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.

NOTA: Tutte le procedure sugli animali descritti in questo protocollo sono state effettuate in conformità con le raccomandazioni della Guida per la cura e l&#39;uso di animali da laboratorio del National Institutes of Health 26. Il protocollo (MO13M391) è stato approvato dall&#39;Università Animal Care and Use Committee Johns Hopkins. Sacrificio 1. neonatale mouse, Dissection, e fissazione <ol><li> Sacrifica un mouse neonatale da CO 2 inalazione. </li><li> Immediatamente tagliare il arti posteriori sopra il ginocchio. Fino al giorno di circa 7 postnatale, le ossa delle gambe sono abbastanza morbido per tagliare con forbici grandi laboratorio o lama affilata e alla successiva sezione un criostato. </li><li> Posizionare il hindlimb in 10 ml di ghiaccio freddo 4% paraformaldeide in tampone fosfato salino (PBS) in una provetta da 15 ml. </li><li> Immersion fissare il arti posteriori in paraformaldeide per 4-24 ore a 4 ° C. Tessuti embrionali richiede solo alcune ores di fissazione. <ol><li> Evitare di fissaggio più di 24 ore, in quanto ciò potrebbe ridurre l&#39;efficienza TUNEL assay. </li></ol></li><li> Cryoprotect il arti posteriori immergendolo in una soluzione di saccarosio. <ol><li> Cambiare paraformaldeide al 10% di saccarosio in PBS nello stesso tubo 15 ml, incubare una notte a 4 ° C. </li><li> Variazione al 30% di saccarosio in PBS, incubare una notte a 4 ° C o fino ai lavandini hindlimb al fondo della provetta. Tissue può essere lasciato nel 30% di saccarosio per un periodo più lungo, a condizione che il saccarosio è stata filtrata sterile. </li></ol></li></ol> 2. Tissue Embedding <ol><li> Riempire uno stampo embedding metà marcato con mezzo di inclusione. </li><li> Inserire hindlimb sopra il mezzo di inclusione, con la parte tagliata il più vicino possibile ad una parete dello stampo. Contrassegnare l&#39;orientamento della arti posteriori sulla parte esterna dello stampo. <ol><li> Incorpora diverse arti posteriori e tagliare nello stesso blocco. Tuttavia, è importante assicurare che tutte hindlimbs nel blocco sono orientate parallelamente tra loro per ottenere sezioni in corrispondenza livelli lungo l&#39;asse dell&#39;arto. </li></ol></li><li> Coprire arti posteriori con mezzo di inclusione. Se zampe posteriori spostano durante il versamento del mezzo, ri-orientare con una pinza a punta smussata o uno stuzzicadenti. </li><li> Lasciare mezzo di inclusione per infiltrarsi tessuto a temperatura ambiente per almeno 15 min. </li><li> Mettere gli stampi direttamente in ghiaccio secco o in isopentano in ghiaccio secco per congelare mezzo di inclusione. Assicurarsi di mantenere il livello di stampi in ogni momento e di evitare lo spostamento del tessuto incorporato. </li><li> Mantenere stampi congelati a -80 ° C fino al momento di taglio, fino a diversi mesi. </li></ol> 3. criosezionamento incorporato Limbs <ol><li> Impostare temperatura della camera criostato a -20 ° C, la temperatura dell&#39;oggetto a -18 ° C; regolare la temperatura dell&#39;oggetto verso il basso, se necessario. </li><li> Portate il blocco di tessuto all&#39;interno della camera criostato per almeno 30 min. Blocco corretta temperaturaerature è fondamentale per la qualità sezione. </li><li> Tagliare sezioni trasversali ad uno spessore di 10 micron o meno. Sezioni spesse non saranno pienamente penetrati da reagenti TUNEL. Utilizzare la piastra anti-rollio, pale, e l&#39;angolo della lama (tipicamente 5 °), raccomandato per il criostato. </li><li> Raccogliere sezioni su gelatin- o vetrini vectabond rivestite a temperatura ambiente. Mettere subito in una scatola di diapositive in ghiaccio secco. Conservare la scatola diapositiva in un contenitore separato con ghiaccio secco posto in prossimità del criostato a portata di mano. <ol><li> Tagliare sezioni trasversali seriali attraverso l&#39;asse dell&#39;arto, saltare 100-200 micron. Raccogliere almeno 3 sezioni adiacenti a