이 작품은 노화의 국립 연구소 (AG04418), 약물 남용에 국립 연구소, 건강 Karolinska Institutet 연구 협력 프로그램의 국립 연구소, Karolinska Institutet, 스웨덴어 연구위원회, 스웨덴어 브레인 파워, 그리고 스웨덴어 뇌 재단에 의해 지원되었다. 대부분의 각각 그림 1 및 2와 창조에 대한 지원 Mattias Karlen 박사 주세페 Coppotelli 덕분에, 기술 지원 Pernold Karin 및 Drs. 배리 J. Hoffer, 많은 도움이 조언과 토론 라스 올슨, 그리고 닐스 - 고란 라슨.

1. cryosectioning을위한 조직 준비 <ol><li> 가능한 윤리적 허가에 따라 자궁 경부 전위 또는 잘린에 의해 동물을 희생. </li><li> 신속 관심 조직 (예 :. 뇌를) 수집, 그리고 신속하게 드라이 아이스 (조직이 최적의 형태를 얻을 액체 질소로 냉각 이소펜탄 또는 프로판의 동결을 요구할 수 있습니다)에 고정. 섹션 -80시 알루미늄 호일의 상점 조직은 ° C까지 준비. </li><li> cryosectioning에 대비하여 냉동 조직을 삽입할 수 있습니다. </li><li> -21에서 14 μm의 그라 이오 스탯 섹션을 모아서 ° C (온도를 조정해야 할 수도 있습니다 ± 1-2 ° C). 사용할 때까지 -20 ° C에서 coverslipping하지 않고 가열 블록 및 저장 슬라이드를 사용하여 슬라이드에 녹여 섹션. </li></ol> 2. COX의 histochemistry <ol><li> 슬라이드는 1 시간 동안 상온에서 건조하도록 허용합니다. 서부 유럽 표준시 필터 종이 슬라이드 얼룩 챔버에 슬라이드를 넣고, 스트립으로 절단. consisten을 얻으려면각 실험에서 t 결과, 그것은 시간 지연을 최소화하기 위해 실험을 한 사람당 10 슬라이드의 최대 처리하는 것이 좋습니다. </li><li> 화학 후드 1X DAB에 따라 준비, 100 μm의는 0.1 M PBS 산도 = 7.0에서 C를 시토크롬. 빠르게 소용돌이. </li><li> 카탈 라 아제 소 2 μg (2 μg ML -1 또는 약 4 IU ML -1) 추가합니다. 카탈 라 아제의 모든 곡식을 깨고 vortexing하여 잘 섞는다. </li><li> 150 적용 - 배양 배지 200 μL를 각 슬라이드에, 골고루 모든 부분에 확산 피펫 팁을 사용합니다. </li><li> 37 40 분 슬라이드를 품어 ° C. </li><li> 슬라이드에서 여분의 솔루션을 제거합니다. 워시는 0.1 M PBS 산도 = 7.0에서 4 번, 매번 10 분, 슬라이드. </li><li> 서부 유럽 표준시 종이 스트립과 함께 슬라이드 얼룩 챔버로 슬라이드를 반환합니다. </li></ol> 3. SDH의 histochemistry <ol><li> 화학 후드 1.5 MM NBT 130 MM 나트륨 호박산, 0.2 MM PMS, 그리고 0.1 M PBS 산도 = 7.0에서 1.0 MM 나트륨 azide에 따라 준비합니다. 받아가를 보호하기 위해주의빛과 PMS. 빠르게 소용돌이. </li><li> 각 슬라이드에 부화 매체 150-200 μL를 적용, 골고루 모든 부분에 확산 피펫 팁을 사용합니다. </li><li> 37 40 분 슬라이드를 품어 ° C. </li><li> 슬라이드에서 여분의 솔루션을 제거합니다. 워시는 0.1 M PBS 산도 = 7.0에서 4 번, 매번 10 분, 슬라이드. </li><li> 70 %, 70 %, 95%, 95 %, 99.5 % 에탄올 다음과 같은 농도에서 2 분 동안 슬라이드를 탈수. 그런 다음 추가 99.5 %의 단계에서 10 분 수 있습니다. </li><li> 장소는 10 분 크실렌에서 슬라이드. Entellan 및 coverslip로 마운트합니다. 