Name: detection and analysis of dna damage in mouse skeletal muscle in situ using the tunel method
Uploaded: 2014-12-16T14:00:00
Description: Watch this Scientific Journal Video about Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method at JoVE.com

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.

MERK: Alle dyr prosedyrer beskrevet i denne protokollen ble utført i samsvar med anbefalingene i Guide for omsorg og bruk av forsøksdyr av National Institutes of Health 26. Protokollen (MO13M391) ble godkjent av Johns Hopkins University Animal Care og bruk komité. 1. Neonatal Mouse Sacrifice, Dissection, og Fiksering <ol><li> Ofre en neonatal musen ved CO 2 innånding. </li><li> Umiddelbart avskåret bakben over kneet. Inntil ca postnatal dag 7 leggbena er...

En fremgangsmåte for å detektere og kvantitativt å analysere DNA-skade-assosierte apoptose i mus skjelettmuskel er beskrevet. Prosedyren omfatter vev høsting, TUNEL farging, digital image oppkjøpet, og bildeanalyse. Vanligste histologiske forsyninger og verktøy er nødvendig, og en spesiell kommersiell TUNEL kit er nødvendig. De essensielle store utstyr elementer som trengs er en kryostat, epifluorescent mikroskop med digitalt bilde evne, og et datasystem for bildeanalyse. Eksperiment...

Terminal deoksynukleotidyltransferase (TdT) dUTP nick end merking (TUNEL) er prosessen med å bruke den TdT enzymet å feste dUTP til 3 &#39;endene av dobbel og enkelttrådet DNA bryter 12,23. Den TUNEL metode for påvisning av apoptose og DNA-skader ble først rapportert over 20 år siden av Gavrieli et al. 1,12,24. Det har siden blitt evaluert og optimalisert i ulike vevspreparater 7,23,27,40. TUNEL har blitt brukt til å studere ischemi-indusert celledød av neuroner 6,14,29 og kardiomyocytter 43,44, eksitotoksisk nevronal celledød 30,31, og som en biomarkør i leddgikt behandling 39. Det har også blitt brukt som en prognostisk faktor og svulst celle markør i ulike humane kreftformer 2,3,15,32,37,38,42. Alternative metoder finnes for DNA-skade og celledød deteksjon, men de har tekniske utfordringer og begrensninger. Southern blotting kan anvendes for å quantify DNA-skader i hele vev lysatene 7,9-11, men denne metoden ikke gir cellenivå oppløsning og er vanskelig å kvantifisere. Kometen analysen er et alternativ celle-basert metode som krever utpakking bevarte kjerner fra celler 4,20,28,36. Selv om kometmetoden fungerer godt på dyrkede isolerte celler, er det mye vanskeligere å forberede intakte kjerner fra skjelettmuskulatur vev 8,21. Som med Southern blot, gjør kometmetoden ikke gi celle-type-spesifikk informasjon fra en hel muskel vevhomogenat. Et annet alternativ til TUNEL-metoden er immunhistokjemi ved hjelp av antistoffer mot enkelt-trådet DNA 25,33,41 eller mot proteiner som deltar i DNA-skade respons og celledødsveier (for eksempel p53, H2AX, og kaspaser) 13,17,22,40. Slike antistoffbaserte metoder krever grundig karakterisering av antistoffer og utmerket antistoffspesifisitet og ga et høyt signal-til-bakgrunnsforhold. Selv når specIFIC antistoffer eksisterer, de kan kreve denaturering av målproteinet gjennom antigen gjenfinning prosedyrer 34,35. Som vi diskuterer her, antigen gjenfinning i muskel vev resulterer i uakseptabel høy autofluorescence. I motsetning til andre metoder, oppnår TUNEL DNA-skade deteksjon med et høyt signal og lav bakgrunn, utmerket spesifisitet som kan testes med enkle positive og negative kontroller, god vevspenetrasjon som ikke krever antigen gjenfinning, og cellulært nivå oppløsning. I tillegg tar TUNEL-metoden ca. 4 timer for å fullføre, mens alternative fremgangsmåter krever vanligvis over natten inkubasjoner. Vi studerer skjelettmuskulatur celledød i en musemodell for spinal muskelatrofi (SMA) 10 som ble generert av Hsieh-Li og kolleger 16. Å kvantifisere apoptotiske celler i muskelen, har vi utviklet en metode for vev forberedelse, flekker, og kvantifisering som fungerer robust tvers av ulike Skeletal muskelgrupper på ulike utviklings tidspunkter i mus. Vi bruker et kommersielt tilgjengelig TUNEL-merking kit og kommersielt tilgjengelig programvare for bildeanalyse. Vi har også med hell brukt TUNEL analysen i kombinasjon med immunofluorescent flekker i ryggmargen 10. Metodene som beskrives her er nyttige for etterforskere som ønsker å vurdere vev patologi, mekanismer for sykdom, mekanismer for aldring og utviklings (pre- og postnatal) celledød i skjelettmuskulatur. Den TUNEL teknikken er spesielt nyttig for studier av DNA-skade og reparasjon og celledød i modellsystemer, hvor bare et delsett av celler som er påvirket og cellenivå oppløsning er nødvendig. Denne videoen beskriver disseksjon, vev behandling, seksjonering, og fluorescens-basert TUNEL merking av mus skjelettmuskulatur. Det beskrives også en fremgangsmåte for semi-automatisert TUNEL signal kvantifisering.

