La cuantificación de la expresión de la proteína y Co-localización Uso de multiplexado inmunohistoquímica y tinción de imágenes multiespectrales
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
La inmunohistoquímica (IHC) es una técnica de laboratorio estándar para la detección de la proteína en el tejido, y IHC es aún ampliamente utilizado en la investigación y diagnóstico de la patología. La evaluación de la tinción IHC es a menudo semi-cuantitativa, la introducción de sesgo potencial en la interpretación de los resultados. Muchos enfoques semi-cuantitativos se han desarrollado que incorporan tanto intensidad de la tinción y el grado de tinción en diagnóstico final 1-4. Otros sistemas incluyen la intensidad de puntuación y la ubicación subcelular con el fin de localizar mejor expresión 5. La incorporación de las puntuaciones medias de varios espectadores se utiliza a menudo con el fin de minimizar los efectos de un solo espectador sesgo 6. A pesar de estos esfuerzos, la subjetividad en el análisis sigue siendo, sobre todo cuando se evalúa el grado de tinción 7. la estandarización del protocolo y de la subjetividad de minimizar la intervención humana es de suma importancia para la creación de resultados precisos y reproducibles IHC.
contenido "> Hay otras opciones además de IHC para determinar la expresión de la proteína dentro de los tejidos. En el marco de la investigación, la inmunohistoquímica ha sido considerado tradicionalmente como un medio para examinar la localización de proteínas 8, mientras que otras técnicas como la inmunotransferencia son vistos como estándar de oro para la investigación de la expresión de proteínas . La determinación de los tejidos o expresión específica para el compartimento celular es difícil sin la incorporación de técnicas avanzadas tales como el fraccionamiento celular o captura por láser microdisección 9,10. El uso de anticuerpos fluorescentes en portaobjetos de tejidos ofrece un compromiso razonable, pero autofluorescencia de fondo debido a NADPH, la lipofuscina, reticular fibras de colágeno, elastina, y pueden hacer difícil cuantificación precisa 11.Plataformas computacionales de patología automatizados son una dirección prometedora para más cuantificación objetiva de la tinción de la patología 12-15. La combinación de imágenes multiespectrales con microarrays de tejidosfacilita el análisis de alto rendimiento de expresión de proteínas en muestras de gran tamaño. Con estas técnicas, el análisis de la proteína co-localización, la heterogeneidad de la tinción, y el tejido y localización subcelular es posible al tiempo que reduce sustancialmente tanto los sesgos y tiempo necesario para el análisis inherentes, mientras que la devolución de datos en un formato continuo en lugar de categórica 16. Por lo tanto, el propósito de este estudio fue demostrar la utilidad de la metodología y para llevar a cabo la inmunohistoquímica multiplexado con el análisis, utilizando software de imagen multiespectral.
Este protocolo está escrito para manual de tinción, múltiplex inmunohistoquímica de una sola sección de tejido de diapositivas con cuatro anticuerpos monoclonales optimizados. Como un experimento representativo, anti-conejo nuclear alfa del receptor de estrógeno (ER) y receptor de andrógenos (AR) se multiplexan con membrana anti-ratón de CD147 y unida a la membrana anti-ratón de E-cadherina. Cualquier anticuerpo de elección puede ser substituted de los anticuerpos mencionados en el presente documento, pero cada combinación de anticuerpos requiere la optimización separada. Pre-tratamiento para todos los anticuerpos debe ser idéntico. Los anticuerpos AR y CD147 deben optimizarse individualmente y luego como un cóctel. Cada anticuerpo se detecta utilizando un sistema de polímero libre de biotina y una de las 4 cromógenos únicas.
Protocol
Representative Results
Discussion
El uso de inmunohistoquímica tradicional para evaluar la expresión de proteínas está limitada por métodos subjetivos, semi-cuantitativos de análisis 22,23. plataformas de avance se han creado para el análisis de alto rendimiento de expresión de biomarcadores y la localización. la segmentación detallada de ambos tejidos y compartimentos subcelulares permite a los usuarios estudiar la expresión de biomarcadores, localización y co-localización con otros marcadores de interés. En estudios previos, s…
Declarações
The authors have nothing to disclose.
Acknowledgements
Los autores agradecen a la Universidad de Wisconsin Iniciativas de Investigación Traslacional en el laboratorio de patología, en parte apoyado por el Departamento de Patología y Medicina de Laboratorio de la Universidad de Washington y la subvención CA014520 UWCCC P30, para el uso de sus instalaciones y servicios.
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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