Immunhistokjemisk analyse i Rat sentralnervesystemet og perifer lymfeknute vevsdelene
Summary
We here present an optimized, detailed protocol for double immunostaining in formalin-fixed, paraffin-embedded rat central nervous system (CNS) and peripheral lymph node (LN) tissue sections.
Abstract
Immunhistokjemi (IHC) gir svært spesifikk, pålitelig og attraktiv protein visualisering. Riktig ytelse og tolkning av en IHC-basert flerfarget merking er utfordrende, spesielt når det brukes for å vurdere sammenhengene mellom target proteiner i vev med høyt fettinnhold som sentralnervesystemet (CNS).
Vår protokoll representerer en videreutvikling av standard immunolabeling teknikk spesielt justert for deteksjon av både strukturelle og løselige proteiner i rotte CNS og perifere lymfeknuter (LN) rammet av nevroinflammasjon. Ikke desto mindre, med eller uten ytterligere modifikasjoner, vår protokoll kan sannsynligvis bli brukt for deteksjon av andre beslektede protein mål, også i andre organer og arter enn her er presentert.
Introduction
Til tross for utnyttelse av avanserte high-throughput analyser utført på methylome, transkriptomet eller proteom- nivå, farging fortsatt gullstandarden for protein deteksjon direkte i vevsprøve, cellekultur eller en celle smøre. Kunnskap om lokalisering / fordelingsmønster, kan immunhistokjemi (IHC) vurdere relative forholdstall og topografiske sammenhengene av target proteiner, og selv indikere deres biologiske aktiviteter. Derfor er IHC mye benyttet for klinisk og forskningsformål, for eksempel, for diagnose, behandling evalueringer, studier av sykdomsmekanismer, funksjonelle og fenotypiske endringer i dyremodeller etc.
I hovedsak omfatter histologi, patologi, biokjemi og immunologi, og IHC betydelig avansert siden 1941, da fluorescensmerkede antistoffer ble benyttet for første gang for å identifisere pneumokokk antigener i infisert vev 1. Visualization av mobilprodukter og komponenter av IHC er basert på binding av antistoffer (ABS) til deres spesifikke antigen (Ag). Dessuten bruker fluoroforen tagget antistoffer, kan immunreaksjoner også visualiseres ved hjelp av enzymer som peroxidase 2,3 eller alkalisk fosfatase 4. Videre er kolloidale gull-merket antistoff 5 som brukes for påvisning av antigen-antistoff-interaksjonen av både lys- og elektronmikroskopi, mens radioaktive markører blir visualisert ved autoradiografi.
Den Ag-Ab-immunoreaksjonsprodukt kan påvises via direkte og indirekte metoder. Den direkte metoden er egentlig raskere og enklere, som den bruker direkte merket primære Abs 6. Men på grunn av betydelig mangel på sensitivitet, blir indirekte metoder å foretrekke til de direkte seg. To-trinns indirekte test prosedyrer krever umerket primær Abs, som den første, og merkede sekundære Abs rettet mot den primære Abs, som det andre lag 7.Signalforsterkning kan oppnås ved å involvere ytterligere, enzym-koblet tertiær Ab (tre-trinns indirekte metode) som binder seg til den sekundære Ab. Vanlig brukte indirekte gjenkjenning metoder er avidin-biotin og peroksidase-antiperoxidase (PAP). Alternativt kan alkalisk fosfatase-antialkaline fosfatase (APAAP) kompleks brukes i stedet for PAP-metoden. Spesielt, alkalisk fosfatase (AP) metoder ser ut til å bli enda mer følsomme enn immunoperoxidase metoder 4. Avidin-biotin kompleks (ABC) en metode som bruker biotinylert sekundært Ab i kombinasjon med enten merket avidin-biotin kompleks (LAB), eller streptavidin-biotin kompleks (plate). Følsomhet kan økes ytterligere ved å involvere avidin merket med peroksidase eller alkalisk fosfatase 8. Andre deteksjonsmetoder i bruk er polymere merking, tyramin forsterkning og immun-rullende sirkel 9. Spesielt, kan forskjellige deteksjonsmetoder kombineres for flere Ag deteksjon i samme vevPrøven som ble rapportert for første gang i 1978 4. Samtidig dobbelt immunofarging presentert her ble utført i formalinfiksert, parafin-innleiret rotte CNS og LN vevssnitt ved hjelp av peroksydase-bundet og AP-konjugerte sekundære antistoffer, henholdsvis. Signalene ble visualisert ved hjelp av 3,3'-diaminobencidine (DAB) kromogen og Fast Blå (FB) APAAP kompleks, henholdsvis.
Protocol
Representative Results
Discussion
Standard IHC prosedyrer ofte krever spesielle tilpasninger for å få et optimalt resultat, som ofte innebærer omfattende erfaring, men også "prøving og feiling" tilnærming. Fra vev fremstilling inntil målet for visualisering, kan nesten hvert trinn i protokollen underkastes individuelt utformet modifikasjoner for å forbedre det endelige utfall. Double flekker protokoll som presenteres her eksemplifiserer IHC-baserte protein targeting spesielt justert for å vurdere sammenhengene mellom målet proteiner …
Disclosures
The authors have nothing to disclose.
Acknowledgements
We thank Hans Lassmann and Jan Bauer for their guidance and support. We also thank Katalin Benedek for excellent technical assistance and Caroline Westerlund for critical and linguistic appraisal.