ciascun livello di serie. </li></ol></li></ol> 4. TUNEL test sulle sezioni degli arti posteriori utilizzando un kit in commercio Disponibile (tutte le misure a temperatura ambiente se non indicato) <ol><li> Scongelare vetrini con sezioni a temperatura ambiente e asciugare durante la notte o durante il fine settimana. </li><li> Reidratare sezioni di immersing vetrini in PBS per 2 x 10 min in una vaschetta Coplin. Asciugare con un laboratorio di pulire, rimuovere quanto più liquido intorno alla sezione possibile. </li><li> Sezioni permeabilize <ol><li> Utilizzare un non-proteolitici, reattivo a base di saponina commerciale oppure 0,5% e il 0,05% Tween20 Triton X100 in PBS. </li><li> Posizionare il vetrino in una camera umida. Una semplice camera umida è una scatola vuota puntale con un coperchio a tenuta, con acqua sufficiente a coprire appena aggiunto il fondo della scatola. </li><li> Pipetta abbastanza reattivo sul vetrino per coprire la sezione (50 ml è sufficiente per gli arti neonatali del mouse). Non pipettare direttamente sulla sezione, come agitazione ripetuta può provocare la sezione per sollevare la diapositiva. </li><li> Utilizzando pinze a punta smussata, posizionare un piccolo rettangolo di parafilm sulla parte superiore della sezione di diffondere la goccia di reagente e coprire completamente la sezione. Se le bolle si formano sotto la parafilm, delicatamente sollevarlo, asciutto, e riapplicare. </li><li> Incubare i vetrini con permeabilireattivo zione per 1 ora in una camera umida coperta. Se sezioni iniziano a sollevarsi dalla diapositiva, tempo di permeabilizzazione può essere ridotto a 30 min. </li><li> Lavare i vetrini in PBS in una vaschetta Coplin 2 x 5 min. I rettangoli parafilm dovrebbero fluttuare verso l&#39;alto e può quindi essere rimosso e scartato. </li><li> Utilizzando un laboratorio strofinata, asciugare quanto più liquido intorno sezioni possibili. </li></ol></li><li> DNA etichettatura pausa TdT-mediata <ol><li> Seguire le istruzioni del kit per fare 1x buffer di etichettatura TdT, pipetta abbastanza buffer di etichettatura sul vetrino per coprire la sezione di tessuto (50 ml è sufficiente per gli arti neonatali del mouse). Incubare nella camera umida per 5 min. In alternativa, immergere il vetrino in buffer di etichettatura TdT in una vaschetta Coplin. </li><li> Seguire le istruzioni del kit per fare TdT mix di etichettatura. Il kit comprende cobalto, magnesio, manganese e cationi; il catione manganese funziona bene su una varietà di tessuti, compresi i muscoli scheletrici, tiss sistema nervosoUES, il fegato, la pelle e ossa. </li><li> Per un controllo positivo, aggiungere DNase (etichetta &quot;nuclease&quot; nel kit) per il mix di etichettatura TdT. In alternativa, pre-trattamento di una sezione di controllo positivo con DNasi in tampone nucleasi per 1 ora a 37 ° C, secondo le istruzioni del kit. Tenere soluzione DNasi e sezioni DNasi trattati da altre sezioni sperimentali per evitare la contaminazione incrociata. </li><li> Per un controllo negativo, omettere TdT enzima dalla miscela etichettatura. </li><li> Utilizzare un laboratorio wipe per rimuovere la maggior quantità di buffer etichettatura TdT dalla diapositiva possibile. Pipettare TdT mix etichettatura sul vetrino (50 microlitri per sezione), coprire con un nuovo rettangolo di parafilm e incubare in camera umida a 37 ° C per 1 ora. </li><li> Seguire le istruzioni del kit per fare tampone arresto 1x. Utilizzare un laboratorio pulire per rimuovere TdT mix etichettatura dalla diapositiva, e aggiungere 200-300 tampone di arresto microlitri per coprire la sezione di tessuto e incubare in camera umida per 5 min. Al<html> ternativa, immergere il vetrino in buffer di arresto in una vaschetta Coplin. </li><li> Lavare il vetrino 2 x 2 min in PBS. Utilizzare un laboratorio strofinata per rimuovere quanto più PBS dal vetrino possibile. </li></ol></li><li> Etichettatura fluorescente <ol><li> Seguire le istruzioni del