Name: visualization of mitochondrial respiratory function using cytochrome c oxidase / succinate dehydrogenase (cox/sdh) double-labeling histochemistry
Uploaded: 2011-11-23T12:00:00
Description: Watch this Scientific Journal Video about Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase COX/SDH Double-labeling Histochemistry at JoVE.com

Dette arbeidet ble støttet av National Institute of Aging (AG04418), National Institute on Drug Abuse, National Institute of Health-Karolinska Institutet Graduate Partnerships Program, Karolinska Institutet, svensk Forskningsrådet, svensk Brain Power, og svensk Brain Foundation. Mange takk til Mattias Karlen og Dr. Giuseppe Coppotelli for kreative støtte med Figur 1 og 2, henholdsvis; Karin Pernold for teknisk assistanse, og Drs. Barry J. Hoffer, Lars Olson og Nils-Göran Larsson for mye nyttige råd og diskusjon.

1. Tissue forberedelse for cryosectioning <ol><li> Offer dyret enten ved cervikal forvridning eller halshogging, i samsvar med tilgjengelige etiske tillatelse. </li><li> Raskt samle vev av interesse (f.eks. Hjernen), og raskt fryse på tørris (vev kan kreve frysing i isopentan eller propan kjølt med flytende nitrogen for å oppnå optimal morfologi). Oppbevar vev i aluminiumsfolie ved -80 ° C til den er klar til seksjonen. </li><li> Bygg frosset vev i forberedelse for cryosectioning. </li><li> Samle 14-mik...

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Den kombinerte COX / SDH histochemical metoden muliggjør visualisering av cellene med mitokondriell dysfunksjon. Denne teknikken, med tidlige studier som kan dateres tilbake til 1968, fortsatt populær, og mange vurderer det &quot;gullstandard&quot; for å identifisere mitokondrielle sykdommer hos pasienter 14,19,26,27. Det er nå ofte brukt for å undersøke mtDNA mutasjon-drevet aldring og aldring-relaterte lidelser 12,13,18,20,21,24. Den COX / SDH dobbel merking metoden er ofte brukt parallelt ...

<table border="1"><tr><td> Navn på reagens </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer (valgfritt) </td></tr><tr><td> Dry Ice </td><td> AGA Gas AB </td><td> blokk skjema </td><td></td></tr><tr><td> Isopentan (2-methylbutane) </td><td> Sigma-Aldrich </td><td> 277258 CAS: 78-78-4 </td><td></td></tr><tr><td> Cyrostat embedding løsning </td><td> Sakura Finetek </td><td> Tissue Tek 4583 </td><td></td></tr><tr><td> Cryostat </td><td> Microm </td><td> Microm Model HM 500M </td><td></td></tr><tr><td> Slides </td><td> Thermo Scientific </td><td> Super Frost Plus Menzel Glaser J1800AMWZ </td><td></td></tr><tr><td> Cover briller Borsilikatglass </td><td> VWR International </td><td> 16004-098 </td><td> 24 x 50 mm </td></tr><tr><td> Filter Paper </td><td> Munktell Filter AB </td><td> Kvalitet: 1350 ArtikkelAntall: 242 001 </td><td> 430 x 430 mm </td></tr><tr><td> 3,3 &#39;-diaminobenzidine tetrahydrochloride (DAB) </td><td> Sigma-Aldrich </td><td> Sigma Flytende Substrat System, D7304 </td><td></td></tr><tr><td> Cytokrom c (Type III, fra equine hjertet) </td><td> Sigma-Aldrich </td><td> C2506 CAS: 9007-43-6 </td><td></td></tr><tr><td> Bovine katalase (fra leveren) </td><td> Sigma-Aldrich </td><td> C9322 CAS: 9001-05-2 </td><td></td></tr><tr><td> Nitroblue tetrazolium (NBT) </td><td> Sigma-Aldrich </td><td> N6876 CAS: 298-83-9 </td><td></td></tr><tr><td> Sodium succinate </td><td> Sigma-Aldrich </td><td> S2378 CAS: 6106-21-4 </td><td></td></tr><tr><td> Phenazine methosulfate (PMS) </td><td> Sigma-Aldrich </td><td> P9625 CAS: 299-11-6 </td><td> PMS er lys sensitive. Skjerme mot lys. </td></tr><tr><td> Natriumazid </td><td> Sigma-Aldrich </td><td> S8032 CAS: 26628-22-8 </td><td></td></tr><tr&gt; <td> Xylen </td><td> VWR International </td><td> EM-XX0060-4 </td><td></td></tr><tr><td> Entellan </td><td> VWR International </td><td> 100503-870 </td><td></td></tr><tr><td> Malonate (Malonic acid) </td><td> Sigma-Aldrich </td><td> M1296 CAS: 141-82-2 </td><td></td></tr></table>

visualization of mitochondrial respiratory function using cytochrome c oxidase / succinate dehydrogenase (cox/sdh) double-labeling histochemistry