슬라이드가 통풍이 지역에서 숙박하거나, 적어도 1-2 시간 건조하도록 허용합니다. </li></ol> 4. mitochondrial 장애의 결정 <ol><li> mitochondrial 장애의 양은 세포 푸른 얼룩의 양을로 표시됩니다. 이 금액을 반 수치, 슬라이드는 코드와 밝은 필드 현미경 아래에서 시각해야합니다. 세미 부량은 눈먼에서 수행되어야합니다<html> 눈금을 사용하여 기초, 0-4 (0, 아니 파란 얼룩, 4, 오직 파란색 얼룩)에서 예를 들어. 그것은 각 과목 / 동물에 대한 의미 가치를 계산하기 위해 주어진 주제 / 동물의 여러 섹션으로 세미 - 부량 이런 종류의를 수행하는 것이 좋습니다. </li><li> 통계는 이러한 맨 - 휘트니 또는 Kruskal - 월리스와 같은 비 파라메 트릭 테스트를 사용하여 수행해야합니다. </li></ol> 5. 적절한 특이성 컨트롤 <ol><li> COX 활동에 대한 특이성 컨트롤 &quot;COX의 histochemistry&quot;단계를 반복하고, 2.5 MM 나트륨 azide, 터미널 호흡 체인 억제제를 추가합니다. </li><li> SDH 활동에 대한 특이성 컨트롤, 나트륨 호박산의 제거 50 MM 말로 네이트, SDH의 경쟁 억제제의 추가와 함께 &quot;SDH의 histochemistry&quot;단계를 반복합니다. </li><li> 씻고 에탄올 시리즈 섹션을 탈수 다음과 같은 단계를 3.4에서 설명하는 슬라이드 마운트 coverslip - 3.6. </li></ol> 6. 대표 결과 : 십t는 &quot;&gt; 야생 유형과 조기 노화 mtDNA의 mutator 마우스의 뇌 부분에서 적절한 COX / SDH 이중 라벨 histochemistry의 COX / SDH 이중 라벨 histochemical 분석 것은 그림 2에서 그림입니다. 대표 예제의 전체 구조는 그림에 표시됩니다 3. 야생 형 생쥐의 어두운 갈색 얼룩 (그림 3, 왼쪽 패널)은 파란색 얼룩으로 표시 호흡 연쇄 결함과 함께 셀을. 정상적인 COX 활동을 보여주 12주 된 mtDNA의 mutator 마우스에 발표되었고,이 결함이되었습니다 46주 (그림 3, 센터 및 오른쪽 패널)에 세 mtDNA의 mutator의 생쥐로 더 널리. 부족 COX 라벨로 인해 야생 형 마우스의 뇌 부분에서 부적 절한 COX / SDH 이중 라벨링의 예는 그림 4에 표시됩니다. 부적 절한 배양 COX 활동의 시연 시간, 또는 부화 중에 슬라이드를 coverslipping하여 분자 산소의 가용성을 줄일 수는 DAB 반응의 감소 증착 결과따라서 이온 제품 및 SDH 보육 동안 파란 formazan 엔드 제품의 형성있었습니다. COX와 SDH 활동도 (왼쪽 그림 5와 센터) 개별적으로 조사 수 있지만, 순차 라벨은 SDH 보육 동안 파란색 침전물의 형성 (그림 3, 센터에 의한 COX의 결함과 세포를 식별 도움이됩니다 오른쪽). COX와 SDH 활동에 대한 특이성 컨트롤도 (그림 5, 오른쪽) 할 수 있습니다. <img alt="그림 1" src="/files/ftp_upload/3266/3266fig1.jpg" /> 그림 1. Mitochondrial 호흡 단지 IV. mitochondrial 호흡 사슬은 내부 멤브레인 내에있는 다섯 단지를 포함하고 있습니다. 호흡 사슬의 목적은 I IV로하고 ATP를 생산하는 복합 V (ATPase)에 의해 사용되는 내부 막에 걸쳐 양성자 그라디언트를 만듭니다 이렇게에서 복잡한에서 전자를 전송하는 것입니다. 레드 hexagons의 represeNT의 subunits는 mtDNA로 인코딩. 