Kvantificering af Protein Expression og Co-lokalisering Brug Multiplexed immunhistokemisk farvede og Multispektral Imaging
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
Immunhistokemi (IHC) er en standard lab teknik til påvisning af protein i væv, og IHC stadig meget udbredt i både forskning og diagnostisk patologi. Evalueringen af IHC farvning ofte semi-kvantitativ, indførelse potentiel skævhed i fortolkning af resultaterne. Der er udviklet mange semi-kvantitative metoder, der inkorporerer både farvning intensitet og farvning udstrækning til endelige diagnose 1-4. Andre systemer omfatter scorings intensitet og subcellulære lokalisering for bedre at lokalisere ekspressionen 5. Inkorporering af gennemsnitlige scores fra flere seere er ofte udnyttes for at minimere virkningerne af enkelt seer partiskhed 6. På trods af disse bestræbelser, subjektivitet i analysen stadig, især når en vurdering af omfanget af farvning 7. Protokol standardisering og minimere subjektivitet fra humant input er altafgørende for at skabe præcise, reproducerbare IHC resultater.
indhold "> Der er andre indstillinger udover IHC til bestemmelse protein ekspression i væv. Inden indstillingen forskning har immunhistokemi traditionelt blevet set som et middel til at undersøge proteinlokalisering 8, mens andre teknikker, såsom immunoblotting betragtes som guldstandard for at undersøge protein-ekspression . Bestemmelse væv eller celler rum-specifikke udtryk er svært uden at inkorporere avancerede teknikker såsom celle fraktionering eller laser capture mikrodissektion 9,10. brugen af fluorescerende antistoffer på væv slides tilbyder et rimeligt kompromis, men baggrunden autofluorescens grundet NADPH, lipofuscins, retikulære fibre, kollagen og elastin kan gøre nøjagtig kvantificering vanskelig 11.Automatiseret beregningsmæssige patologi platforme er en lovende retning for mere objektiv kvantificering af patologi farvning 12-15. Kombinere Multispektralbilledteknik med væv microarraysletter high-throughput analyse af protein-ekspression i store stikprøvestørrelser. Med disse teknikker, analyse af protein co-lokalisering, farvning heterogenitet, og væv og subcellulære lokalisering er muligt, mens væsentligt reducere både iboende fordomme og nødvendige tid til analyse, mens returnering af data i en kontinuerlig snarere end kategorisk format 16. Derfor er formålet med denne undersøgelse var at demonstrere anvendeligheden af og metode til at udføre multiplex immunhistokemi med analyse, ved hjælp af multispektral imaging software.
Denne protokol er skrevet til manuel, multiplex immunhistokemisk farvning af en enkelt vævssnit slide med fire optimerede monoklonale antistoffer. Som repræsentativt forsøg, er nukleare anti-kanin østrogenreceptor alfa (ERa) og androgen receptor (AR) multiplekset med membranbundet anti-muse CD147 og membranbundet anti-muse E-cadherin. Enhver antistof valg kan substituted for antistofferne anført heri, men hver kombination af antistoffer kræver separat optimering. Forbehandling for alle antistofferne skal være identiske. De AR og CD147-antistoffer bør optimeres individuelt og derefter som en cocktail. Hvert antistof påvises ved anvendelse af et biotin-fri polymer system og et af 4 unikke chromogener.
Protocol
Representative Results
Discussion
Anvendelsen af traditionelle immunhistokemi til evaluering proteinekspression er begrænset af subjektive, semikvantitative analysemetoder 22,23. Advance platforme er skabt til high-throughput analyse af biomarkør udtryk og lokalisering. Detaljeret segmentering af både væv og subcellulære rum giver brugerne mulighed for at studere biomarkør udtryk, lokalisering, og co-lokalisering med andre markører af interesse. I tidligere undersøgelser har vi vist anvendeligheden af IHC og Multispektralbilledteknik,…
Divulgations
The authors have nothing to disclose.
Acknowledgements
Forfatterne takker University of Wisconsin Implementeringsforskning initiativer i Patologi laboratorium, delvist støttet af UW Patologisk Institut og Laboratoriet Medicin og UWCCC tilskud P30 CA014520, til brug af sine faciliteter og services.
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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