多重化された免疫組織化学染色およびマルチスペクトルイメージングを用いたタンパク質発現と共局在の定量
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
免疫組織化学（IHC）は、組織内のタンパク質の検出のための標準的な実験室の技術であり、IHCは、まだ広く研究および診断病理学の両方で使用されています。 IHC染色の評価は、結果の解釈に潜在的なバイアスを導入、多くの場合、半定量的です。多くの半定量的アプローチは、最終的な診断1-4に染色強度および染色程度の両方を組み込んだ開発されています。他のシステムは、より良い表現5をローカライズするために、スコアリング強度と細胞内局在を含みます。複数の視聴者からの平均スコアの組み込みは、多くの場合、単一のビューアバイアス6の影響を最小限にするために利用されます。 7染色の程度を評価する際に、これらの努力にもかかわらず、分析中の主観性はまだ特に、残っています。プロトコルの標準化と人間の入力から主観性を最小限に抑えるには、正確で再現性のIHCの結果を作成するための最も重要です。
コンテンツ">組織内のタンパク質の発現を決定するためのIHC以外の他のオプションがあります。そのような免疫ブロット法などの他の技術は、タンパク質の発現を調査するためのゴールドスタンダードとして見ている間に研究環境の中で、免疫組織化学は、伝統的に、タンパク質の局在8を検討するための手段と見られています。組織または細胞コンパートメント特異的な発現を決定することは、このような細胞分画またはレーザーキャプチャーマイクロダイセクション9,10などの高度な技術を組み込むことなく、困難である。組織スライド上の蛍光抗体の使用は、合理的な妥協点を提供していますが、NADPHによるバックグラウンド自己蛍光、リポフスチン、網状繊維、コラーゲン、およびエラスチンは11正確な定量が困難になることができます。自動計算病理プラットフォームは病理染色12-15のより客観的な定量化のための有望な方向です。組織マイクロアレイを用いてマルチスペクトルイメージングを組み合わせます大きなサンプルサイズのタンパク質発現のハイスループット分析を容易にします。カテゴリ形式16ではなく、連続してデータを戻しながら、実質的に、固有のバイアスおよび分析のために必要な時間の両方を低減しながら、これらの技術を用いて、タンパク質の共局在化、染色不均一性、ならびに組織および細胞内局在の解析が可能です。したがって、この研究の目的は、マルチスペクトル画像化ソフトウェアを使用して、分析を多重免疫組織化学を実施するための有用性と方法論を実証することでした。
このプロトコルは、4つの最適化されたモノクローナル抗体を用いた単一の組織切片スライドのマニュアル、多重免疫組織化学染色のために書かれています。代表的な実験として、核抗ウサギエストロゲン受容体α（ERα）およびアンドロゲン受容体（AR）は、膜に結合した抗マウスCD147および膜に結合した抗マウスE-カドヘリンと多重化されています。選択した任意の抗体がsubstitutすることができます本明細書に記載された抗体について編が、抗体の各組合せは、別々の最適化を必要とします。すべての抗体のための前処理は同じでなければなりません。 ARとCD147抗体は、カクテルとして個別に、その後、最適化されなければなりません。各抗体は、ビオチンを含まないポリマー系と4ユニークな色原体のいずれかを使用して検出されます。
Protocol
Representative Results
Discussion
タンパク質の発現を評価するための伝統的な免疫組織化学の使用は、分析22,23の主観的、半定量的な方法によって制限されます。アドバンス・プラットフォームは、バイオマーカーの発現と局在のハイスループット分析のために作成されています。組織および細胞内区画の両方の詳細なセグメンテーションは、ユーザーが関心のある他のマーカーとバイオマーカーの発現、局在化、お…
Declarações
The authors have nothing to disclose.
Acknowledgements
著者は、その設備とサービスの利用のために、病理検査医学のUW省によってサポートされている部分とUWCCCグラントP30のCA014520で、病理検査室にウィスコンシントランスレーショナルリサーチの取り組みの大学に感謝します。
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 |
Referências
- Valdman, A., et al. Expression of redox pathway enzymes in human prostatic tissue. Anal Quant Cytol Histol. 31 (6), 367-374 (2009).
- Rimm, D. L., Camp, R. L., Charette, L. A., Olsen, D. A., Provost, E. Amplification of tissue by construction of tissue microarrays. Exp Mol Pathol. 70 (3), 255-264 (2001).
- Jonmarker, S., et al. Expression of PDX-1 in prostate cancer, prostatic intraepithelial neoplasia and benign prostatic tissue. APMIS. 116 (6), 491-498 (2008).