kit per la preparazione soluzione di etichettatura fluoresceina etichettare dNTP a rotture del DNA. Soluzione Pipettare sul vetrino (50 microlitri per sezione), coprire con un nuovo rettangolo di parafilm e incubare in camera umida per 20 min. </li><li> Lavare il vetrino per 2 minuti in PBS in una vaschetta Coplin. Utilizzare un laboratorio strofinata per rimuovere il più possibile dal PBS diapositiva. </li><li> Diluire Hoechst 33258 colorazione nucleare a 1 mg / ml in PBS e pipetta sul vetrino (100-200 ml è sufficiente a coprire la sezione). Incubare nella camera umida per 5 min. </li><li> Lavare 3x per 5 min in PBS. </li><li> Coverslip con Antifade mezzi di montaggio fluorescenza e sigillare i bordi con smalto. </li></ol></li></ol>jove_title &quot;&gt; 5. Acquisizione Immagine Digitale <ol><li> Acquisire le immagini utilizzando un microscopio a fluorescenza convenzionale con DAPI, FITC, e filtri Texas rossi (o equivalenti). Utilizzare un obiettivo più alto ingrandimento (20X o 40X) per verificare che l&#39;etichettatura era successo e nuclei (intatti o frammentati) sono stati etichettati. Un obiettivo 10X è sufficiente per la quantificazione di puncta TUNEL-positive. </li><li> Raccogliere immagini della colorazione Hoechst, TUNEL colorazione, e il segnale autofluorescenti come tre canali a falsi colori separati uniti in una sola immagine. Se l&#39;etichetta fluoroforo TUNEL è verde, segnale di immagine autofluorescenti nel filtro rosso, e viceversa. <ol><li> Mantenere tutte le impostazioni di imaging equivalente tra le diapositive per la successiva soglia e quantificazione. Per ogni filtro, i tempi prestabiliti e di esposizione di registrazione, il guadagno, e binning (senza binning è meglio); non utilizzare le impostazioni di esposizione o di guadagno automatico. </li><li> Usa-prove ed errori per trovare esposizione e guadagno impostazioni che meglio catturare la gamma di intenpiazzole in tutti i vetrini colorati. Assicurarsi che non vi siano pixel saturi nelle immagini acquisite, in modo da pregiudizi quantificazione. </li></ol></li><li> Prendere le immagini di tutto il gruppo muscolare di interesse. Questo può richiedere più immagini da &quot;cuciti&quot; insieme. </li></ol> Analisi 6. Immagine <ol><li> Utilizzare i programmi di analisi delle immagini disponibili in commercio per analizzare le immagini digitali di TUNEL colorazione. Analisi con software commerciale qui è mostrato. In alternativa, il software ImageJ esente da NIH può essere utilizzato per analizzare TUNEL colorazione. </li><li> Macchia una sezione adiacente alla sezione TUNEL macchiato con ematossilina eosina (H &amp; E) e prendere un full-sezione di immagine digitale con un microscopio campo chiaro convenzionale. Usa questa immagine H &amp; E in combinazione con un atlante di anatomia 5,18,19 tracciare le strutture anatomiche da quantificare. </li><li> Con il canale TUNEL spento, tracciare manualmente il contorno dell&#39;area da quantificare, utilizzandoHoechst e canali di autofluorescenza di orientamento. Convertire l&#39;area tracciata in una maschera (un insieme di pixel selezionato coordinate da analizzare). </li><li> Attivare il canale TUNEL. Utilizzando la funzione di intensità thresholding nel programma di analisi delle immagini, impostare la soglia inferiore di escludere tutti i segnali autofluorescenza. </li><li> Applicare le stesse impostazioni di soglia a tutte le immagini del set di studio. Convertire le selezioni thresholded in maschere. </li><li> Per ogni immagine, combinare la maschera zona muscolare e la maschera TUNEL. La nuova maschera combinazione rappresenterà segnale TUNEL all&#39;interno solo l&#39;area del muscolo tracciato. </li><li> Eseguire maschera quantificazione sulla maschera combinazione per determinare il numero di oggetti e l&#39;area del segnale TUNEL all&#39;interno dell&#39;area muscolare tracciata oggetto. Eseguire questo utilizzando la funzione Particle Analyzer in ImageJ. </li></ol>

