Kvantifiering av proteinuttryck och samlokalisering Använda Multiplex Immuno-histokemisk färgning och multispektrala
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
Immunohistokemi (IHC) är en standard lab teknik för detektering av protein i vävnad, och IHC fortfarande används i stor utsträckning både forskning och diagnostiska patologi. Utvärderingen av IHC färgning är ofta halvkvantitativ, införa eventuell bias i tolkningen av resultaten. Många halv kvantitativa metoder har utvecklats som innefattar både färgningsintensitet och färgning utsträckning i slutlig diagnos 1-4. Andra system inkluderar poäng intensitet och subcellulär plats för att bättre lokalisera uttryck 5. Inkorporering av den genomsnittliga poängen från flera tittare används ofta för att minimera effekterna av enstaka betraktaren partiskhet 6. Trots dessa ansträngningar, fortfarande subjektivitet i analysen, särskilt vid bedömningen av omfattningen av färgning 7. Protokoll standardisering och minimera subjektivitet från mänsklig ingången är av största vikt att skapa exakta, reproducerbara IHC resultat.
innehåll "> Det finns andra alternativ förutom IHC för att bestämma proteinuttryck i vävnader. Inom forskningsmiljön har immunohistokemi traditionellt setts som ett sätt att undersöka protein lokalisering 8, medan andra tekniker såsom immunoblotting betraktas som gold standard för att undersöka proteinuttryck . Fastställande vävnad eller cellfack specifikt uttryck är svårt utan att införliva avancerade tekniker såsom cellfraktionering eller laser capture microdissection 9,10. användningen av fluorescerande antikroppar på vävnadsglas ger en rimlig kompromiss, men bakgrunden autofluorescens på grund av NADPH, lipofuscins, retikulär fibrer, kollagen och elastin kan göra exakt kvantifiering svårt 11.Automatiserade beräknings patologi plattformar är en lovande riktning för mer objektiv kvantifiering av patologi färgning 12-15. Kombinera multispektrala avbildning med vävnads microarraysunderlättar hög genomströmning analys av proteinuttryck i stora provstorlekar. Med dessa tekniker är möjlig analys av protein samlokalisering, färgning heterogenitet, och vävnad och subcellulär lokalisering medan avsevärt minska både inneboende fördomar och tid som behövs för analys, medan återvänder data i en kontinuerlig snarare än kategoriska format 16. Därför är syftet med denna studie var att visa nyttan av och metod för att utföra multiplex immunohistokemi med analys, med hjälp av multispektral bildbehandlingsprogram.
Detta protokoll är skriven för manuell, multiplex immunhistokemisk färgning av en enda vävnadssnitt glida med fyra optimerade monoklonala antikroppar. Som ett representativt experiment, är kärnantikanin östrogenreceptor-alfa (ERa) och androgenreceptorn (AR) multiplexeras med membranbundna anti-mus-CD147 och membranbundna anti-mus-E-cadherin. Alla antikropp val kan utbytbarheted för antikropparna som anges häri, men varje kombination av antikroppar kräver separat optimering. Pre-behandling för alla antikropparna måste vara identiska. AR och CD147-antikroppar bör optimeras individuellt och sedan som en cocktail. Varje antikropp detekteras med hjälp av en biotin-fri polymersystemet och en av 4 unika kromogener.
Protocol
Representative Results
Discussion
Användning av traditionella immunohistokemi för utvärdering proteinuttryck är begränsat av subjektiva, halv kvantitativa analysmetoder 22,23. Advance plattformar har skapats för hög genomströmning analys av biomarkörer uttryck och lokalisering. Detaljerad segmentering av både vävnad och subcellulära fack tillåter användare att studera biomarkör uttryck, lokalisering, och samlokalisering med andra markörer av intresse. I tidigare studier har vi visat användbarheten av IHC och multispektral avb…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Författarna tackar University of Wisconsin translationell forskning Initiativ i patologi laboratorium, delvis med stöd av UW Institutionen för patologi och laboratoriemedicin och UWCCC bidrag P30 CA014520, för användning av sina anläggningar och tjänster.
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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