화이트 hexagons는 핵 DNA (단지 II가 완전히 핵 게놈에서 인코딩됩니다)로 인코딩된 subunits을 나타냅니다. 따라서, mitochondrial 게놈의 돌연변이가 호흡 체인 단지의 subunits의 돌연변이에 의한 호흡 사슬의 장애를 일으킬 수 있습니다. <img alt="그림 2" src="/files/ftp_upload/3266/3266fig2.jpg" /> 그림 2. COX / SDH 이중 라벨링 histochemical 분석의 플로우 차트. 관심의 장기를 해부하다, 빠르게 드라이 아이스에 조직을 동결하고,에 그들을 저장할 - 80 ° C를 그라 이오 스탯 섹션을 수집하고 감시 - 20 ° C까지 사용할 수 있습니다. 1 시간 동안 실온에서 공기 건조에 섹션을 허용합니다. COX의 histochemistry에 대한 배양 매체를 준비, 슬라이드에 적용, 37 40 분 품어 ° C. 십분마다 세척을 위해 PBS에서 4 번 섹션을 씻으십시오. SDH의 histochemistry에 대한 배양 매체를 준비, SLI에 적용DES는 다시 37 ° C. 40 분 알을 품다 PBS에 다시 섹션을 씻고, 에탄올 시리즈 탈수, 그리고 마운트와 슬라이드를 coverslip. COX / SDH를 두 번 표시 부분은 1-2 시간 이내에 밝은 필드 현미경 아래에서 볼 준비가되었습니다. <img alt="그림 3" src="/files/ftp_upload/3266/3266fig3.jpg" /> 그림 3. COX / SDH 이중 라벨의 대표 사례. 야생 유형 및 조기 노화 mtDNA의 mutator 마우스의 뇌 부분이 순차적으로 COX와 SDH 활동에 분류했다. (스케일 바. 200 μm의) 일반 COX 활동 (어두운 갈색 색상으로 표시) 야생 형 마우스 (왼쪽)에서 해마에 표시되었다. COX의 결함은 (파란색으로 표시) mtDNA mutator 마우스 (중앙 오른쪽)에서 해마에서 공개되었습니다. mtDNA mutator 마우스의 나이 46주에 의해 COX 활동에 더욱 감소는 호흡 사슬 기능 장애의 확산 악화를 제안,가 발생했습니다. 관찰된 미토mtDNA mutator 생쥐 12 chondrial 장애는 mtDNA 포인트 돌연변이뿐만 아니라 직선 삭제 5 증가 레벨 높은 수준의로 인해 발생합니다. <img alt="그림 4" src="/files/ftp_upload/3266/3266fig4.jpg" /> 그림 4. 부적 절한 COX / SDH 이중 라벨의 예. 야생 형 마우스의 뇌 부분이 순차적으로 COX와 SDH 활동에 분류했다. (스케일 바. 200 μm의) COX 활동의 시범에 대한 불충 분한 부화 시간은 (10 25 분) 40 분 배양 시간 (왼쪽 센터)에 비해, 갈색 DAB 반응 생성물의 감소 증착 결과. SDH 보육 동안 파란 formazan 엔드 제품의 형성 허용 단축 부화 시간은 misleadingly COX의 결함과 세포의 존재를 몰랐다. COX 보육 동안 슬라이드를 Coverslipping하면 DAB 반응의 부정확 형성 및 증착 결과이온 제품 (오른쪽). <img alt="그림 5" src="/files/ftp_upload/3266/3266fig5.jpg" /> 그림 5. I ndividual COX와 SDH 라벨 및 특이성 제어. 야생 형 마우스에서 뇌 섹션이 별도로 어두운 갈색 색상과 블루 색상, 각각 (왼쪽과 가운데)로 표시 COX와 SDH 활동에 대한 분류했다. COX와 SDH 활동 개별적으로 분류 될 수 있지만, 순차 상표는 mitochondrial 장애와 세포를 찾는데 유리한 것으로 입증되었습니다. 야생 형 마우스 뇌에서 COX와 SDH 활동을위한 특이성 컨트롤의 예제는 상표의 부재를 (오른쪽)했다. (스케일 바. 200 μm의)

