Цитохром с оксидазы / натрия дегидрогеназы (ЦОГ / SDH) двойной маркировки метод позволяет для прямой визуализации митохондриальных дыхательных ферментов недостатки в свежезамороженных срезах тканей. Это просто гистохимические техники и полезен в расследовании митохондриальных болезней, старения и связанных со старением заболеваний.
Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of aging suggests a role for mtDNA mutations, which can alter bioenergetics homeostasis and cellular function, in the aging process 3. A wealth of evidence has been compiled in support of this theory 1,4, an example being the mtDNA mutator mouse 5; however, the precise role of mtDNA damage in aging is not entirely understood 6,7.
Observing the activity of respiratory enzymes is a straightforward approach for investigating mitochondrial dysfunction. Complex IV, or cytochrome c oxidase (COX), is essential for mitochondrial function. The catalytic subunits of COX are encoded by mtDNA and are essential for assembly of the complex (Figure 1). Thus, proper synthesis and function are largely based on mtDNA integrity 2. Although other respiratory complexes could be investigated, Complexes IV and II are the most amenable to histochemical examination 8,9. Complex II, or succinate dehydrogenase (SDH), is entirely encoded by nuclear DNA (Figure 1), and its activity is typically not affected by impaired mtDNA, although an increase might indicate mitochondrial biogenesis 10-12. The impaired mtDNA observed in mitochondrial diseases, aging, and age-related diseases often leads to the presence of cells with low or absent COX activity 2,12-14. Although COX and SDH activities can be investigated individually, the sequential double-labeling method 15,16 has proved to be advantageous in locating cells with mitochondrial dysfunction 12,17-21.
Many of the optimal constitutions of the assay have been determined, such as substrate concentration, electron acceptors/donors, intermediate electron carriers, influence of pH, and reaction time 9,22,23. 3,3′-diaminobenzidine (DAB) is an effective and reliable electron donor 22. In cells with functioning COX, the brown indamine polymer product will localize in mitochondrial cristae and saturate cells 22. Those cells with dysfunctional COX will therefore not be saturated by the DAB product, allowing for the visualization of SDH activity by reduction of nitroblue tetrazolium (NBT), an electron acceptor, to a blue formazan end product 9,24. Cytochrome c and sodium succinate substrates are added to normalize endogenous levels between control and diseased/mutant tissues 9. Catalase is added as a precaution to avoid possible contaminating reactions from peroxidase activity 9,22. Phenazine methosulfate (PMS), an intermediate electron carrier, is used in conjunction with sodium azide, a respiratory chain inhibitor, to increase the formation of the final reaction products 9,25. Despite this information, some critical details affecting the result of this seemly straightforward assay, in addition to specificity controls and advances in the technique, have not yet been presented.
Комбинированный ЦОГ / SDH гистохимические метод позволяет визуализации клеток с митохондриальной дисфункцией. Этот метод, с ранних исследований, начиная с 1968 года, остается популярным, и многие считая его «золотым стандартом» для выявления митохондриальных заболеваний у пациентов, 1…
The authors have nothing to disclose.
Работа выполнена при поддержке Национального института старения (AG04418), Национальный институт по злоупотреблению наркотиками, Национальный Институт Здоровья-Каролинского института Высшей Программа партнерства Каролинского института, шведского исследовательского совета, шведский Питание мозга, и шведский фонд мозга. Большое спасибо Маттиас Карлен и доктор Джузеппе Coppotelli для творческой поддержки с рисунком 1 и 2 соответственно; Карин Pernold для оказания технической помощи, а также д-ра. Барри Дж. Хоффер, Ларс Олсон, и Нильс-Горан Ларссон на протяжении большей полезные советы и обсуждения.
Name of the reagent | Company | Catalogue number | Comments (optional) |
Dry Ice | AGA Gas AB | block form | |
Isopentane (2-methylbutane) | Sigma-Aldrich | 277258 CAS: 78-78-4 |
|
Cyrostat embedding solution | Sakura Finetek | Tissue Tek 4583 | |
Cryostat | Microm | Microm Model HM 500M | |
Slides | Thermo Scientific | Super Frost Plus Menzel Gläser J1800AMWZ |
|
Cover glasses Borosilicate glass |
VWR International | 16004-098 | 24 x 50 mm |
Filter Paper | Munktell Filter AB | Quality: 1350 Article Number: 242 001 |
430 x 430 mm |
3,3′-diaminobenzidine tetrahydrochloride (DAB) | Sigma-Aldrich | Sigma Liquid Substrate System, D7304 | |
Cytochrome c (Type III, from equine heart) | Sigma-Aldrich | C2506 CAS: 9007-43-6 |
|
Bovine catalase (from liver) | Sigma-Aldrich | C9322 CAS: 9001-05-2 |
|
Nitroblue tetrazolium (NBT) | Sigma-Aldrich | N6876 CAS: 298-83-9 |
|
Sodium succinate | Sigma-Aldrich | S2378 CAS: 6106-21-4 |
|
Phenazine methosulfate (PMS) | Sigma-Aldrich | P9625 CAS: 299-11-6 |
PMS is light sensitive. Shield from light. |
Sodium azide | Sigma-Aldrich | S8032 CAS: 26628-22-8 |
|
Xylene | VWR International | EM-XX0060-4 | |
Entellan | VWR International | 100503-870 | |
Malonate (Malonic acid) |
Sigma-Aldrich | M1296 CAS: 141-82-2 |