Immunofluorescerende mærkning i næseslimhindevævssektioner af allergiske rhinitisrotter via flerfarvet immunoassay
Summary
Denne protokol beskriver en flerfarvet immunofluorescensteknik til evaluering af rottemodellen for allergisk rhinitis.
Abstract
Allergisk rhinitis (AR) er en kronisk, ikke-infektiøs inflammatorisk sygdom i næseslimhinden, primært medieret af specifikt immunglobulin E (IgE), der påvirker ca. 10% -20% af verdens befolkning. Mens immunfluorescens (IF) farvning længe har været en standardteknik til påvisning af sygdomsspecifik proteinekspression, er konventionelle IF-teknikker begrænsede i deres evne til at detektere ekspressionsniveauerne for tre eller flere proteiner i den samme prøve. Derfor er flerfarvede IF-teknikker blevet udviklet i de senere år, som muliggør samtidig mærkning af flere mål i celler eller væv.
Denne protokol giver et omfattende overblik over processen til etablering af en rottemodel af AR, opnåelse af næseslimhindeprøver og de tekniske procedurer for flerfarvet immunofluorescens. Alle rotter i AR-gruppen udviste typiske symptomer som nysen, løbende næse og kløende næse, hvor adfærdsmæssige observationer scorede ≥5 point. Hæmatoxylin og eosin (H&E) farvning afslørede øgede inflammatoriske celletal og forstyrret nasal slimhindeintegritet i AR-gruppen. Flerfarvet immunfluorescens (mIF) viste øget ekspression af RORγt og TICAM-1, mens Foxp3-ekspression faldt i næseslimhindevævet hos AR-rotter.
Introduction
Allergisk rhinitis (AR) er en kronisk, ikke-infektiøs inflammatorisk sygdom i næseslimhinden, primært medieret af specifikt immunglobulin E (IgE)1,2. Det er kendetegnet ved symptomer som nysen, løbende næse, næsestop og nasal kløe. Med industrialisering og urbanisering øges forekomsten af AR gradvist og påvirker ca. 10% -20% af verdens befolkning1. Immunofluorescensteknikken (IF) er en fluorescerende farvningsmetode, der anvender en antistof-antigenbindingsreaktion. Det kan anvendes til at detektere og kvantificere fordelingen og ekspressionsniveauerne af specifikke proteiner i biologiske væv eller celler. I AR-forskning kan IF samtidig detektere flere mål, herunder AR-relaterede cytokiner, inflammatoriske celler, receptorer og mere, hvilket letter udforskningen af AR-patogenese og virkningerne af lægemidler 3,4,5,6.
Den flerfarvede immunfluorescens (mIF) farvningsproces ligner meget traditionel IF med tilsætning af et antistofelueringstrin under hver farvningsrunde. Denne ændring muliggør samtidig påvisning af flere biomarkører på samme vævssektion gennem sekventiel enkeltmærkning og flere runder af genfarvning. mIF er baseret på tyramidsignalforstærkning (TSA), hvilket muliggør gentagne cyklusser med TSA-fluorescerende farvning og brug af mikrobølgeopvarmning til at fjerne antistoffer og samtidig bevare fluorescerende signaler 7,8. Sammenlignet med konventionel IF tilbyder mIF flere fordele: (1) det kan detektere svagt udtrykte antigener, der er udfordrende at identificere med konventionel IF 9,10; (2) det giver farvning af høj kvalitet med et forbedret signal-støj-forhold; 3) det gør det muligt at kvantificere vævsspecifikke strukturer og interesseområder11 (4) multiplexing af flere veje udnytter effektivt væv og bevarer begrænsede patologiske ressourcer12; (5) Multiparameteranalyse gennem mIF giver dybere indsigt i væv og afdækker skjult biologisk information13.
Samlet set tillader mIF observation af forskellige antigenekspressioner og distributioner inden for samme prøve, hvilket letter undersøgelsen af målproteiner. I fremtiden vil forskere, der søger at forstå ekspressionen og fordelingen af flere målproteiner, finde denne teknik et værdifuldt valg. Denne undersøgelse demonstrerer anvendelsen af mIF til farvning af næseslimhindeprøver fra rotter med AR og evaluerer etableringen af en rottemodel af AR.
Protocol
Representative Results
Discussion
Allergisk rhinitis (AR) er en ikke-infektiøs inflammatorisk sygdom i næseslimhinden som følge af en kombination af miljømæssige og genetiske faktorer. Det er blevet et globalt sundhedsproblem, der påvirker arbejdseffektiviteten, mindsker livskvaliteten, forringer søvn, kognitiv funktion og forårsager irritabilitet og træthed. AR påvirker 10-20 % af verdens befolkning¹ og medfører betydelige økonomiske omkostninger og forårsager årlige tab på op til 30-50 milliarder euro i EU-landene18</s…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Dette arbejde blev støttet af Sichuan Provincial Department of Science and Technology (2021YJ0175).