Mitokondrie DNA (mtDNA) mangler er en viktig årsak til sykdom, og kan ligge til grunn for aldring og aldring-relaterte endringer 1,2. Mitokondrie teori om aldring antyder en rolle for mtDNA mutasjoner som kan endre bioenergi homeostase og cellulære funksjon, i aldringsprosessen 3. Et vell av bevis er utarbeidet til støtte for denne teorien 1,4, et eksempel er den mtDNA mutator musen 5, men er den presise rolle mtDNA skader i aldring ikke helt forstått 6,7. Observere aktiviteten av respiratorisk enzymer er en grei metode for å undersøke mitokondriell dysfunksjon. Kompleks IV, eller cytokrom c oksidase (COX), er avgjørende for mitochondrial funksjon. Den katalytiske subenheter av COX er kodet av mtDNA og er viktig for montering av komplekset (figur 1). Dermed er riktig syntese og funksjon i stor grad basert på mtDNA integritet 2.Selv om andre respiratoriske komplekser kan bli etterforsket, Komplekser IV og II er mest mottagelig for histochemical undersøkelse 8,9. Complex II, eller succinate dehydrogenase (SDH), er helt kodet av kjerne-DNA (figur 1), og om aktiviteten er vanligvis ikke påvirket av svekket mtDNA, men en økning kan indikere mitochondrial biogenesis 10-12. Den svekket mtDNA observert i mitokondrie sykdommer, aldring og aldersrelaterte sykdommer som ofte fører til tilstedeværelsen av celler med lav eller fraværende COX aktivitet 2,12-14. Selv om COX og SDH aktiviteter kan undersøkes individuelt, har den sekvensielle dobbel merking metode 15,16 viste seg å være en fordel i å finne celler med mitokondriedysfunksjon 12,17-21. Mange av optimal forfatninger av analysen vært fastsatt, slik som substrat konsentrasjon, elektron akseptorer / givere, middels elektron bærere, påvirkning av pH, og reaksjonen tIME 9,22,23. 3,3 &#39;-diaminobenzidine (DAB) er en effektiv og pålitelig elektron donor 22. I celler med fungerende COX, vil den brune indamine polymer produktet lokalisere i mitochondrial cristae og mette celler 22. Disse cellene med dysfunksjonelle COX vil derfor ikke bli mettet av DAB produktet, noe som åpner for visualisering av SDH aktivitet ved reduksjon av nitroblue tetrazolium (NBT), et elektron akseptor, til en blå formazanforbindelsen sluttprodukt 9,24. Cytokrom c og natrium succinate underlag legges til normalisere endogene nivåer mellom kontroll og syke / muterte vev 9. Catalase legges til som en forholdsregel for å unngå mulig forurensing reaksjoner fra peroksidase aktivitet 9,22. Phenazine methosulfate (PMS), en mellomliggende elektron carrier, brukes i forbindelse med natriumazid, en respiratoriske kjede hemmer, for å øke dannelsen av den endelige reaksjonen produktene 9,25. Til tross for dette informereasjon, noen kritiske detaljer som påvirker resultatet av denne høvisk enkle analysen, i tillegg til spesifisitet kontroller og fremskritt i teknikken, ennå ikke har blitt presentert.

Watch this Scientific Journal Video about Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase COX/SDH Double-labeling Histochemistry at JoVE.com

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase COX/SDH Double-labeling Histochemistry (Video) | JoVE

Cytokrom c oksidase / natrium dehydrogenase (COX / SDH) dobbel merking metode gjør det mulig for direkte visualisering av mitokondrie luftveiene enzym mangler i fersk-frosset vev seksjoner. Dette er en grei histochemical teknikk og er nyttig i å undersøke mitokondrie sykdommer, aldring og aldring-relaterte lidelser.

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Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of ...

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Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase (COX/SDH) Double-labeling Histochemistry

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