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A., Lightowlers, R. N., James, O. F., Turnbull, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+mitochondrial+DNA+mutations+in+human+aging:+implications+for+the+central+nervous+system+and+muscle.">Role of mitochondrial DNA mutations in human aging: implications for the central nervous system and muscle.</a> Ann. Neurol. 43, 217-223 (1998).</li><li>Borthwick, G. M., Johnson, M. A., Ince, P. G., Shaw, P. J., Turnbull, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+enzyme+activity+in+amyotrophic+lateral+sclerosis:+implications+for+the+role+of+mitochondria+in+neuronal+cell+death.">Mitochondrial enzyme activity in amyotrophic lateral sclerosis: implications for the role of mitochondria in neuronal cell death.</a> Ann. Neurol. 46, 787-790 (1999).</li><li>Gellerich, F. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+respiratory+rates+and+activities+of+respiratory+chain+complexes+correlate+linearly+with+heteroplasmy+of+deleted+mtDNA+without+threshold+and+independently+of+deletion+size.">Mitochondrial respiratory rates and activities of respiratory chain complexes correlate linearly with heteroplasmy of deleted mtDNA without threshold and independently of deletion size.</a> Biochim. Biophys. Acta. 1556, 41-52 (2002).</li><li>Larsson, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+transcription+factor+A+is+necessary+for+mtDNA+maintenance+and+embryogenesis+in+mice.">Mitochondrial transcription factor A is necessary for mtDNA maintenance and embryogenesis in mice.</a> Nat. Genet. 18, 231-236 (1998).</li><li>Ekstrand, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+parkinsonism+in+mice+with+respiratory-chain-deficient+dopamine+neurons.">Progressive parkinsonism in mice with respiratory-chain-deficient dopamine neurons.</a> Proc. Natl. Acad. Sci. U. S. A. 104, 1325-1330 (2007).</li><li>Seligman, A. M., Karnovsky, M. J., Wasserkrug, H. L., Hanker, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nondroplet+ultrastructural+demonstration+of+cytochrome+oxidase+activity+with+a+polymerizing+osmiophilic+reagent,+diaminobenzidine+(DAB).">Nondroplet ultrastructural demonstration of cytochrome oxidase activity with a polymerizing osmiophilic reagent, diaminobenzidine (DAB).</a> J. Cell. Biol. 38, 1-14 (1968).</li><li>Dubowitz, V., Brooke, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+Biopsy:+A+Modern+Approach.">Muscle Biopsy: A Modern Approach.</a> , (1973).</li><li>Cottrell, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytochrome+c+oxidase+deficient+cells+accumulate+in+the+hippocampus+and+choroid+plexus+with+age.">Cytochrome c oxidase deficient cells accumulate in the hippocampus and choroid plexus with age.</a> Neurobiol. Aging. 22, 265-272 (2001).</li><li>Blanco, C. E., Sieck, G. C., Edgerton, V. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+histochemical+determination+of+succinic+dehydrogenase+activity+in+skeletal+muscle+fibres.">Quantitative histochemical determination of succinic dehydrogenase activity in skeletal muscle fibres.</a> Histochem. J. 20, 230-243 (1988).</li><li>Moraes, C. T., Ricci, E., Bonilla, E., DiMauro, S., Schon, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mitochondrial+tRNA(Leu(UUR))+mutation+in+mitochondrial+encephalomyopathy,+lactic+acidosis,+and+strokelike+episodes+(MELAS):+genetic,+biochemical,+and+morphological+correlations+in+skeletal+muscle.">The mitochondrial tRNA(Leu(UUR)) mutation in mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes (MELAS): genetic, biochemical, and morphological correlations in skeletal muscle.</a> Am. J. Hum. Genet. 50, 934-949 (1992).</li><li>Petruzzella, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extremely+high+levels+of+mutant+mtDNAs+co-localize+with+cytochrome+c+oxidase-negative+ragged-red+fibers+in+patients+harboring+a+point+mutation+at+nt+3243.">Extremely high levels of mutant mtDNAs co-localize with cytochrome c oxidase-negative ragged-red fibers in patients harboring a point mutation at nt 3243.</a> Hum. Mol. Genet. 3, 449-454 (1994).</li><li>Tulinius, M. H., Holme, E., Kristiansson, B., Larsson, N. G., Oldfors, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+encephalomyopathies+in+childhood.+I.+Biochemical+and+morphologic+investigations.">Mitochondrial encephalomyopathies in childhood. I. Biochemical and morphologic investigations.</a> J. Pediatr. 119, 242-250 (1991).</li><li>Haas, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+in-depth+evaluation+of+suspected+mitochondrial+disease.">The in-depth evaluation of suspected mitochondrial disease.</a> Mol. Genet. Metab. 94, 16-37 (2008).</li></ol>