- McCarty, K. S., Miller, L. S., Cox, E. B., Konrath, J., McCarty, K. S. Estrogen receptor analyses. Correlation of biochemical and immunohistochemical methods using monoclonal antireceptor antibodies. Arch Pathol Lab Med. 109 (8), 716-721 (1985).
- Volante, M., et al. Somatostatin receptor type 2A immunohistochemistry in neuroendocrine tumors: a proposal of scoring system correlated with somatostatin receptor scintigraphy. Mod Pathol. 20 (11), 1172-1182 (2007).
- Muris, J. J., et al. Immunohistochemical profiling of caspase signaling pathways predicts clinical response to chemotherapy in primary nodal diffuse large B-cell lymphomas. Blood. 105 (7), 2916-2923 (2005).
- Jaraj, S. J., et al. Intra- and interobserver reproducibility of interpretation of immunohistochemical stains of prostate cancer. Virchows Arch. 455 (4), 375-381 (2009).
- Nakane, P. K., Pierce, G. B. Enzyme-labeled antibodies: preparation and application for the localization of antigens. J Histochem Cytochem. 14 (12), 929-931 (1966).
- Peters, T. J. Investigation of tissue organelles by a combination of analytical subcellular fractionation and enzymic microanalysis: a new approach to pathology. J Clin Pathol. 34 (1), 1-12 (1981).
- Emmert-Buck, M. R., et al. Laser Capture Microdissection. Science. 274 (5289), 998-1001 (1996).
- Waters, J. C. Accuracy and precision in quantitative fluorescence microscopy. J Cell Biol. 185 (7), 1135-1148 (2009).
- Huang, W., Hennrick, K., Drew, S. A colorful future of quantitative pathology: validation of Vectra technology using chromogenic multiplexed immunohistochemistry and prostate tissue microarrays. Hum Pathol. 44 (1), 29-38 (2013).
- Rimm, D. L. C-Path: A Watson-Like Visit to the Pathology Lab. Science Translational Medicine. 3 (108), (2011).
- Fiore, C., et al. Utility of multispectral imaging in automated quantitative scoring of immunohistochemistry. J Clin Pathol. 65 (6), 496-502 (2012).
- Stack, E. C., Wang, C., Roman, K. A., Hoyt, C. C. Multiplexed immunohistochemistry, imaging, and quantitation: A review, with an assessment of Tyramide signal amplification, multispectral imaging and multiplex analysis. Methods. 70 (1), 46-58 (2014).
- Rizzardi, A. E., et al. Quantitative comparison of immunohistochemical staining measured by digital image analysis versus pathologist visual scoring. Diagn Pathol. 7, 42 (2012).
- Bauman, T. M., et al. Characterization of fibrillar collagens and extracellular matrix of glandular benign prostatic hyperplasia nodules. PLoS One. 9 (10), e109102 (2014).
- Bauman, T. M., et al. Beta-catenin is elevated in human benign prostatic hyperplasia specimens compared to histologically normal prostate tissue. Am J Clin Exp Urol. 2 (4), 313-322 (2014).
- Bauman, T. M., Ewald, J. A., Huang, W., Ricke, W. A. CD147 expression predicts biochemical recurrence after prostatectomy independent of histologic and pathologic features. BMC Cancer. 15 (1), 549 (2015).
- Bauman, T. M., et al. Finasteride treatment alters tissue specific androgen receptor expression in prostate tissues. Prostate. 74 (9), 923-932 (2014).
- Nicholson, T. M., Sehgal, P. D., Drew, S. A., Huang, W., Ricke, W. A. Sex steroid receptor expression and localization in benign prostatic hyperplasia varies with tissue compartment. Differentiation. 85 (4-5), 140-149 (2013).
- Taylor, C. R., Levenson, R. M. Quantification of immunohistochemistry–issues concerning methods, utility and semiquantitative assessment II. Histopathology. 49 (4), 411-424 (2006).
- Matos, L. L., Trufelli, D. C., de Matos, M. G., da Silva Pinhal, M. A. Immunohistochemistry as an important tool in biomarkers detection and clinical practice. Biomark Insights. 5, 9-20 (2010).
- Kononen, J., et al. Tissue microarrays for high-throughput molecular profiling of tumor specimens. Nat Med. 4 (7), 844-847 (1998).
- Ong, C. W., et al. Computer-assisted pathological immunohistochemistry scoring is more time-effective than conventional scoring, but provides no analytical advantage. Histopathology. 56 (4), 523-529 (2010).