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<li>Portera-Cailliau, C., Price, D. L., Martin, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Excitotoxic+neuronal+death+in+the+immature+brain+is+an+apoptosis-necrosis+morphological+continuum%5BTitle%5D)+AND+%22J.+Comp+Neurol%22%5BJournal%5D)">Excitotoxic neuronal death in the immature brain is an apoptosis-necrosis morphological continuum.</a> J. Comp Neurol. 378 (1), 70-87 (1997).</li>
<li>Portera-Cailliau, C., Price, D. L., Martin, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Non-NMDA+and+NMDA+receptor-mediated+excitotoxic+neuronal+deaths+in+adult+brain+are+morphologically+distinct%3A+further+evidence+for+an+apoptosis-necrosis+continuum%5BTitle%5D)+AND+%22J.+Comp+Neurol%22%5BJournal%5D)">Non-NMDA and NMDA receptor-mediated excitotoxic neuronal deaths in adult brain are morphologically distinct: further evidence for an apoptosis-necrosis continuum.</a> J. Comp Neurol. 378 (1), 88-104 (1997).</li>
<li>Ravi, D., Ramadas, K., Mathew, B. S., Nalinakumari, K. R., Nair, M. K., Pillai, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((De+novo+programmed+cell+death+in+oral+cancer%5BTitle%5D)+AND+%22Histopathology%22%5BJournal%5D)">De novo programmed cell death in oral cancer.</a> Histopathology. 34 (3), 241-249 (1999).</li>
<li>Sakaki, T., Kohmura, E., Kishiguchi, T., Yuguchi, T., Yamashita, T., Hayakawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Loss+and+apoptosis+of+smooth+muscle+cells+in+intracranial+aneurysms.+Studies+with+in+situ+DNA+end+labeling+and+antibody+against+single-stranded+DNA%5BTitle%5D)+AND+%22Acta+Neurochir.%28Wien%29%22%5BJournal%5D)">Loss and apoptosis of smooth muscle cells in intracranial aneurysms. Studies with in situ DNA end labeling and antibody against single-stranded DNA.</a> Acta Neurochir.(Wien). 139 (5), 469-474 (1997).</li>
<li>Shi, S. R., Cote, R. J., Taylor, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antigen+retrieval+immunohistochemistry%3A+past%2C+present%2C+and+future%5BTitle%5D)+AND+%22J.+Histochem.+Cytochem%22%5BJournal%5D)">Antigen retrieval immunohistochemistry: past, present, and future.</a> J. Histochem. Cytochem. 45 (3), 327-343 (1997).</li>
<li>Shi, S. R., Imam, S. A., Young, L., Cote, R. J., Taylor, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antigen+retrieval+immunohistochemistry+under+the+influence+of+pH+using+monoclonal+antibodies%5BTitle%5D)+AND+%22J.+Histochem.+Cytochem%22%5BJournal%5D)">Antigen retrieval immunohistochemistry under the influence of pH using monoclonal antibodies.</a> J. Histochem. Cytochem. 43 (2), 193-201 (1995).</li>
<li>Singh, N. P., McCoy, M. T., Tice, R. R., Schneider, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+simple+technique+for+quantitation+of+low+levels+of+DNA+damage+in+individual+cells%5BTitle%5D)+AND+%22Exp.+Cell+Res%22%5BJournal%5D)">A simple technique for quantitation of low levels of DNA damage in individual cells.</a> Exp. Cell Res. 175 (1), 184-191 (1988).</li>
<li>Sirvent, J. J., Aguilar, M. C., Olona, M., Pelegri, A., Blazquez, S., Gutierrez, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prognostic+value+of+apoptosis+in+breast+cancer+pT1-pT2%29.+A+TUNEL%2C+p53%2C+bcl-2%2C+bag-1+and+Bax+immunohistochemical+study%5BTitle%5D)+AND+%22Histol.Histopathol%22%5BJournal%5D)">Prognostic value of apoptosis in breast cancer pT1-pT2). A TUNEL, p53, bcl-2, bag-1 and Bax immunohistochemical study.</a> Histol.Histopathol. 19 (3), 759-770 (2004).</li>
<li>Skyrlas, A., Hantschke, M., Passa, V., Gaitanis, G., Malamou-Mitsi, V., Bassukas, I. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expression+of+apoptosis-inducing+factor+%28AIF%29+in+keratoacanthomas+and+squamous+cell+carcinomas+of+the+skin%5BTitle%5D)+AND+%22Exp.+Dermatol%22%5BJournal%5D)">Expression of apoptosis-inducing factor (AIF) in keratoacanthomas and squamous cell carcinomas of the skin.</a> Exp. Dermatol. 20 (8), 674-676 (2011).</li>
<li>Smith, M. D., Weedon, H., Papangelis, V., Walker, J., Roberts-Thomson, P. J., Ahern, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Apoptosis+in+the+rheumatoid+arthritis+synovial+membrane%3A+modulation+by+disease-modifying+anti-rheumatic+drug+treatment%5BTitle%5D)+AND+%22Rheumatology.%28Oxford%29%22%5BJournal%5D)">Apoptosis in the rheumatoid arthritis synovial membrane: modulation by disease-modifying anti-rheumatic drug treatment.</a> Rheumatology.(Oxford). 49 (5), 862-875 (2010).</li>
<li>Stadelmann, C., Lassmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Detection+of+apoptosis+in+tissue+sections%29%5BTitle%5D)+AND+%22Cell+Tissue+Res%22%5BJournal%5D)">Detection of apoptosis in tissue sections).</a> Cell Tissue Res. 301 (1), 19-31 (2000).</li>
<li>Schans, G. P., van Loon, A. A., Groenendijk, R. H., Baan, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Detection+of+DNA+damage+in+cells+exposed+to+ionizing+radiation+by+use+of+anti-single-stranded+DNA+monoclonal+antibody%5BTitle%5D)+AND+%22Int.+J.+Radiat.+Biol%22%5BJournal%5D)">Detection of DNA damage in cells exposed to ionizing radiation by use of anti-single-stranded DNA monoclonal antibody.</a> Int. J. Radiat. Biol. 55 (5), 747-760 (1989).</li>
<li>Watanabe, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Detection+of+apoptotic+cells+in+human+colorectal+cancer+by+two+different+in+situ+methods%3A+antibody+against+single-stranded+DNA+and+terminal+deoxynucleotidyl+transferase-mediated+dUTP-biotin+nick+end-labeling+%28TUNEL%29+methods%5BTitle%5D)+AND+%22Jpn.+J.+Cancer+Res%22%5BJournal%5D)">Detection of apoptotic cells in human colorectal cancer by two different in situ methods: antibody against single-stranded DNA and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) methods.</a> Jpn. J. Cancer Res. 90 (2), 188-193 (1999).</li>
<li>Watanabe, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Apoptosis+signal-regulating+kinase+1+is+involved+not+only+in+apoptosis+but+also+in+non-apoptotic+cardiomyocyte+death%5BTitle%5D)+AND+%22Biochem.+Biophys.+Res.+Commun%22%5BJournal%5D)">Apoptosis signal-regulating kinase 1 is involved not only in apoptosis but also in non-apoptotic cardiomyocyte death.</a> Biochem. Biophys. Res. Commun. 333 (2), 562-567 (2005).</li>
<li>Yaoita, H., Ogawa, K., Maehara, K., Maruyama, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Attenuation+of+ischemia%2Freperfusion+injury+in+rats+by+a+caspase+inhibitor%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">Attenuation of ischemia/reperfusion injury in rats by a caspase inhibitor.</a> Circulation. 97 (3), 276-281 (1998).</li>
</ol>

