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Abstract
Introduction
Protocol
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Biology

Detektion og analyse af DNA-skader i Mouse Skeletmuskel<em> In Situ</em> Brug af TUNEL Method

Published: December 16, 2014
doi:
10.3791/52211
,
1Division of Neuropathology, Department of Pathology, Pathobiology Graduate Program,Johns Hopkins School of Medicine

Summary

This video describes dissection, tissue processing, sectioning, and fluorescence-based TUNEL labeling of mouse skeletal muscle. It also describes a method for semi-automated analysis of TUNEL labeling.

Abstract

Terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) is the method of using the TdT enzyme to covalently attach a tagged form of dUTP to 3’ ends of double- and single-stranded DNA breaks in cells. It is a reliable and useful method to detect DNA damage and cell death in situ. This video describes dissection, tissue processing, sectioning, and fluorescence-based TUNEL labeling of mouse skeletal muscle. It also describes a method of semi-automated TUNEL signal quantitation. Inherent normal tissue features and tissue processing conditions affect the ability of the TdT enzyme to efficiently label DNA. Tissue processing may also add undesirable autofluorescence that will interfere with TUNEL signal detection. Therefore, it is important to empirically determine tissue processing and TUNEL labeling methods that will yield the optimal signal-to-noise ratio for subsequent quantitation. The fluorescence-based assay described here provides a way to exclude autofluorescent signal by digital channel subtraction. The TUNEL assay, used with appropriate tissue processing techniques and controls, is a relatively fast, reproducible, quantitative method for detecting apoptosis in tissue. It can be used to confirm DNA damage and apoptosis as pathological mechanisms, to identify affected cell types, and to assess the efficacy of therapeutic treatments in vivo.

Introduction

Terminal deoxynucleotidyltransferase (TdT) dUTP nick endemærkning (TUNEL) er den proces, ved hjælp af TdT enzym til at fastgøre dUTP til 3'-enderne af dobbelt og enkelt-strenget DNA bryder 12,23. TUNEL metode til påvisning af apoptose og DNA-skader blev først rapporteret mere end 20 år siden af Gavrieli et al. 1,12,24. Det er siden blevet vurderet og optimeret i forskellige vævspræparater 7,23,27,40. TUNEL er blevet anvendt til at undersøge iskæmi-induceret celledød af neuroner 6,14,29 og cardiomyocytter 43,44, excitotoksisk neuronal celledød 30,31, og som biomarkør i arthritis behandling 39. Det er også blevet brugt som en prognostisk faktor og tumorcellemarkøren i forskellige humane cancere 2,3,15,32,37,38,42.

Der findes alternative metoder til DNA-skader og celledød afsløring, men de har tekniske udfordringer og forbehold. Southern blotting kan anvendes til quantify DNA-skader i hele væv lysater 7,9-11, men denne metode giver ikke cellulært niveau opløsning og er vanskelig at kvantificere. Kometen assay er en alternativ celle-baserede metode, der kræver at udvinde bevarede kerner fra celler 4,20,28,36. Selvom comet assay fungerer godt på dyrkede isolerede celler, er det langt mere vanskeligt at fremstille intakte kerner fra skeletmuskelvæv 8,21. Som med Southern blot, er kometen assay ikke celle-typespecifikke oplysninger fra en hel muskel vævshomogenat. Et andet alternativ til TUNEL metode er immunhistokemi ved anvendelse af antistoffer mod enkeltstrenget DNA 25,33,41 eller mod proteiner, der deltager i DNA beskadigelse respons og celledødsveje (f.eks p53, H2AX og caspaser) 13,17,22,40. Sådanne antistof-baserede fremgangsmåder kræver grundig karakterisering af antistoffer og fremragende antistofspecificitet til opnåelse af et højt signal-til-baggrund-forholdet. Selv når specSp ec ifik antistoffer eksisterer, kan de kræve denaturering af målproteinet via antigen modtagelsesprocedurerne 34,35. Som vi diskuterer her, antigen hentning i muskelvæv resulterer i uacceptabelt høj autofluorescens. I modsætning til de alternative metoder, TUNEL opnår DNA-skader detektion med et højt signal og lav baggrund, fremragende specificitet, der kan testes med enkle positive og negative kontroller, god vævspenetration, der ikke kræver antigen hentning og cellulært niveau opløsning. Desuden TUNEL metode tager omkring 4 timer at gennemføre, mens alternative metoder kræver typisk overnattende inkubationer.

Vi studerer skeletmuskel celledød i en musemodel af spinal muskulær atrofi (SMA) 10, der blev genereret af Hsieh-Li og kolleger 16. For at kvantificere apoptotiske celler i musklen, har vi udviklet en fremgangsmåde til vævspræparat, farvning og kvantificering, der virker robust over forskellige SkeleTal muskelgrupper på forskellige udviklingsmæssige tidspunkter i mus. Vi bruger en kommercielt tilgængelig TUNEL-mærkning kit og kommercielt tilgængelig billedanalyse software. Vi har også med held anvendt TUNEL assay i kombination med immunfluorescensfarvning i rygmarven 10.

De her beskrevne metoder er nyttige for forskere, der ønsker at vurdere væv patologi, mekanismer på sygdom, mekanismer for aldring og udviklingsmæssige (præ- og postnatal) celledød i skeletmuskulatur. TUNEL teknik er især nyttig til undersøgelser af DNA-skader og reparation og celledød i modelsystemer, hvor kun en undergruppe af celler påvirkes og celleniveau opløsning er nødvendig.

Denne video beskriver dissektion, vævsbehandling, sektionering og fluorescens-baserede TUNEL mærkning af mus skeletmuskulatur. Det beskriver også en metode til halvautomatisk TUNEL signal kvantificering.