Materials
| Al(OH)3 | Sollerbauer Biotechnology Co., Ltd | A7130 | |
| 75% ethanol | Anhui Yiren An Co., Ltd | 20210107 | |
| Ammonia | Chengdu Kolon Chemical Co., Ltd | 2021070101 | |
| Anhydrous ethanol | Chengdu Kolon Chemical Co., Ltd | 2022070501 | |
| Anti-fluorescence quenching sealer | SouthernBiotech | 0100-01 | |
| Automatic dyeing machine | Thermo scientific | Varistain Gemini ES | |
| Carrier slides | Nantong Mei Wei De Experimental Equipment Co., Ltd | 220518001 | |
| Citrate-phosphate buffer | Servicebio biotechnology co., Ltd | G1201 | |
| Citric acid antigen repair solution (PH 6.0) | Xavier Biotechnology Co., Ltd | G1201 | |
| Coverslip | Nantong Mei Wei De Experimental Equipment Co. | 220518001 | |
| Coverslip | Nantong Mewtech Life Science Co., Ltd | CS01-2450 | |
| CY3-Tyramide | Sawell Biotechnology Co., Ltd | G1223-50UL | |
| DAPI | Sawell Biotechnology Co., Ltd | G1012 | |
| Decoloring shaker | SCILOGEX | S1010E | |
| EDTA decalcification solution | Wuhan Xavier Biotechnology Co., Ltd | CR2203047 | |
| Electric heating blast dryer | Shanghai Yiheng Scientific Instruments Co., Ltd | DHG-9240A | |
| Embedding box marking machine | Thermo scientific | PrintMate AS | |
| Embedding machine | Wuhan Junjie Electronics Co., Ltd | JB-P5 | |
| Fast tissue dewatering machine | Thermo scientific | STP420 ES | |
| Film sealer | Thermo scientific | Autostainer 360 | |
| FITC-Tyramide | Sawell Biotechnology Co., Ltd | G1222-50UL | |
| Fluorescence microscope | Sunny Optical Technology Co.Ltd | CX40 | |
| Foxp3 | Affinity Biosciences Co., Ltd | bs-10211R | |
| Freezing table | Wuhan Junjie Electronics Co., Ltd | JB-L5 | |
| Goat Anti-Rabbit IgG H&L (HRP) | Liankebio Co., Ltd | GAR0072 | |
| Goat serum | Biosharp | BL210A | |
| H&E staining kit | Leagene | DH0020 | |
| Hemostatic forceps | Shanghai Medical Devices Co., Ltd | J31010 | |
| Hydrochloric acid | Sichuan Xilong Science Co., Ltd | 210608 | |
| Immunohistochemical pen | Biosharp | BC004 | |
| Microwave oven | Midea | M1-L213B | |
| Neutral gum | Sinopharm Group Chemical Reagent Co., Ltd | 10004160 | |
| Ovalbumin | Sollerbauer Biotechnology Co., Ltd | A804010 | |
| Oven | Shanghai Yiheng Scientific Instruments Co., Ltd | DHG-9240A | |
| Palm centrifuge | SCILOGEX | D1008E | |
| Paraformaldehyde | Beyotime Biotechnology Co., Ltd | P0099-100ml | |
| Pathology section scanner | 3DHISTECH Kft | Pannoramic SCAN | |
| PBS buffer | Biosharp | G4202 | |
| Pipette | Dragon | KE0003087/KA0056573 | |
| Rorγt | Affinity Biosciences Co., Ltd | DF3196 | |
| Scalpel | Quanzhou Excellence Medical Co., Ltd | 20170022 | |
| Self-fluorescent quenching agent Sudan Black B | Bioengineering Co., Ltd | A602008-0025 | |
| Slicer | Thermo scientific | HM325 | |
| Slicing machine | Thermo scientific | HM325 | |
| Slide | Nantong Mewtech Life Science Co., Ltd | PC2-301 | |
| Sprague Dawley rats | Sichuan Academy of Traditional Chinese Medicine | SYX  2023-0100 |
|
| TICAM-1 | Affinity Biosciences Co., Ltd | DF6289 | |
| Tissue scissors | Shanghai Medical Devices Co., Ltd | J22120 | |
| Tissue spreading baking sheet machine | Wuhan Junjie Electronics Co., Ltd | JK-6 | |
| TYR-690 fluorescent dyes | Shanghai Rutron Biotechnology Co., Ltd | RC0086-34RM | |
| Vortex mixer | SCILOGEX | SLK-O3000-S | |
| Water bath-slide drier | Wuhan Junjie Electronics Co., Ltd | JK-6 | |
| Wax trimmer | Wuhan Junjie Electronics Co., Ltd | JXL-818 | |
| Xylene | Chengdu Kolon Chemical Co., Ltd | 2022051901 |
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