관심 없음 충돌 선언하지 않습니다.

통합 COX / SDH histochemical 방법은 mitochondrial 장애와 세포의 시각화 수 있습니다. 이 기법은, 초기 연구는 1968까지 거슬러 올라가는와 함께, 많은이에게 환자 14,19,26,27에서 mitochondrial 질병을 식별에 대한 &quot;황금 표준&quot;을 고려와 함께 인기가 남아있다. 그것은 지금 자주 mtDNA 변이 기반 노화와 노화 관련 질환 12,13,18,20,21,24을 조사하는 데 사용됩니다. COX / SDH 이중 라벨 부착 방법은 ...

<table border="1"><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 (옵션) </td></tr><tr><td> 드라이 아이스 </td><td> AGA 가스 AB </td><td> 블록 형태 </td><td></td></tr><tr><td> 이소펜탄 (2 methylbutane) </td><td> 시그마 - 올드 리치 </td><td> 277,258 CAS : ​​78-78-4 </td><td></td></tr><tr><td> Cyrostat의 삽입 솔루션 </td><td> 사쿠라 Finetek </td><td> 조직 테크 4583 </td><td></td></tr><tr><td> 그라 이오 스탯 </td><td> Microm </td><td> Microm 모델 HM 500 미터 </td><td></td></tr><tr><td> 슬라이드 </td><td> 써모 과학 </td><td> 슈퍼 프로스트 플러스 Menzel Gläser J1800AMWZ </td><td></td></tr><tr><td> 커버 안경 Borosilicate 유리 </td><td> VWR 인터내셔널 </td><td> 16004-098 </td><td> 24 X 50mm </td></tr><tr><td> 필터 용지 </td><td> Munktell 필터 AB </td><td> 품질 : 1350 기사번호 : 242 001 </td><td> 430 X 430mm </td></tr><tr><td> 3,3 &#39;- diaminobenzidine의 tetrahydrochloride (DAB) </td><td> 시그마 - 올드 리치 </td><td> 시그마 액체 기판 시스템, D7304 </td><td></td></tr><tr><td> 시토크롬 C (유형 III, 말 마음에서) </td><td> 시그마 - 올드 리치 </td><td> C2506 CAS : ​​9007-43-6 </td><td></td></tr><tr><td> (간부터) 카탈 라 아제 소 </td><td> 시그마 - 올드 리치 </td><td> C9322 CAS : ​​9001-05-2 </td><td></td></tr><tr><td> Nitroblue tetrazolium (NBT) </td><td> 시그마 - 올드 리치 </td><td> N6876 CAS : ​​298-83-9 </td><td></td></tr><tr><td> 나트륨 호박산 </td><td> 시그마 - 올드 리치 </td><td> S2378 CAS : ​​6106-21-4 </td><td></td></tr><tr><td> Phenazine의 methosulfate (PMS) </td><td> 시그마 - 올드 리치 </td><td> P9625 CAS : ​​299-11-6 </td><td> PMS는 빛을 구분합니다. 빛과 방패. </td></tr><tr><td> 나트륨 azide </td><td> 시그마 - 올드 리치 </td><td> S8032 CAS : ​​26628-22-8 </td><td></td></tr><tR&gt; <td> 크실렌 </td><td> VWR 인터내셔널 </td><td> EM - XX0060 - 4 </td><td></td></tr><tr><td> Entellan </td><td> VWR 인터내셔널 </td><td> 100503-870 </td><td></td></tr><tr><td> 말로 네이트 (Malonic 산) </td><td> 시그마 - 올드 리치 </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

Mitochondrial DNA (mtDNA) 결함이 질병의 중요한 원인이며, 변경의 1,2를 노화와 노화 관련 기초 수 있습니다. 노화의 mitochondrial 이론은 노화 3, bioenergetics의 항상성 및 세포 기능을 변경할 수 있습니다 mtDNA 변이에 대한 역할을 제안합니다. 증거가 풍부한이 이론 1,4, mtDNA mutator 마우스 오되는 예제를 지원하도록 컴파일되어 있지만, 노화의 mtDNA 손상의 정확한 역할은 6,7 완전히 이해되지 않습니다. 호흡 효소의 활동을 관찰하는 것은 mitochondrial 장애를 조사하는 간단한 방법입니다. 복잡한 IV 또는 시토크롬 C 산화 효소 (COX)는 mitochondrial 기능에 대한 필수적입니다. COX의 촉매 subunits는 mtDNA에 의해 인코딩과 복잡한 조립 (그림 1) 필수입니다. 그러므로, 적절한 합성과 기능을 크게 mtDNA 무결성 2 기반으로합니다.다른 호흡 단지가 조사 될 수 있지만, 단지 IV와 II는 histochemical 시험 8,9 가장 의무가 있습니다. 증가 mitochondrial biogenesis 10-12을 나타내는 수도 있지만, 복잡한 II, 또는 호박산 탈수소 효소 (SDH)는, 완전히 핵 DNA (그림 1)에 의해 인코딩되어, 그 활동은 일반적으로 장애인 mtDNA에 의해 영향을받지 않습니다. 장애 mtDNA는 노화, mitochondrial 질환에서 관찰하고, 연령 관련 질병은 종종 낮은 값이나 COX 활동 2,12-14과 함께 세포의 존재에 이르게한다. COX와 SDH 활동 개별적으로 조사 수 있지만, 순차적 이중 라벨 부착 방법 15,16은 mitochondrial 장애 12,17-21와 세포를 찾는데 유리한 것으로 입증되었습니다. 분석의 최적의 체질의 대부분은 이러한 기판 농도, 전자 acceptors / 기증자, 중급 전자 통신 사업자, 산도의 영향, 그리고 반응 t으로 결정되었습니다IME 9,22,23. 3,3 &#39;- diaminobenzidine (DAB)은 효과적이고 신뢰할 수있는 전자 기증자 22. COX 기능과 세포에서 갈색 indamine 폴리머 제품은 mitochondrial cristae에 집중하고 세포에게 22 포화 것입니다. 장애 COX있는 사람 세포 따라서 파란색 formazan 엔드 제품 9,24에 nitroblue tetrazolium (NBT), 전자 수용체의 감소에 의한 SDH 활동의 시각화에 대한 허용, DAB 제품에서 포화되지 않습니다. 시토크롬 C와 나트륨 호박산 기판은 제어와 돌연변이 / 병에 걸린 조직 구 사이의 내생적인 수치를 정상화에 추가됩니다. 카탈 라 아제 퍼옥시데이즈 활동 9,22에서 가능한 오염 반응을 피하기 위해 예방책으로 추가됩니다. Phenazine의 methosulfate (PMS), 중간 전자 운반체는 최종 반응 제품 9,25의 형성을 증가 나트륨 azide, 호흡 사슬 억제제와 함께 사용됩니다. 이 통보에도 불구하고ation,이 알맞은 간단 분석의 결과에 영향을 미치는 몇 가지 중요한 세부 사항은, 특이성 컨트롤과 기술의 진보뿐만 아니라, 아직 제시되지 않았습니다.