This study was supported by grants from Biogen Idec, the Wenner-Gren Foundation, the Swedish Research Council, the Swedish Association of Persons with Neurological Disabilities, Swedish Brain Foundation, the EU 6TH Framework EURATools (LSHG-CT-2005-019015) and Neuropromise (LSHM-CT-2005-018637). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Materials
| Reagent | |||
| Isoflurane (Isofluran): 1-Chlor-2,2,2-trifluorethyl (difluormethyl ether) 2-Chlor-2-(difluormethoxy)-1,1,1-trifluorethan (C3H2ClF5O) | Baxter | 1001936060 | Eye irradiation. Probably influences fertility and damages baby in uterus. Administer only in adequately equipped anesthetizing environment. |
| Sodiumchloride (NaCl) | Merck | 1.0604 | |
| Phosphate Buffer Saline (PBS), tablet | Sigma-Aldrich | P4417 | |
| Paraformaldehyde (OH(CH2O)nH (n = 8 – 100), 4% in 1x PBS | Apl pharma | 34 24 28 | Health hazard. Corrosive. Flamable. Acute toxicity. |
| Ethanol ≥99.5%, absolute (CH3CH2OH) | Sigma-Aldrich | 459844-1L | Flamable. |
| Xylene (Xylenes, histological grade; C6H4(CH3)2 | Sigma-Aldrich | 534056 | Flamable. Acute toxicity. |
| Histo-Comp Paraffin Wax | Tissue-Tek | V0-5-1001 | |
| Adhesive Microscope Slides | Starfrost | MIC-1040-W | |
| Xylol substitute XEM-200 | Vogel GmbH | ND-HS-200 | Respiratory sensitization. Carcinogenicity. |
| α- CD8 (Ox-8) mouse anti-rat, primary antibody | AbD Serotec | MCA48G | |
| α- Iba1 (AIF1) mouse anti-rat, primary antibody | Millipore | MABN92 | |
| α- CD68 (ED1) mouse anti-rat, primary antibody | AbD Serotec | MCA341R | |
| α- eotaxin C-19 (CCL11) goat anti-rat, primary antibody | Santa Cruz BT | SC-6181 | |
| Alkaline phosphatase (AP)-conjugated secondary antibody | Dakopatts, Denmark | D0314 | |
| Biotinylated secondary antibody | Amersham Biotech | RPN1025 | |
| Avidin- horseradish peroxidase complex (HRP) | Sigma-Aldrich | A3151 | |
| Naphthol AS-MX phosphate (C19H18NO5P) | Sigma-Aldrich | N4875-1G | Acute toxicity. |
| Fast Blue RR Salt, Azoic Diazo No. 24 (C15H14ClN3O3 x 1/2 ZnCl2) | Sigma-Aldrich | FBS25 | |
| Levamisol hydrochloride (C11H13ClN2S) | Sigma-Aldrich | 31742 | Acute toxicity. |
| 3,3′-Diaminobenzidine tetrahydrochloride (DAB Chromogen) | DAKO | S3000 | Highly flammable. Toxic. |
| Copper sulphate (CuSO4) | Merck | 1.02791 | Acute toxicity. Environmental hazzard. |
| GelTol Aqueous Mounting Medium | Thermo Electron Corporation | 230100 | |
| Hydrogen peroxide, 30% (H2O2) | Merck | 107210 | Acute toxicity. |
| Methanol (CH3OH) | Fluka | 65543 | Acute toxicity. Respiratory sensitization. Carcinogenicity. Flammable. |
| Tris (hydroxymethyl) aminomethane, TRIS base (C4H11NO3 ) | AppliChem | A1379 | Skin and eye irritation. |
| Tris Buffered Saline (TBS), tablet | Sigma-Aldrich | T5030 | |
| Di- Sodium hydrogen phosphate dihydrate (Na2HPO4 x 2H2O) | Merck | 1.0658 | |
| Sodium dihydrogen phosphate monohydrate (NaH2PO4 x 1H2O) | Merck | 1.06346 | |
| Fetal calf serum (FCS) | Cambrex BioScience | DE-14-802F | |
| DAKO cytomation wash buffer 10x | DAKO | S3006 | Should be stored at 2-8 °C to inhibit bacterial growth. Avoid foaming. |
| N,N- Dimethylformamide; DMF (C3H7NO) | Fluka | 40250 | Flammable. Acute toxicity. |
| Glas coverslips 24 x 36 mm | Menzel-Gläser | BB024036A1 | |
| Hydrochloric acid, conc. (HCl) | Sigma-Aldrich | 30721 | Corrosive, irritant, permeator. Lung sensitizer (as acid mist). Toxic. |
| Hydrochloric acid solution volumetric, 2M HCl (2N) | Fluka | 71826 | Corrosive, irritant, permeator. Lung sensitizer (as acid mist). Toxic. |
| Sodium nitrite, ReagentPlus, ≥99.0% (NaNO2) | Sigma-Aldrich | S2252 | Oxidant. Toxic. Dangerous for the environment. |
| Ethylenedinitrilotetraacetic acid disodium salt dihydrate (EDTA; C10H14N2Na2O8 x 2H2O) | Merck | 1.08454 | Oral exposures may cause reproductive and developmental effects. |
| Equipment | |||
| Tissue-Tek V.I.P Vacuum Infiltration Processor | Sakura | 5902 VIP Jr. 115 V, 60 Hz | |
| Hacker-Bright 8000 Series Base Sledge Microtome | Hacker instruments | ||
| Household food steamer | Braun | MultiGourmet FS 20 | |
| Light microscope | Leica Polyvar 2 |
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