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			<td height="21" style="height:21px;width:216px;">Name of Reagent/ Equipment</td>
			<td style="width:121px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:460px;">Comments/Description</td>
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			<td height="147" style="height:147px;width:216px;">4% Paraformaldehyde in phosphate buffered saline</td>
			<td style="width:121px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">19202</td>
			<td style="width:460px;">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 &#176;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.</td>
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			<td height="42" style="height:42px;width:216px;">Sucrose</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:119px;">S0389</td>
			<td style="width:460px;">Used for cryoprotecting tissue before freezing. Sucrose from any supplier may be used.</td>
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			<td height="21" style="height:21px;width:216px;">O.C.T. compound&#160;</td>
			<td style="width:121px;">Tissue-Tek</td>
			<td style="width:119px;">4583</td>
			<td>Embedding medium for cryosectioning.</td>
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			<td height="42" style="height:42px;width:216px;">Cryostat</td>
			<td style="width:121px;">Leica</td>
			<td style="width:119px;">CM 3050S</td>
			<td style="width:460px;">A Leica CM3050S cryostat was used for the preparations described here. Any cryostat capable of cutting 10 &#956;m sections may be used.</td>
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			<td height="21" style="height:21px;width:216px;">Glass slides, 25 x 75 x 1 mm</td>
			<td style="width:121px;">Fisher</td>
			<td style="width:119px;">12-552-3</td>
			<td>Slides from any supplier may be used.</td>
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			<td height="105" style="height:105px;width:216px;">Gelatin</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:119px;">G-9391</td>
			<td style="width:460px;">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 &#176;C, dip slides for several seconds, and let dry. Gelatin from any supplier may be used. Alternatively, gelatin-precoated slides may be purchased.</td>
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			<td height="84" style="height:84px;width:216px;">Chromium(III) potassium sulfate dodecahydrate (chrome alum)</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:119px;">243361</td>
			<td style="width:460px;">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.</td>
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			<td height="42" style="height:42px;width:216px;">Vectabond tissue adhesion reagent</td>
			<td style="width:121px;">Vector Labs</td>
			<td style="width:119px;">SP-1800</td>
			<td style="width:460px;">Optional substrate for better tissue adhesion to glass slides; gellatin-coated slides may be used instead.</td>
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			<td height="42" style="height:42px;width:216px;">Tween20</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:119px;">P9416</td>
			<td style="width:460px;">A detergent used to permeabilize tissue. Tween20 from any supplier may be used.</td>
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			<td height="42" style="height:42px;width:216px;">Triton X100</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:119px;">T8787</td>
			<td style="width:460px;">A detergent used to permeabilize tissue. Triton X100 from any supplier may be used.</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TACS 2 TdT fluorescein in situ apoptosis detection kit</td>
			<td style="width:121px;">Trevigen</td>
			<td style="width:119px;">4812-30-K</td>
			<td>Commercial kit for fluorescence-based TUNEL labeling.</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DNase/nuclease</td>
			<td style="width:121px;">Trevigen</td>
			<td style="width:119px;">4812-30-K</td>
			<td>(included with kit)</td>
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			<td height="21" style="height:21px;width:216px;">DNase/nuclease buffer</td>
			<td style="width:121px;">Trevigen</td>
			<td style="width:119px;">4812-30-K</td>
			<td>(included with kit)</td>
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			<td height="42" style="height:42px;width:216px;">10x phosphate buffered saline (PBS), pH 7.4</td>
			<td style="width:121px;">Amresco</td>
			<td style="width:119px;">780</td>
			<td style="width:460px;">Make 1x PBS for washes and dilutions. PBS from any supplier may be used.</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">DNase-free water</td>
			<td style="width:121px;">Quality Biologicals</td>
			<td style="width:119px;">351-029-131</td>
			<td>Water from any supplier may be used.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Hoechst 33258</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:119px;">94403</td>
			<td style="width:460px;">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.</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Parafilm M</td>
			<td style="width:121px;">multiple</td>
			<td style="width:119px;">807</td>
			<td style="width:460px;">Any other hydrophobic film or cover slip may be used. Available from multiple suppliers.&#160;</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Fluorescent microscope with digital camera</td>
			<td style="width:121px;">&#160;--</td>
			<td style="width:119px;">&#160;--</td>
			<td style="width:460px;">Any fluorescent microscope capable of digitally capturing red, green, and blue fluorescence in separate channels may be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vectashield antifade media</td>
			<td style="width:121px;">Vector Labs</td>
			<td style="width:119px;">H-1000</td>
			<td>Antifade media from any supplier may be used.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">glass coverslips, No.1 thickness</td>
			<td style="width:121px;">Brain Research Labs</td>
			<td style="width:119px;">2222-1</td>
			<td style="width:460px;">Cover slips from any supplier may be used. The smallest size of 22x22 mm is sufficient for neonatal mouse leg sections.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Nail polish</td>
			<td style="width:121px;">Ted Pella</td>
			<td style="width:119px;">114-8</td>
			<td style="width:460px;">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.&#160;</td>
		</tr>
	</tbody>
</table>

detection and analysis of dna damage in mouse skeletal muscle in situ using the tunel method

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&#8217; 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.

Med vellykket flekker, bør TUNEL-positive signal være sterke nok til å isolere fra autofluorescence ved å sette intensitet terskler. TUNEL-positive objekter ved lav forstørrelse kan vises som lyse uregelmessige fragmenter i skjelettmuskulatur (figur 1A). Men ved høyere forstørrelse, noe TUNEL-positive objekter med den klassiske apoptotisk morfologi må overholdes, hvis celledød typen som er involvert er apoptose (figur 1B). Den positive kontrollen (DNase lagt til) skal fremvise ...

Watch this Scientific Journal Video about Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method at JoVE.com

Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

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.

Deteksjon og analyse av DNA Skade i Mouse Skeletal Muscle<em&gt; In Situ</em&gt; Bruke TUNEL Method

Terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) is the method of using the TdT enzyme to covalently ...

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Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

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