The authors have nothing to disclose.

Un metodo per rilevare e analizzare quantitativamente DNA danni associata apoptosi nel topo muscoli scheletrici è descritto. La procedura prevede la raccolta dei tessuti, TUNEL colorazione, l&#39;acquisizione di immagini digitali, e l&#39;analisi delle immagini. Sono necessarie forniture istologiche comuni e strumenti, e uno speciale kit di TUNEL commerciale è necessario. Gli elementi essenziali necessari grandi apparecchiature sono un criostato, microscopio epifluorescente con capacità di immagine digitale, e un sis...

Etichettatura fine nick Terminal deossinucleotidil transferasi (TdT) dUTP (TUNEL) è il processo di utilizzo dell&#39;enzima TdT allegare dUTP al 3 &#39;estremità del doppio e singolo filamento di DNA rompe 12,23. Il metodo TUNEL per il rilevamento di apoptosi e danni al DNA è stato segnalato prima oltre 20 anni fa da Gavrieli et al. 1,12,24. Da allora è stato valutato e ottimizzate in diverse preparazioni di tessuto 7,23,27,40. TUNEL è stato utilizzato per studiare ischemia indotta morte cellulare dei neuroni 6,14,29 e cardiomiociti 43,44, eccitotossico morte neuronale 30,31, e come biomarker nel trattamento dell&#39;artrite 39. E &#39;stato anche utilizzato come fattore prognostico e marker delle cellule tumorali in diversi tumori umani 2,3,15,32,37,38,42. Esistono metodi alternativi per il danno al DNA e la rilevazione morte cellulare, ma hanno sfide tecniche e avvertimenti. Southern blotting può essere utilizzato per quantifdanno al DNA y in tutto lisati tissutali 7,9-11, ma questo metodo non fornisce la risoluzione-livello cellulare ed è difficile da quantificare. Il Comet assay è un metodo a base di cellule alternativa che richiede l&#39;estrazione di nuclei conservati dalle cellule 4,20,28,36. Sebbene il saggio comet funziona bene su cellule isolate coltivate, è molto più difficile preparare nuclei intatti dal tessuto muscolare scheletrico 8,21. Come con la macchia del sud, il test della cometa non fornisce informazioni cell-tipo-specifica da un intero tessuto omogenato muscolare. Un&#39;altra alternativa al metodo TUNEL è immunoistochimica utilizzando anticorpi contro il DNA a singolo filamento 25,33,41 o contro le proteine ​​che partecipano a percorsi di DNA di risposta danno e morte cellulare (ad es p53, H2AX, e caspasi) 13,17,22,40. Tali metodi a base di anticorpi richiedono caratterizzazione completa di anticorpi ed eccellente anticorpo specificità per produrre un elevato rapporto segnale-background. Anche quando specEsistono anticorpi IFIC, possono richiedere la denaturazione delle proteine ​​bersaglio mediante procedure di recupero antigene 34,35. Mentre discutiamo qui, il recupero antigene risultati tessuto muscolare in inaccettabilmente alto autofluorescenza. A differenza dei metodi alternativi, TUNEL raggiunge DNA rilevamento dei danni con un segnale alto e basso fondo, eccellente specificità che può essere testato con semplici controlli positivi e negativi, buona penetrazione tissutale, che non richiedono il recupero dell&#39;antigene, e la risoluzione a livello cellulare. Inoltre, il metodo TUNEL dura circa 4 ore per completare, mentre i metodi alternativi in ​​genere richiedono incubazione durante la notte. Studiamo la morte delle cellule del muscolo scheletrico in un modello murino di atrofia muscolare spinale (SMA) 10 che è stato generato da Hsieh-Li e colleghi 16. Per quantificare cellule apoptotiche nel muscolo, abbiamo sviluppato un metodo di preparazione dei tessuti, la colorazione, e quantificazione che funziona robusto tra i diversi Skelegruppi muscolari Tal in diversi momenti dello sviluppo nei topi. Usiamo un kit TUNEL etichettatura disponibile in commercio e disponibile in commercio software di analisi dell&#39;immagine. Abbiamo anche utilizzato con successo il test TUNEL in combinazione con immunofluorescenza nel midollo spinale 10. I metodi descritti sono utili per gli investigatori che vogliono valutare la patologia dei tessuti, dei meccanismi della malattia, i meccanismi di invecchiamento, e di sviluppo (pre e post-natale) la morte cellulare nel muscolo scheletrico. La tecnica TUNEL è particolarmente utile per gli studi di danno e riparazione del DNA e morte cellulare in sistemi modello in cui solo un sottoinsieme di celle è colpita e risoluzione livello cellulare è necessaria. Questo video descrive la dissezione, processazione dei tessuti, sezionamento, e l&#39;etichettatura TUNEL basato sulla fluorescenza del mouse del muscolo scheletrico. Viene inoltre descritto un metodo di semi-automatico del segnale quantificazione TUNEL.