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

시토크롬 C 산화 효소 / 나트륨 탈수소 효소 (COX / SDH)를 두 번 라벨링 방법은 신선한 - 냉동 조직 섹션에서 mitochondrial 호흡 효소 결함의 직접적인 시각화를위한 수 있습니다. 이것은 간단한 histochemical 기법입니다 mitochondrial 질병, 노화, 그리고 노화 관련 질환을 조사하는 데 유용합니다.

시토크롬를 사용 Mitochondrial 호흡 기능의 시각화 C 데이스 / 호박산 탈수소 효소 (COX / SDH)를 두 번 라벨 Histochemistry

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

visualization-mitochondrial-respiratory-function-using-cytochrome-c

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Biology

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

36.5K 조회수.  Karolinska Institutet. 시토크롬 C 산화 효소 / 나트륨 탈수소 효소 (COX / SDH)를 두 번 라벨링 방법은 신선한 - 냉동 조직 섹션에서 mitochondrial 호흡 효소 결함의 직접적인 시각화를위한 수 있습니다. 이것은 간단한 histochemical 기법입니다 mitochondrial 질병, 노화, 그리고 노화 관련 질환을 조사하는 데 유용합니다.

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

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

Microinjection을 사용하여 안정적인 트렌스​​ 제닉 C. 엘레간스의 생성

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

연구

생물학

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

시토크롬의 세포질 Microinjection 의한 Apoptosis의 활성화 C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

The isolated microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of factors that control vessel diameter, and thus, perfusion resistance1-5. This is a classic experimental preparation that was, in large measure, initially described by Uchida et al.15 several decades ago. This initial description provided the basis for the techniques that was extensively modified and enhanced, primarily in the laboratory of Dr. Brian Duling at the University of Virginia6-8, and we present a current approach in the following pages. This preparation will specifically refer to the gracilis arteriole in a rat as the microvessel of choice, but the basic preparation can readily be applied to vessels isolated from nearly any other tissue or organ across species9-13. Mechanical (i.e., dimensional) changes in the isolated microvessels can easily be evaluated in response to a broad array of physiological (e.g., hypoxia, intravascular pressure, or shear) or pharmacological challenges, and can provide insight into mechanistic elements comprising integrated responses in an intact, although ex vivo, tissue. The significance of this method is that it allows for facile manipulation of the influences on the integrated regulation of microvessel diameter, while also allowing for the control of many of the contributions from other sources, including intravascular pressure (myogenic), autonomic innervation, hemodynamic (e.g., shear stress), endothelial dependent or independent stimuli, hormonal, and parenchymal influences, to provide a partial list. Under appropriate experimental conditions and with appropriate goals, this can serve as an advantage over in vivo or in situ tissue/organ preparations, which do not readily allow for the facile control of broader systemic variables. 
The major limitation of this preparation is essentially the consequence of its strengths. By definition, the behavior of these vessels is being studied under conditions where many of the most significant contributors to the regulation of vascular resistance have been removed, including neural, humoral, metabolic, etc. As such, the investigator is cautioned to avoid over-interpretation and extrapolation of the data that are collected utilizing this preparation. The other significant area of concern with regard to this preparation is that it can be very easy to damage cellular components such as the endothelial lining or the vascular smooth muscle, such that variable source of error can be introduced. It is strongly recommended that the individual investigator utilize appropriate measurements to ensure the quality of the preparation, both at the initiation of the experiment and periodically throughout the course of a protocol.

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

An ex vivo preparation is described for isolation of the largest gracilis muscle resistance arterioles for interrogation of both vascular responses to vasoactive stimuli and the assessment of basic structural properties via passive wall mechanics.

예 생체내</em혈관 반응성 조사를위한> 절연 골격 Microvessel 준비

The isolated  microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of  factors that control vessel ...

Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC regulates mitochondrial function and cellular energy status. Numerous reports demonstrated that the activation of PKC-a and PKC-&epsilon; improves mitochondrial function in the ischemic heart and mediates cardioprotection. In contrast, we have demonstrated that PKC-&alpha; and PKC-&epsilon; are involved in nephrotoxicant-induced mitochondrial dysfunction and cell death in kidney cells. Therefore, the goal of this study was to develop an in vitro model of renal cells maintaining active mitochondrial functions in which PKC isozymes could be selectively activated or inhibited to determine their role in regulation of oxidative phosphorylation and cell survival. Primary cultures of renal proximal tubular cells (RPTC) were cultured in improved conditions resulting in mitochondrial respiration and activity of mitochondrial enzymes similar to those in RPTC in vivo. Because traditional transfection techniques (Lipofectamine, electroporation) are inefficient in primary cultures and have adverse effects on mitochondrial function, PKC-&epsilon; mutant cDNAs were delivered to RPTC through adenoviral vectors. This approach results in transfection of over 90% cultured RPTC.
Here, we present methods for assessing the role of PKC-&epsilon; in: 1. regulation of mitochondrial morphology and functions associated with ATP synthesis, and 2. survival of RPTC in primary culture. PKC-&epsilon; is activated by overexpressing the constitutively active PKC-&epsilon; mutant. PKC-&epsilon; is inhibited by overexpressing the inactive mutant of PKC-&epsilon;. Mitochondrial function is assessed by examining respiration, integrity of the respiratory chain, activities of respiratory complexes and F0F1-ATPase, ATP production rate, and ATP content. Respiration is assessed in digitonin-permeabilized RPTC as state 3 (maximum respiration in the presence of excess substrates and ADP) and uncoupled respirations. Integrity of the respiratory chain is assessed by measuring activities of all four complexes of the respiratory chain in isolated mitochondria. Capacity of oxidative phosphorylation is evaluated by measuring the mitochondrial membrane potential, ATP production rate, and activity of F0F1-ATPase. Energy status of RPTC is assessed by determining the intracellular ATP content. Mitochondrial morphology in live cells is visualized using MitoTracker Red 580, a fluorescent dye that specifically accumulates in mitochondria, and live monolayers are examined under a fluorescent microscope. RPTC viability is assessed using annexin V/propidium iodide staining followed by flow cytometry to determine apoptosis and oncosis.
These methods allow for a selective activation/inhibition of individual PKC isozymes to assess their role in cellular functions in a variety of physiological and pathological conditions that can be reproduced in in vitro.

The effects of activation of protein kinase C (PKC) isozymes on mitochondrial functions associated with respiration and oxidative phosphorylation and on cell viability are described. The approach adapts adenoviral technique to selectively overexpress PKC isozymes in primary cell culture and a variety of assays to determine mitochondrial functions and energy status of the cell.

신장 세포 Overexpressing 단백질 키나제 C Isozymes에 Mitochondrial 기능 및 세포 생존 능력의 평가

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC ...

Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, convergence and extension of facial prominences, and maturation of the craniofacial skeleton. To study the contribution of the cranial neural crest to specific regions of the zebrafish palate a sox10: kaede transgenic zebrafish line was generated. Sox10 provides lineage restriction of the kaede reporter protein to the neural crest, thereby making the cell labeling a more precise process than traditional dye or reporter mRNA injection. Kaede is a photo-convertible protein that turns from green to red after photo activation and makes it possible to follow cells precisely. The sox10: kaede transgenic line was used to perform lineage analysis to delineate CNC cell populations that give rise to maxillary versus mandibular elements and illustrate homology of facial prominences to amniotes. This protocol describes the steps to generate a live time-lapse video of a sox10: kaede zebrafish embryo. Development of the ethmoid plate will serve as a practical example. This protocol can be applied to making a time-lapse confocal recording of any kaede or similar photoconvertible reporter protein in transgenic zebrafish. Furthermore, it can be used to capture not only normal, but also abnormal development of craniofacial structures in the zebrafish mutants.

Visualization of experimental data has become a key element in presenting results to the scientific community. Generation of live time-lapse recording of growing embryos contributes to better presentation and understanding of complex developmental processes. This protocol is a step-by-step guide to cell labeling via photoconversion of kaede protein in zebrafish.

sox10의 두개 안면 개발의 시각화 : 단풍 나무 형질 전환 제브라 피쉬의 선은 시간 경과 공 초점 현미경을 사용하여

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, ...

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well when a small number of genes are targeted for knockdown, large scale feeding screens show variable knockdown efficiencies, which limits their utility. We have deconstructed previously published RNAi knockdown protocols and found that the primary source of the reduced knockdown can be attributed to the loss of dsRNA-encoding plasmids from the bacteria fed to the animals. Based on these observations, we have developed a dsRNA feeding protocol that greatly reduces or eliminates plasmid loss to achieve efficient, high throughput knockdown. We demonstrate that this protocol will produce robust, reproducible knock down of C. elegans genes in multiple tissue types, including neurons, and will permit efficient knockdown in large scale screens. This protocol uses a commercially available dsRNA feeding library and describes all steps needed to duplicate the library and perform dsRNA screens. The protocol does not require the use of any sophisticated equipment, and can therefore be performed by any C. elegans lab.

