Die Quantifizierung der Proteinexpression und Co-Lokalisation Mit Multiplexed immunhistochemischen Anfärbung und Multispektraltechnik
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
Immunhistochemie (IHC) ist eine Standardlaborverfahren zum Nachweis von Protein in Gewebe und IHC ist immer noch weit verbreitet in Forschung und Diagnose der Pathologie verwendet. Die Auswertung der IHC-Färbung ist oft semi-quantitative, Potentialvorspannung in die Interpretation der Ergebnisse einzuführen. Viele semi-quantitative Ansätze wurden die 1-4 sowohl Färbeintensität und Färbung Ausmaß in die endgültige Diagnose integrieren entwickelt. Andere Systeme sind Scoring – Intensität und subzellulären Ort, um eine bessere Ausdruck 5 lokalisieren. Der Einbau von Durchschnittswerte von mehreren Zuschauern wird häufig genutzt , um die Auswirkungen der einzelnen Betrachter Bias 6 zu minimieren. Trotz dieser Bemühungen bei der Analyse, Subjektivität noch bleibt, vor allem bei der Bewertung des Ausmaßes der 7 – Färbung. Protokoll Standardisierung und zur Minimierung der Subjektivität von menschlichen Input ist von größter Bedeutung genaue, reproduzierbare IHC Ergebnisse zu schaffen.
Inhalt "> Es gibt andere Optionen neben IHC für die Proteinexpression in Geweben zu bestimmen. Im Rahmen des Forschungs Einstellung hat Immunhistochemie traditionell als Mittel angesehen Proteinlokalisierung 8, während andere Techniken wie Immunoblotting angesehen werden als Goldstandard für die Untersuchung von Protein – Expression zu untersuchen , . Bestimmung Gewebe oder Zellkompartiment spezifische Expression ist schwierig , ohne fortgeschrittene Techniken wie Zell Fraktionierung oder Laser Mikrodissektion 9,10 enthält. Die Verwendung von fluoreszierenden Antikörpern auf Gewebeschnitten einen vernünftigen Kompromiß bietet, aber aufgrund NADPH Hintergrund – Autofluoreszenz, Lipofuscine, retikuläre Fasern, Kollagen und Elastin kann 11 genaue Quantifizierung erschweren.Automatisierte Rechen Pathologie Plattformen sind eine vielversprechende Richtung für mehr objektive Quantifizierung der Pathologie Färbung 12-15. Die Kombination von multispektrale Bild mit Gewebe-Mikroarrayserleichtert Hochdurchsatz-Analyse der Proteinexpression in großen Stichprobengrößen. Mit diesen Techniken Analyse von Protein – Co-Lokalisation, Färbungs Heterogenität und Gewebe und subzelluläre Lokalisierung ist möglich , während im Wesentlichen sowohl inhärent vorspannt und die Zeit , die zur Analyse zu reduzieren, während die Daten in einem kontinuierlichen und nicht kategorische Format 16 zurückkehrt. Daher war das Ziel dieser Studie zur Durchführung einer Multiplex-Immunhistochemie mit Analyse die Nützlichkeit und die Methodik zu veranschaulichen, multispektrale Abbildungssoftware.
Dieses Protokoll wird für die manuelle, multiplex immunhistochemische Färbung eines einzigen Gewebeschnitt Schlitten mit vier optimierte monoklonalen Antikörpern geschrieben. Als ein repräsentatives Experiment werden Kern anti-Kaninchen-Östrogenrezeptor alpha (ER & agr;) und Androgen-Rezeptor (AR) gemultiplext mit membrangebundenen anti-Maus-CD147 und membrangebundenen anti-Maus-E-Cadherin. Jeder Antikörper der Wahl kann Substituted für die aufgeführten Antikörper hier, aber jede Kombination von Antikörpern erfordert getrennte Optimierung. Vorbehandlung für alle Antikörper müssen identisch sein. Die AR und CD147-Antikörper sollten einzeln und dann als Cocktail optimiert werden. Jeder Antikörper erkannt wird ein Biotin freie Polymersystems und eines von 4 einzigartigen Chromogene verwendet wird.
Protocol
Representative Results
Discussion
Die Verwendung von traditionellen Immunhistochemie zur Bewertung der Proteinexpression durch subjektive, semi-quantitative Methoden der Analyse 22,23 begrenzt. Advance-Plattformen sind für Hochdurchsatz-Analyse von Biomarkern Expression und Lokalisation erstellt. Detaillierte Segmentierung der beiden Gewebe und subzellulärer Kompartimente ermöglicht es Benutzern, Biomarker Ausdruck zu studieren, Lokalisierung und Co-Lokalisation mit anderen Markern von Interesse. In früheren Studien haben wir die Brauchba…
Declarações
The authors have nothing to disclose.
Acknowledgements
Die Autoren danken der University of Wisconsin Translational Forschungsinitiativen in der Pathologie Labor, teilweise durch die UW Abteilung für Pathologie und Labormedizin und UWCCC Zuschuss P30 CA014520 unterstützt, für die Benutzung ihrer Einrichtungen und Dienstleistungen.
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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