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

Con la colorazione di successo, il segnale TUNEL-positivi dovrebbe essere abbastanza luminoso per isolare da autofluorescenza impostando soglie di intensità. Oggetti TUNEL-positive a basso ingrandimento possono apparire frammenti irregolari come brillanti nel muscolo scheletrico (Figura 1A). Tuttavia, a più alto ingrandimento, si devono osservare alcuni oggetti TUNEL-positivi con la classica morfologia apoptotica, se il tipo di morte cellulare in questione è l&#39;apoptosi (Figura 1B).</stron...

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.

Rilevamento e analisi del danno al DNA nel muscolo scheletrico mouse<em&gt; In Situ</em&gt; Utilizzo del metodo TUNEL

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

detection-analysis-dna-damage-mouse-skeletal-muscle-situ-using-tunel

Research

JoVE Journal

Biology

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

16.2K Views.  Johns Hopkins School of Medicine. 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. 

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

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs 1-6. Yet the development of artificial molecular machines centers on devices capable of directional motion, i.e. molecular motors, and on their scaled-down mechanical parts (wheels, axels, pendants etc) 7-9. This imitates the macro-machines, even though the physical properties essential for these devices, such as inertia and momentum conservation, are not usable in the nanoworld environments 10. Alternative designs, which do not follow the mechanical macromachines schemes and use mechanisms developed in the evolution of biological molecules, can take advantage of the specific conditions of the nanoworld. Besides, adapting actual biological molecules for the purposes of nano-design reduces potential dangers the nanotechnology products may pose. Here we demonstrate the assembly and application of one such bio-enabled construct, a semi-artificial molecular device which combines a naturally-occurring molecular machine with artificial components. From the enzymology point of view, our construct is a designer fluorescent enzyme-substrate complex put together to perform a specific useful function. This assembly is by definition a molecular machine, as it contains one 12. Yet, its integration with the engineered part - fluorescent dual hairpin - re-directs it to a new task of labeling DNA damage12.
Our construct assembles out of a 32-mer DNA and an enzyme vaccinia topoisomerase I (VACC TOPO). The machine then uses its own material to fabricate two fluorescently labeled detector units (Figure 1). One of the units (green fluorescence) carries VACC TOPO covalently attached to its 3'end and another unit (red fluorescence) is a free hairpin with a terminal 3'OH. The units are short-lived and quickly reassemble back into the original construct, which subsequently recleaves. In the absence of DNA breaks these two units continuously separate and religate in a cyclic manner. In tissue sections with DNA damage, the topoisomerase-carrying detector unit selectively attaches to blunt-ended DNA breaks with 5'OH (DNase II-type breaks)11,12, fluorescently labeling them. The second, enzyme-free hairpin formed after oligonucleotide cleavage, will ligate to a 5'PO4 blunt-ended break (DNase I-type breaks)11,12, if T4 DNA ligase is present in the solution 13,14 . When T4 DNA ligase is added to a tissue section or a solution containing DNA with 5'PO4 blunt-ended breaks, the ligase reacts with 5'PO4 DNA ends, forming semi-stable enzyme-DNA complexes. The blunt ended hairpins will interact with these complexes releasing ligase and covalently linking hairpins to DNA, thus labeling 5'PO4 blunt-ended DNA breaks.
This development exemplifies a new practical approach to the design of molecular machines and provides a useful sensor for detection of apoptosis and DNA damage in fixed cells and tissues.

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

We demonstrate the assembly and application of a molecular-scale device powered by a topoisomerase protein. The construct is a bio-molecular sensor which labels two major types of DNA breaks in tissue sections by attaching two different fluorophores to their ends.

In Assemblea in vitro di semi-artificiale della macchina molecolare e il suo utilizzo per il rilevamento del danno al DNA

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs 1-6. Yet the development of artificial molecular ...

