TUNEL染色,(3)使用荧光显微镜成像部分,最后(4)分析和量化结果。准备和冷冻硬组织没有去钙化的协议也描述了13。这些方法针对颅面组织进行了优化。它们也适用于不同年龄的样品的其他组织,并作适当的修改。
Protocol
Representative Results
Discussion
在这里,我们提供了一个详细的协议,用于制备小鼠头和未去切骨组织,以及用于细胞增殖、细胞死亡和BMP信号标记的免疫染色的冷冻切片。我们还详细介绍了从免疫荧光图像获取定量数据的策略。这些方法也适用于具有适当修改的其他组织。
组织制备的条件因组织的大小和类型而异。固定和冷冻保护时间通常需要几个小时到一夜之间。固定后,组织也可以嵌入石蜡和切片与微托<…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了国家卫生研究院(R01DE020843至Y.M.)、国际FOP协会(Y.M.)和中国国家自然科学基金资助(31500788至J.Y.)的支持。
Materials
| Adhesive tape | Leica | #39475214 | |
| Alexa fluor 488-goat anti-Rabbit secondary antibody | Invitrogen | A-11034 | |
| Antifade Mountant with DAPI | Invitrogen | P36931 | |
| Bovine serum albumin | Sigma | A2153 | |
| Coverslips | Fisher Brand | 12-545-E | |
| Cryostat | Leica | CM1850 | |
| EDTA | Sigma | E6758 | |
| Fluorescence microscope | Olympus | BX51 | |
| Gelatin | Sigma | G1890 | |
| In Situ Cell Death Detection Kit | Millipore | S7165 | |
| Microscope slides | Fisher Brand | 12-550-15 | |
| OCT Compound | Fisher Healthcare | 23-730-571 | |
| Paraformaldehyde (PFA) | Sigma | P6148 | |
| Phosphate buffered saline (PBS) | Sigma | P4417 | |
| Polyethylene glycol tert-octylphenyl ether | Sigma | T9284 | Triton X-100 |
| Proteinase K | Invitrogen | AM2542 | |
| Rabbit anti-Ki67 antibody | Cell Signaling Technology | 9129 | Lot#:3; RRID:AB_2687446 |
| Rabbit anti-pSmad1/5/9 antibody | Cell Signaling Technology | 13820 | Lot#:3; RRID:AB_2493181 |
| Sodium citrate | Sigma | 1613859 | |
| Sucrose | Sigma | S9378 | |
| Tris | Sigma | 10708976001 |
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