蛋白表达和共定位使用复用免疫组织化学染色和多光谱成像定量
Summary
Immunohistochemistry is a powerful lab technique for evaluating protein localization and expression within tissues. Current semi-automated methods for quantitation introduce subjectivity and often create irreproducible results. Herein, we describe methods for multiplexed immunohistochemistry and objective quantitation of protein expression and co-localization using multispectral imaging.
Abstract
Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within tissues. Many semi-quantitative systems have been developed for scoring expression using immunohistochemistry, but inherent subjectivity limits reproducibility and accuracy of results. Furthermore, the investigation of spatially overlapping biomarkers such as nuclear transcription factors is difficult with current immunohistochemistry techniques. We have developed and optimized a system for simultaneous investigation of multiple proteins using high throughput methods of multiplexed immunohistochemistry and multispectral imaging. Multiplexed immunohistochemistry is performed by sequential application of primary antibodies with secondary antibodies conjugated to horseradish peroxidase or alkaline phosphatase. Different chromogens are used to detect each protein of interest. Stained slides are loaded into an automated slide scanner and a protocol is created for automated image acquisition. A spectral library is created by staining a set of slides with a single chromogen on each. A subset of representative stained images are imported into multispectral imaging software and an algorithm for distinguishing tissue type is created by defining tissue compartments on images. Subcellular compartments are segmented by using hematoxylin counterstain and adjusting the intrinsic algorithm. Thresholding is applied to determine positivity and protein co-localization. The final algorithm is then applied to the entire set of tissues. Resulting data allows the user to evaluate protein expression based on tissue type (ex. epithelia vs. stroma) and subcellular compartment (nucleus vs. cytoplasm vs. plasma membrane). Co-localization analysis allows for investigation of double-positive, double-negative, and single-positive cell types. Combining multispectral imaging with multiplexed immunohistochemistry and automated image acquisition is an objective, high-throughput method for investigation of biomarkers within tissues.
Introduction
免疫组织化学（IHC）是组织中检测蛋白质的标准实验室技术，IHC仍广泛用于研究和诊断病理学。 IHC染色的评价往往是半定量，引入潜在的偏见到结果的解释。许多半定量方法已经开发了包含两个染色强度和染色程度成最终诊断1-4。其它系统包括，以更好地定位表达5得分强度和亚细胞定位。从多个观看者平均分数的掺入通常被用于以最小化单个观察者偏倚6的影响。尽管有这些努力，主观性分析仍然存在，评估染色7的程度时尤为如此。协议的标准化和减少主观性的人力投入是极为重要的创造准确，重现IHC结果。
内容“>还有除了IHC其他选项来确定组织中的表达。在研究设置，免疫组化历来被视为检验蛋白定位8的一种手段，而其他技术，如免疫印迹被视为黄金标准调查的表达。确定组织或细胞特异性车厢表达很难不采用先进的技术，如细 胞分馏或激光捕获显微切割9,10。在组织切片中使用荧光抗体提供了一个合理的妥协，但背景自发荧光由于NADPH，lipofuscins，网状纤维，胶原和弹性蛋白可以作出准确的定量困难11。自动计算病理平台是病理染色12-15的更客观的定量一个有前途的方向。结合组织芯片多光谱成像便于在大样本蛋白表达的高通量分析。用这些技术，蛋白的共定位，染色不均匀性，以及组织和亚细胞定位的分析是可能的，同时基本上降低了两个固有偏差和必要的分析时间，同时在连续返回数据，而不是绝对的格式16。因此，本研究的目的是为了展示与分析进行复用免疫组化，采用多光谱成像软件工具和方法。
该协议具有四个优化单克隆抗体单一组织切片上的手册，复用免疫组化染色写的。作为一个代表性实验，核抗兔雌激素受体α（ERα）和雄激素受体（AR）被复用膜结合的抗小鼠CD147和膜结合的抗小鼠E-钙粘蛋白。选择的任何抗体可substitut编为本文所列的抗体，但是抗体的每一个组合都需要单独的最优化。前处理的所有抗体必须相同。 AR和CD147抗体应单独再优化为鸡尾酒。每种抗体是使用无生物素的聚合物系统和4个独特的色原一个检测到。
Protocol
Representative Results
Discussion
使用用于评估蛋白表达的传统免疫组织化学被分析22,23的主观的，半定量法的限制。提前平台已经为生物标志物的表达和定位的高通量分析创建。两个组织和亚细胞区室的详细分割允许用户来研究生物标志物表达，定位，和共定位与其他感兴趣的标记。在以前的研究中，我们已经证明IHC和多光谱成像，用于研究定位于相同的细胞隔室18,20,21的蛋白质尤其是当的效用。当组织芯片结?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
作者感谢威斯康星转化研究倡议的大学病理实验室，由病理及实验室医学系UW支持一部分，UWCCC授予P30 CA014520，使用其设施和服务。
Materials
