细胞系定量免疫印迹作为常规组织样本中生物标志物蛋白定量免疫荧光定量的标准
Summary
我们描述了使用定量免疫印迹来验证免疫荧光组织学结合图像分析作为一种手段, 定量的蛋白质感兴趣的福尔马林固定, 石蜡嵌入 (ffpe) 组织样本。我们的研究结果证明了免疫荧光组织学在确定常规活检样本中生物标志物蛋白的相对数量方面的作用。
Abstract
福尔马林固定、石蜡包埋 (ffpe) 组织样品中感兴趣的蛋白质的定量在临床和研究应用中具有重要意义。定量的最佳方法是准确的, 具有广泛的线性动态范围, 并保持样品的结构完整性, 以便识别单个细胞类型。目前的方法, 如免疫组织化学 (ihc), 质谱和免疫印迹每个不符合这些规定, 由于其分类性质或需要使样品同质化。作为另一种方法, 我们建议使用免疫荧光 (if) 和图像分析来确定 ffpe 组织中感兴趣的蛋白质的相对丰度。在此基础上, 我们证明了该方法易于优化, 产生了广泛的动态范围, 并与定量免疫印迹的黄金标准相比是线性可量化的。此外, 这种方法允许保持样品的结构完整性, 并允许区分各种细胞类型, 这在诊断应用中可能是至关重要的。总体而言, 这是一种用于 ffpe 样品中蛋白质相对定量的可靠方法, 可轻松适应临床或研究需求。
Introduction
在许多临床领域都需要量化福尔马林固定、石蜡包埋 (ffpe) 组织活检样本中的蛋白质。例如, 使用常规活检标本中生物标志物蛋白的定量来阐明预后, 并为癌症患者的治疗提供信息1。然而, 目前的方法通常是主观的, 缺乏验证。
免疫组织化学 (ihc) 通常用于病理实验室, 通常依赖于针对目标蛋白的初级抗体和与酶标签 (如辣根过氧化物酶 2) 结合的二级抗体.常规 ihc 是敏感的, 可以利用微小样本, 并保持组织样本的形态完整性, 从而允许评估其相关的组织学背景下的蛋白质表达。然而, 由于 ihc 产生的显色信号是减法的, 它的动态范围相对狭窄, 多路复用 2,3,4的潜力有限。矩阵辅助激光去光/电离质谱成像 (aldi-msi) 保留了形态完整性。然而, 这种开发的技术与适度的形态分辨率相关, 需要进行大量的校准和归一化, 从而损害了其常规临床使用的可行性 5, 6,7.在组织样本中量化蛋白质的替代技术包括免疫印迹8、质谱法 9、10、11和酶联免疫吸附法 (elisa)12, 每个其中开始于样品组织的均质裂解物。主要组织样本是异质性的, 因为它们包含多种细胞类型。因此, 需要对样品进行同质化的技术不允许对特定细胞群 (如癌细胞) 中的蛋白质进行定量。
与 ihc 一样, if 适用于小型 ffpe 样品, 并允许保留组织学完整性13。然而, 由于荧光信号的加性, if 适用于多种原生抗体和荧光标签的应用。因此, 感兴趣的蛋白质可以在使用其他抗体定义的特定细胞或细胞隔间 (例如, 细胞核与细胞质) 中相对量化。荧光信号也具有更大的动态范围13,14的优势。研究了if在ffpe 样品中的优越性、重现性和复用潜力。
在此, 我们描述了使用定量免疫印迹使用已建立的细胞系作为一个黄金标准, 以确定 if 的定量性质, 并结合计算机辅助图像分析, 以确定感兴趣的蛋白质的相对丰度ffpe 组织样本中的组织学切片。我们已经成功地应用了这种方法在多方法量化生物标志物蛋白在临床活检样本16,17,18.