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

While dsRNA feeding in C. elegans is a powerful tool to assess gene function, current protocols for large scale feeding screens result in variable knockdown efficiencies. We describe an improved protocol for performing large scale RNAi feeding screens that results in highly efficacious and reproducible knockdown of gene expression.

대규모 유전자 넉다운에<em&gt; C. 엘레</em&gt; 강력한 기능 상실 표현형을 생성하는 dsRNA를 먹이는 라이브러리 사용

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well ...

Measuring Respiratory Function in Mice Using Unrestrained Whole-body Plethysmography

Respiratory dysfunction is one of the leading causes of morbidity and mortality in the world and the rates of mortality continue to rise. Quantitative assessment of lung function in rodent models is an important tool in the development of future therapies. Commonly used techniques for assessing respiratory function including invasive plethysmography and forced oscillation. While these techniques provide valuable information, data collection can be fraught with artefacts and experimental variability due to the need for anesthesia and/or invasive instrumentation of the animal. In contrast, unrestrained whole-body plethysmography (UWBP) offers a precise, non-invasive, quantitative way by which to analyze respiratory parameters. This technique avoids the use of anesthesia and restraints, which is common to traditional plethysmography techniques. This video will demonstrate the UWBP procedure including the equipment set up, calibration and lung function recording. It will explain how to analyze the collected data, as well as identify experimental outliers and artefacts that results from animal movement. The respiratory parameters obtained using this technique include tidal volume, minute volume, inspiratory duty cycle, inspiratory flow rate and the ratio of inspiration time to expiration time. UWBP does not rely on specialized skills and is inexpensive to perform. A key feature of UWBP, and most appealing to potential users, is the ability to perform repeated measures of lung function on the same animal.

The assessment of respiratory physiology has traditionally relied upon techniques, which require restraint or sedation of the animal. Unrestrained whole-body plethysmography, however, provides precise, non-invasive, quantitative analysis of respiratory physiology in animal models. In addition, the technique allows repeated respiratory assessment of mice allowing for longitudinal studies.

무제한 전신 혈량 측정법을 사용하여 마우스의 호흡 기능을 측정

Respiratory dysfunction is one of the leading causes of morbidity and mortality in the world and the rates of mortality continue to rise. Quantitative ...

High-resolution Respirometry to Assess Mitochondrial Function in Permeabilized and Intact Cells

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in biological samples (intact and permeabilized cells, tissues or isolated mitochondria). The high-resolution oxygraph device is equipped with two chambers and uses polarographic oxygen sensors to measure oxygen concentration and calculate oxygen consumption within each chamber. Oxygen consumption rates are calculated using software and expressed as picomoles per second per number of cells. Each high-resolution oxygraph chamber contains a stopper with injection ports, which makes it ideal for substrate-uncoupler-inhibitor titrations or detergent titration protocols for determining effective and optimum concentrations for plasma membrane permeabilization. The technique can be applied to measure respiration in a wide range of cell types and also provides information on mitochondrial quality and integrity, and maximal mitochondrial respiratory electron transport system capacity.

High-resolution respirometry is used to determine mitochondrial oxygen consumption. This is a straightforward technique to determine mitochondrial respiratory chain complexes&#39; (I-IV) respiratory rates, maximal mitochondrial electron transport system capacity, and mitochondrial outer membrane integrity.

고해상도 호흡율은 투과성이 그대로 세포에서 미토콘드리아 기능을 평가하는

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in ...

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Fluorescence microscopy is an indispensable tool for the direct assessment of mitochondrial structure and function in live cells and for studying the mitochondrial structure-function relationship, which is primarily modulated by the molecules governing fission and fusion events between mitochondria. This paper describes and demonstrates specific methods for studying mitochondrial structure and function in live as well as in fixed tissue in the model organism Drosophila melanogaster. The tissue of choice here is the Drosophila ovary, which can be isolated and made amenable for ex vivo live confocal microscopy. Furthermore, the paper describes how to genetically manipulate the mitochondrial fission protein, Drp1, in Drosophila ovaries to study the involvement of Drp1-driven mitochondrial fission in modulating the mitochondrial structure-function relationship. The broad use of such methods is demonstrated in already-published as well as in novel data. The described methods can be further extended towards understanding the direct impact of nutrients and/or growth factors on the mitochondrial properties ex vivo. Given that mitochondrial dysregulation underlies the etiology of various diseases, the described innovative methods developed in a genetically tractable model organism, Drosophila, are anticipated to contribute significantly to the understanding of the mechanistic details of the mitochondrial structure-function relationship and to the development of mitochondria-directed therapeutic strategies.

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Specific methods for studying mitochondrial structure and function in live and fixed Drosophila ovaries are described and demonstrated in this paper.

에서 미토콘드리아의 구조와 기능을 연구<em&gt; 초파리</em&gt; 난소

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.