Ricerca

Bioingegneria

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and pathological processes, such as cancer and ageing. Recently, senescence has also been implicated in tissue repair and regeneration. Therefore, it has become increasingly critical to identify senescent cells in vivo. Senescence-associated &#946;-galactosidase (SA-&#946;-Gal) assay is the most widely used assay to detect senescent cells both in culture and in vivo. This assay is based on the increased lysosomal contents in the senescent cells, which allows the histochemical detection of lysosomal &#946;-galactosidase activity at suboptimum pH (6 or 5.5). In comparison with other assays, such as flow cytometry, this allows the identification of senescent cells in their resident environment, which offers valuable information such as the location relating to the tissue architecture, the morphology, and the possibility of coupling with other markers via immunohistochemistry&#160;(IHC). The major limitation of the SA-&#946;-Gal assay is the requirement of fresh or frozen samples.
Here, we present a detailed protocol to understand how cellular senescence promotes cellular plasticity and tissue regeneration in vivo. We use SA-&#946;-Gal to detect senescent cells in the skeletal muscle upon injury, which is a well-established system to study tissue regeneration. Moreover, we use&#160;IHC to detect Nanog, a marker of pluripotent stem cells, in a transgenic mouse model. This protocol enables us to examine and quantify cellular senescence in the context of induced cellular plasticity and in vivo reprogramming.

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Here we present a detailed protocol to detect both senescent and pluripotent stem cells in the skeletal muscle upon injury while inducing in vivo reprogramming. This method is suitable for evaluating the role of cellular senescence during tissue regeneration and reprogramming in vivo.

Valutazione delle lesioni-indotta senescenza e In Vivo riprogrammazione nel muscolo scheletrico

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and ...

Biologia dello sviluppo

Identificazione del danno al DNA negli ovociti arrestati con profase di topi

Detection of DNA Double-Stranded Breaks in Mouse Oocytes

Oocytes are amongst the biggest and most long-lived cells in the female body. They are formed in the ovaries during embryonic development and remain arrested at the prophase of meiosis I. The quiescent state may last for years until the oocytes receive a stimulus to grow and obtain the competency to resume meiosis. This protracted state of arrest makes them extremely susceptible to accumulating DNA-damaging insults, which affect the genetic integrity of the female gametes and, therefore, the genetic integrity of the future embryo. 
Consequently, the development of an accurate method to detect DNA damage, which is the first step for the establishment of DNA damage response mechanisms, is of vital importance. This paper describes a common protocol to test the presence and progress of DNA damage in prophase-arrested oocytes during a period of 20 h. Specifically, we dissect mouse ovaries, retrieve the cumulus-oocyte complexes (COCs), remove the cumulus cells from the COCs, and culture the oocytes in &#924;2 medium containing 3-isobutyl-1-methylxanthine to maintain the state of arrest. Thereafter, the oocytes are treated with the cytotoxic, antineoplasmic drug, etoposide, to engender double-strand breaks (DSBs). 
By using immunofluorescence and confocal microscopy, we detect and quantify the levels of the core protein &#947;H2AX, which is the phosphorylated form of the histone H2AX. H2AX becomes phosphorylated at the sites of DSBs after DNA damage. The inability to restore DNA integrity following DNA damage in oocytes can lead to infertility, birth defects, and increased rates of spontaneous abortions. Therefore, the understanding of DNA damage response mechanisms and, at the same time, the establishment of an intact method for studying these mechanisms are essential for reproductive biology research.

Autore in primo piano: Comprendere la risposta al danno al DNA negli ovociti dei mammiferi e negli embrioni preimpianto 

Maintaining oocyte genome integrity is necessary to ensure the genetic fidelity in the resulting embryo. Here, we present an accurate protocol for detecting DNA double-strand breaks in mammalian female germ cells.

Rilevamento di rotture a doppio filamento di DNA in ovociti di topo

Oocytes are amongst the biggest and most long-lived cells in the female body. They are formed in the ovaries during embryonic development and remain ...

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

Repair of acute injury to the cell membrane is an elemental process of normal cellular physiology, and defective membrane repair has been linked to many degenerative human diseases. The recent discovery of MG53 as a key component of the membrane resealing machinery allows for a better molecular understanding of the basic biology of tissue repair, as well as for potential translational applications in regenerative medicine. Here we detail the experimental protocols for exploring the in vivo function of MG53 in repair of muscle injury using treadmill exercise protocols on mouse models, for testing the ex vivo membrane repair capacity by measuring dye entry into isolated muscle fibers, and for monitoring the dynamic process of MG53-mediated vesicle trafficking and cell membrane repair in cultured cells using live cell confocal microscopy.

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

Described here are protocols used to visualize the dynamic process of MG53-mediated cell membrane repair in whole animals and at the cellular level. These methods can be applied to investigate the cell biology of plasma membrane resealing and regenerative medicine.

Visualizzazione di MG53-mediata di riparazione della membrana cellulare Utilizzando In vivo E In vitro Sistemi

Repair of acute injury to the cell membrane is an elemental process of normal cellular physiology, and defective membrane repair has been linked to many ...