Protocol
使用初级人体组织样本的申请获得了女王大学卫生科学和附属教学医院研究伦理委员会 (hsreb) 的批准。 1. 建立细胞线组织微阵列 (tma) 收获和清洗细胞。注: 该议定书已在各种已建立的永生细胞系 (例如hela、jurkat、rch-acv) 上进行了测试。 对于粘附细胞, 一旦细胞达到约 80%的融合, 就会收获大约 1.3 x 10 7 细胞。使用适合该细胞系的试剂分离细胞。<b…
Representative Results
该方案用于确定 if 在构建 ffpe 组织块的细胞系中确定抗凋亡蛋白 bcl-2 的相对数量的能力。在癌细胞中选择性地量化 bcl-2 可以阐明致癌机制, 并可用于病理诊断和为临床管理决策提供信息 24.更具体地说, bcl-2 在适当的 b 淋巴细胞发育中起着重要作用, 其表达通常在淋巴瘤25、26、27的背?…
Discussion
我们已经描述了一种方法, 利用定量免疫印迹 (ib) 来证明免疫荧光 (if) 的作用, 以确定在 ffpe 组织样本中的目标蛋白的相对丰度。目前的蛋白质定量方法受到其分类性质的限制, 如显色性 ihc2,3, 或需要对样品进行同质化, 防止对样本结构和细胞群的调查, 如与 ib 和质谱法8,9,10,<s…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作的部分资金来自弗雷德里克·班廷和查尔斯最佳加拿大研究生奖学金 (上午)。
Materials
| 697 | DSMZ | ACC 42 | Cell line |
| JeKo-1 | ATCC | CRL-3006 | Cell line |
| Jurkat | ATCC | TIB-152 | Cell line |
| RCH-ACV | DSMZ | ACC 548 | Cell line |
| Granta-519 | DSMZ | ACC 342 | Cell line |
| REH | DSMZ | ACC 22 | Cell line |
| Raji | ATCC | CCL-86 | Cell line |
| HeLa | ATCC | CCL-2 | Cell line |
| Trypsin/EDTA solution | Invitrogen | R001100 | For detaching adhesive cells |
| Fetal bovine serum (FBS) | Wisent Inc. | 81150 | To neutralize trypsin |
| Neutral Buffered Formalin | Protocol | 245-684 | For fixing cell pellets |
| UltraPure low melting point agarose | Invitrogen | 15517-022 | For casting cell pellets |
| Mouse monoclonal anti-human Bcl-2 antibody, clone 124 | Dako (Agilent) | cat#: M088729-2, RRID: 2064429 | To detect Bcl-2 by immunoflourencence and immunoblot (lot#: 00095786 |
| Ventana Discovery XT | Roche | – | For automation of immunofluorescence staining |
| EnVision+ System- HRP labelled polymer (anti-mouse) | Dako (Agilent) | K4000 | For immunofluorescence signal amplification |
| EnVision+ System- HRP labelled polymer (anti-rabbit) | Dako (Agilent) | K4002 | For immunofluorescence signal amplification |
| Cyanine 5 tyramide reagent | Perkin Elmer | NEL745001KT | For immunofluorescence signal amplification |
| Aperio ImageScope | Leica Biosystems | – | To view scanned slides |
| HALO image analysis software | Indica Labs | – | For quantification of immunofluorescence |
| Protease inhibitors (Halt protease inhibitor cocktail, 100X) | Thermo Scientific | 1862209 | To add to RIPA buffer |
| Ethylenediaminetetraacetic acid (EDTA) | BioShop | EDT001 | For RIPA buffer |
| NP-40 | BDH Limited | 56009 | For RIPA buffer |
| Sodium deoxycholate | Sigma-Aldrich | D6750 | For RIPA buffer |
| Glycerol | FisherBiotech | BP229 | For Laemlli buffer |
| Bromophenol blue | BioShop | BRO777 | For Laemlli buffer |
| Dithiothreitol (DTT) | Bio-Rad | 161-0611 | For Laemlli buffer |
| Bovine serum albumin (BSA) | BioShop | ALB001 | For immunoblot washes |
| Protein ladder (Precision Plus Protein Dual Color Standards) | Bio-Rad | 161-0374 | For running protein gel |
| Filter paper (Extra thick blot paper) | Bio-Rad | 1703969 | For blotting transfer |
| Nitrocellulose membrane | Bio-Rad | 162-0115 | For blotting transfer |
| Trans-blot SD semi-dry transfer cell | Bio-Rad | 1703940 | For semi-dry transfer |
| Skim milk powder (Nonfat dry milk) | Cell Signaling Technology | 9999S | For blocking buffer |
| Tween 20 | BioShop | TWN510 | For wash buffer |
| GAPDH rabbit monoclonal antibody | Epitomics | 2251-1 | Primary antibody of control protein (lot#: YE101901C) |
| Goat anti-mouse IgG (HRP conjugated) antibody | abcam | cat#: ab6789, RRID: AB_955439 | Secondary antibody for immunoblot |
| Goat anti-rabbit IgG (HRP conjugated) antibody | abcam | cat#: ab6721, RRID: AB_955447 | Secondary antibody for immunoblot (lot#: GR3192725-5) |
| Clarity Western ECL substrates | Bio-Rad | 1705060 | For immunoblot signal detection |
| Amersham Imager 600 | GE Healthcare Life Sciences | 29083461 | For immunoblot signal detection |
| ImageJ software | Freeware, NIH | – | For densitometry analysis |
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Citer Cet Article
Moore, A. M., Boudreau, L. R., Virk, S., LeBrun, D. P. Quantitative Immunoblotting of Cell Lines as a Standard to Validate Immunofluorescence for Quantifying Biomarker Proteins in Routine Tissue Samples. J. Vis. Exp. (143), e58735, doi:10.3791/58735 (2019).