| Xylene | Fisher Chemical | X3F1GAL | NFPA rating:Health – 2, Fire – 3 , Reactivity-0 |
| Ethyl Alcohol-200 proof | Fisher Scientific | 4355223 | NFPA rating:Health – 0, Fire – 3 , Reactivity-0 |
| Tris Base | Fisher Scientific | BP152-500 | NFPA rating:Health – 2, Fire – 0 , Reactivity-0 |
| Tris Hydroxymethyl aminomethane HCl | Fisher Scientific | BP153-1 | NFPA rating:Health – 2, Fire – 0 , Reactivity-0 |
| Tween 20 | Chem-Impex | 1512 | NFPA rating:Health – 0, Fire –1 , Reactivity-0 |
| Phosphate-buffered saline | Fisher Scientific | BP2944-100 | NFPA rating:Health – 1, Fire –0 , Reactivity-0 |
| Peroxidazed | Biocare Medical | PX968 | Avoid contact with skin and eyes. May cause skin irritation and eye damage. |
| Diva Decloaker | Biocare Medical | DV2004 | This product has been classified as non‐hazardous based on the physical and/or chemical nature and/or concentration of ingredients. |
| Estrogen Receptor alpha | Thermo Fisher Scientific-Labvision | RM9101 | Not classified as hazardous |
| Androgen Receptor | SCBT | sc-816 | Not classified as hazardous |
| CD147 | Biodesign | P87535M | Not classified as hazardous |
| E-cadherin | Dako | M3612 | Not classified as hazardous |
| Renoir Red Andibody Diluent | Biocare Medical | PD904 | It is specially designed to work with Tris-based antibodies |
| DeCloaking Chamber | Biocare Medical | Model DC2002 | Take normal precautions for using a pressure cooker |
| Barrier pen-Immuno Edge | Vector Labs | H-4000 | |
| Denaturing Kit-Elution step | Biocare Medical | DNS001H | Not classified as hazardous |
| Mach 2 Goat anti-Rabbit HRP Polymer | Biocare Medical | RHRP520 | Not classified as hazardous |
| Mach 2 Goat anti-Rabbit AP Polymer | Biocare Medical | RALP525 | Not classified as hazardous |
| Mach 2 Goat anti-Mouse HRP Polymer | Biocare Medical | M3M530 | Not classified as hazardous |
| Betazoid DAB Chromogen Kit | Biocare Medical | BDB2004 | 1. DAB is known to be a suspected carcinogen. 2. Do not expose DAB components to strong light or direct sunlight. 3. Wear appropriate personal protective equipment and clothing. 4. DAB may cause sensitization of skin. Avoid contact with skin and eyes. 5. Observe all federal, state and local environmental regarding disposal |
| Warp Red Chromogen Kit | Biocare Medical | WR806 | Corrosive. Acid that may cause skin irritation or eye damage. |
| Vina Green Chromogen Kit | Biocare Medical | BRR807 | Harmful if swallowed |
| Bajoran Purple Chromogen Kit | Biocare Medical | BJP807 | Flammable liquid. Keep away from heat, flames and sparks. Harmful by ingestion or absorption. Avoid contact with skin or eyes, and avoid inhalation. |
| Cat Hematoxylin | Biocare Medical | CATHE | Purple solution with a mild acetic acid (vinegar) scent. May be irritating to skin or eyes. Avoid contact with skin and eyes. Avoid ingestion. |
| XYL Mounting Media | Richard Allen | 8312-4 | NFPA rating:Health – 2, Fire – 3 , Reactivity-0 |
| 1.5 Coverslips | Fisher Brand | 22266858 | Sharp edges |
| Incubation (Humidity)Chamber | obsolete | obsolete | Multiple vendors available |
| Convection Oven | Stabil- Therm | C-4008-Q | |
| Background Punisher Blocking Reagent | Biocare Medical | BP974 | This product is not classified as hazardous. |
| inForm software | PerkinElmer | CLS135781 | Primary multispectral imaging software used in manuscript |
| Nuance software | PerkinElmer | NUANCEEX | Software used for making spectral libraries within manuscript |
| Vectra microscope and slide scanner | PerkinElmer | VECTRA | Automated slide scanner and microscope for obtaining IM3 image cubes |
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