Biologia

Profilazione dei segni istonici genomici nei mioblasti di topo utilizzando CUT&#38;Tag

Using Cleavage Under Targets and Tagmentation (CUT&#38;Tag) Assay in Mouse Myoblast Research

This protocol paper aims to provide the new researchers with the full details of using Cleavage Under Targets and Tagmentation (CUT&amp;Tag) to profile the genomic locations of chromatin binding factors, histone marks, and histone variants. CUT&amp;Tag protocols function very well with mouse myoblasts and freshly isolated muscle stem cells (MuSCs). They can easily be applied to many other cell types as long as the cells can be immobilized by Concanavalin-A beads. Compared to CUT&amp;Tag, chromatin immunoprecipitation (ChIP) assays are time-consuming experiments. ChIP assays require the pre-treatment of chromatin before the chromatic material can be used for immunoprecipitation. In cross-linking ChIP (X-ChIP), pre-treatment of chromatin involves cross-linking and sonication to fragment the chromatin. In the case of native ChIP (N-ChIP), the fragmented chromatins are normally achieved by Micrococcal nuclease (MNase) digestion. Both sonication and MNase digestion introduce some bias to the ChIP experiments. CUT&amp;Tag assays can be finished within fewer steps and require much fewer cells compared to ChIPs but provide more unbiased information on transcription factors or histone marks at various genomic locations. CUT&amp;Tag can function with as few as 5,000 cells. Due to its higher sensitivity and lower background signal than ChIPs, researchers can expect to obtain reliable peak data from merely several millions of reads after sequencing.

Autore in primo piano: Affrontare le sfide e le innovazioni negli studi sulle cellule staminali muscolari

Researchers new to the epigenetic field will find CUT&amp;Tag a significantly easier alternative to ChIP assays. CUT&amp;Tag has tremendously benefited the epigenetic studies on rare and primary cell populations, generating high-quality data from very few cells. This protocol describes performing H3K4me1 CUT&amp;Tag assays on mouse myoblasts isolated from mouse hindlimb muscles.

Utilizzo del test Cleavage Under Targets and Tagmentation (CUT&#38;Tag) nella ricerca sui mioblasti di topo

This protocol paper aims to provide the new researchers with the full details of using Cleavage Under Targets and Tagmentation (CUT&amp;Tag) to profile ...

Cryosectioning of Contiguous Regions of a Single Mouse Skeletal Muscle for Gene Expression and Histological Analyses

With this method, consecutive cryosections are collected to enable both microscopy applications for tissue histology and enrichment of RNA for gene expression using adjacent regions from a single mouse skeletal muscle. Typically, it is challenging to achieve adequate homogenization of small skeletal muscle samples because buffer volumes may be too low for efficient grinding applications, yet without sufficient mechanical disruption, the dense tissue architecture of muscle limits penetration of buffer reagents, ultimately causing low RNA yield. By following the protocol reported here, 30 &#956;m sections are collected and pooled allowing cryosectioning and subsequent needle homogenization to mechanically disrupt the muscle, increasing the surface area exposed for buffer penetration. The primary limitations of the technique are that it requires a cryostat, and it is relatively low throughput. However, high-quality RNA can be obtained from small samples of pooled muscle cryosections, making this method accessible for many different skeletal muscles and other tissues. Furthermore, this technique enables matched analyses (e.g., tissue histopathology and gene expression) from adjacent regions of a single skeletal muscle so that measurements can be directly compared across applications to reduce experimental uncertainty and to reduce replicative animal experiments necessary to source a small tissue for multiple applications.

Consecutive cryo-sections are collected to enable histological applications and enrichment of RNA for gene expression measurements using adjacent regions from a single mouse skeletal muscle. High-quality RNA is obtained from 20 - 30 mg of pooled cryosections and measurements are directly compared across applications.

Criosezionamento di regioni contigue di un muscolo scheletrico mouse unico per l&#39;espressione genica e analisi istologiche

With this method, consecutive cryosections are collected to enable both microscopy applications for tissue histology and enrichment of RNA for gene ...

Rilevamento e analisi del danno al DNA nel muscolo scheletrico mouse

Riepilogo

Esplora Altri Video

In Assemblea in vitro di semi-artificiale della macchina molecolare e il suo utilizzo per il rilevamento del danno al DNA

Valutazione delle lesioni-indotta senescenza e In Vivo riprogrammazione nel muscolo scheletrico

Rilevamento di rotture a doppio filamento di DNA in ovociti di topo

Rilevamento di rotture a doppio filamento di DNA in ovociti di topo

Rilevamento di rotture a doppio filamento di DNA in ovociti di topo

Visualizzazione di MG53-mediata di riparazione della membrana cellulare Utilizzando In vivo E In vitro Sistemi

Utilizzo del test Cleavage Under Targets and Tagmentation (CUT&Tag) nella ricerca sui mioblasti di topo

Utilizzo del test Cleavage Under Targets and Tagmentation (CUT&Tag) nella ricerca sui mioblasti di topo

Utilizzo del test Cleavage Under Targets and Tagmentation (CUT&Tag) nella ricerca sui mioblasti di topo

Criosezionamento di regioni contigue di un muscolo scheletrico mouse unico per l'espressione genica e analisi istologiche