Protocol

BEMÆRK: Alle dyr, der er beskrevet i denne protokol blev gennemført i overensstemmelse med anbefalingerne i vejledningen for pleje og anvendelse af forsøgsdyr i National Institutes of Health 26. Protokollen (MO13M391) blev godkendt af Johns Hopkins University Animal Care og brug Udvalg. 1. Neonatal Mouse Sacrifice, Dissektion og fiksering Ofre en neonatal mus af CO 2 indånding. Umiddelbart afbrød bagbenet over knæet. Indtil ca. postna…

Representative Results

Med vellykket farvning, bør TUNEL-positive signal være lyse nok til at isolere fra autofluorescens ved at indstille intensitet tærskler. TUNEL-positive ting ved lav forstørrelse kan fremstå som lyse uregelmæssige fragmenter i skeletmuskulatur (figur 1A). Men ved højere forstørrelse, bør observeres nogle TUNEL-positive objekter med klassiske apoptotisk morfologi, hvis celledød typen involveret er apoptose (figur 1B). Den positive kontrol (DNase tilføjet) bør udvise rigelige T…

Discussion

En metode til at påvise og kvantitativt analysere DNA-beskadigelse-associeret apoptose i mus skeletmuskel er beskrevet. Proceduren omfatter væv høst, TUNEL farvning, billedoptagelse digitalt, og billedanalyse. Fælles histologiske leverancer og værktøjer er nødvendige, og en særlig kommerciel TUNEL kit er nødvendig. De væsentlige store udstyrsdele nødvendige er en kryostat, epifluorescerende mikroskop med digital billedbehandling kapacitet, og et computersystem til billedanalyse.

F…

Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was supported by NIH-NINDS grant RO1-NS065895 and NIH-NINDS grant 5-F31-NS076250-02.

We thank JHU SOM Microscope Facility for the use of the cryostat.

Materials

Name of Reagent/ Equipment Company Catalog Number Comments/Description
4% Paraformaldehyde in phosphate buffered saline Electron Microscopy Sciences 19202 For procedures described here, 4% solution was prepared fresh from powder. Paraformaldehyde from any supplier may be used. Prepared formaldehyde solution should be stored at 4 °C and should not be used after its expiration date (up to several months). Paraformaldehyde is a carcinogen and a toxin by inhalation and skin contact. Please follow precautions specified in the MSDS when handling paraformaldehyde.
Sucrose Sigma S0389 Used for cryoprotecting tissue before freezing. Sucrose from any supplier may be used.
O.C.T. compound  Tissue-Tek 4583 Embedding medium for cryosectioning.
Cryostat Leica CM 3050S A Leica CM3050S cryostat was used for the preparations described here. Any cryostat capable of cutting 10 μm sections may be used.
Glass slides, 25 x 75 x 1 mm Fisher 12-552-3 Slides from any supplier may be used.
Gelatin Sigma G-9391 Gelatin is used to promote tissue section adhesion to glass slides. To coat glass slides with gelatin, dissolve 2.75 g gelatin and 0.275 g chrome alum in 500 mL distilled water, warm to 60 °C, dip slides for several seconds, and let dry. Gelatin from any supplier may be used. Alternatively, gelatin-precoated slides may be purchased.
Chromium(III) potassium sulfate dodecahydrate (chrome alum) Sigma 243361 Chrome alum is added to gelatin solution to promote tissue adhesion on glass slides. It is a possible carcinogen and a toxin by inhalation and skin contact. Please follow precautions specified in the MSDS when handling chrome alum.
Vectabond tissue adhesion reagent Vector Labs SP-1800 Optional substrate for better tissue adhesion to glass slides; gellatin-coated slides may be used instead.
Tween20 Sigma P9416 A detergent used to permeabilize tissue. Tween20 from any supplier may be used.
Triton X100 Sigma T8787 A detergent used to permeabilize tissue. Triton X100 from any supplier may be used.
TACS 2 TdT fluorescein in situ apoptosis detection kit Trevigen 4812-30-K Commercial kit for fluorescence-based TUNEL labeling.
DNase/nuclease Trevigen 4812-30-K (included with kit)
DNase/nuclease buffer Trevigen 4812-30-K (included with kit)
10x phosphate buffered saline (PBS), pH 7.4 Amresco 780 Make 1x PBS for washes and dilutions. PBS from any supplier may be used.
DNase-free water Quality Biologicals 351-029-131 Water from any supplier may be used.
Hoechst 33258 Sigma 94403 Nuclear dye. Any blue fluorescent nuclear dye may be used. As a DNA-binding dye, Hoechst is a suspected carcinogen and should be handled with protective equipment to minimize skin contact.
Parafilm M multiple 807 Any other hydrophobic film or cover slip may be used. Available from multiple suppliers. 
Fluorescent microscope with digital camera  –  – Any fluorescent microscope capable of digitally capturing red, green, and blue fluorescence in separate channels may be used.
Vectashield antifade media Vector Labs H-1000 Antifade media from any supplier may be used.
glass coverslips, No.1 thickness Brain Research Labs 2222-1 Cover slips from any supplier may be used. The smallest size of 22×22 mm is sufficient for neonatal mouse leg sections.
Nail polish Ted Pella 114-8 Used to seal coverslips. Nail polish from any supplier (including regular retailers) may be used. Avoid using nail polish with color or additives that may reflect light during fluorescent imaging. 

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TUNELDNA DamageMouseSkeletal MuscleFluorescenceApoptosisQuantitation

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Fayzullina, S., Martin, L. J. Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method. J. Vis. Exp. (94), e52211, doi:10.3791/52211 (2014).
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