멀티 플렉스 면역 조직 화학적 염색 및 멀티 스펙트럼 영상을 사용하여 단백질 발현 및 공동 현지화의 정량
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
면역 조직 화학 (IHC)는 조직 내 단백질의 검출을위한 표준 실험실 기술이며, IHC 여전히 널리 연구 및 진단 병리학 모두에 사용된다. IHC 염색의 평가 결과의 해석에 잠재적 인 편견을 도입, 자주 반 정량적이다. 많은 반 정량적 인 접근 방식은 최종 진단 1-4로 염색 강도 및 염색 정도를 모두 통합하는 개발되었다. 다른 시스템은 더 나은 식 (5)를 국산화하기 위해 채점 강도와 세포 내 위치를 포함한다. 다수 시청자의 평균 점수의 혼입은 종종 단일 뷰어 바이어스 (6)의 효과를 최소화하기 위해 이용된다. 이러한 노력에도 불구하고, 분석 주관 여전히 7 오염의 정도를 평가하는 특히, 남아있다. 인간의 입력에서 프로토콜 표준화 및 최소화 주관 정확하고 재현성 IHC 결과를 만드는 중요합니다.
콘텐츠 "> 조직 내 단백질 발현을 결정하기위한 IHC 이외에 다른 옵션이있다. 이러한 면역과 같은 다른 기술은 단백질 발현을 조사하는 금 표준으로 간주되는 동안 연구 설정 내 면역 전통적 단백질 지역화 8을 조사하는 수단으로 간주되어 . 결정 조직 또는 세포 구획 별 발현 조직 슬라이드 형광 항체의 사용은 합리적인 타협을 제공한다. 이러한 세포 분획 또는 레이저 캡처 미세 절제 9,10와 같은 고급 기법을 도입하지 않고 어렵지만 의한 NADPH에 배경 형광도, lipofuscins, 망상 섬유, 콜라겐과 엘라스틴은 11 정확한 정량 어렵게 만들 수 있습니다.자동 계산 병리 플랫폼은 병리 염색 12-15의보다 객관적인 정량을위한 유망 방향입니다. 조직 마이크로 어레이와 멀티 스펙트럼 영상을 결합큰 샘플 크기에서 단백질 발현의 높은 처리량 분석을 용이하게한다. 범주 형 포맷 (16)보다 연속적으로 데이터를 리턴하는 동안 실질적으로 고유 편향 및 분석에 필요한 시간의 양을 줄이면서 이러한 기술로 단백질 공동 지역화 염색 얼룩이, 조직 및 세포 내 편재 분석이 가능하다. 따라서, 본 연구의 목적은 멀티 스펙트럼 영상 소프트웨어를 사용하여 분석 면역 다중화를 수행하는 유틸리티의 방법을 설명 하였다.
이 프로토콜은 네 개의 최적화 된 단일 클론 항체와 단일 조직 섹션 슬라이드 설명서, 멀티 플렉스 면역 조직 화학 염색을 위해 작성되었습니다. 대표 실험으로서, 핵 항 – 토끼 에스트로겐 수용체 알파 (ERα) 및 안드로겐 수용체 (AR)은 막 – 결합 항 – 마우스 CD147 및 막 – 결합 항 – 마우스 E-cadherin과 함께 다중화된다. 선택의 항체는 SUBSTITUT 할 수있다여기에 나와있는 항체 에드하지만, 항체의 각 조합은 별도의 최적화가 필요합니다. 모든 항체에 대한 사전 처리는 동일해야합니다. 아칸소와 CD147 항체 칵테일로 개별적으로하고 최적화해야합니다. 각 항체는 비오틴없는 중합체 시스템 및 4 고유 발색제 중 하나를 사용하여 검출된다.
Protocol
Representative Results
Discussion
단백질 발현을 평가하기위한 기존의 면역 조직 화학의 사용은 분석 22,23 주관적 반 정량 방법에 의해 제한된다. 사전 플랫폼은 바이오 마커의 발현 및 지역화의 높은 처리량 분석을 위해 만들어졌습니다. 조직과 세포 내 구획 모두의 상세한 분할은 사용자가 관심의 다른 마커와 바이오 마커의 발현, 현지화 및 공동 현지화를 연구 할 수 있습니다. 이전의 연구에서, 우리는 같은 세포 구획 <…
Disclosures
The authors have nothing to disclose.
Acknowledgements
저자는 시설과 서비스 사용을 위해, 병리 및 진단 검사 의학의 UW 부에 의해 지원되는 부분 UWCCC 부여 P30의 CA014520에, 병리 실험실에서 위스콘신 중개 연구 이니셔티브의 대학 감사합니다.
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 |
References
- 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).
