Description
Observing the activity of respiratory enzymes is a straightforward approach for investigating mitochondrial dysfunction. Cytochrome c oxidase (COX) is essential for mitochondrial function. Succinate dehydrogenase (SDH) is entirely encoded by nuclear DNA and its activity is typically not affected by impaired mitochondrial DNA (mtDNA). Combining the COX with the SDH can demonstrate COX negative fibers. These may be SDH positive and may be ragged red fibers. Three of the 13 subunits of COX are encoded by mitochondrial DNA whereas SDH is encoded by nuclear DNA. Therefore SDH is not affected by mitochondrial DNA mutations. Ragged red fibers in mitochondrial myopathies are generally COX negative except in Mitochondrial Encephalopathy, Lactic acidosis, and Stroke-like episodes (MELAS). Intra fiber mosaicisms (mixture of bluish COX deficient and brownish COX positive mitochondria within the same fiber) are also well demonstrated by this combined technique. 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.
Kit Components
| SKU# | Reagent | Size |
| VB-3022-1 | COX A Solution | 1ml×5 |
| VB-3022-2 | COX B Solution | 1ml×5 |
| VB-3022-3 | Succinate Solution | 0.3ml×5 |
| VB-3022-4 | Yellow SDH Incubation Medium | 1.5 ml×5 |
| VB-3022-5 | COX Inhibitor Solution | 1ml×2 |
| VB-3022-6 | SDH Inhibitor Solution | 1ml×2 |
- Storage
Store at -20°C
Protocol
- Tissue preparation for cryosectioning
- Sacrifice the animal by either cervical dislocation or decapitation, in accordance with available ethical permit.
- Quickly collect tissues of interest without fixation, and rapidly freeze on dry ice (tissues may require freezing in isopentane) or propane chilled with liquid nitrogen to obtain optimal morphology).
- Store tissues in aluminum foil at -80 °C until ready to section.
- Embed frozen tissue in preparation for cryosectioning.
- Collect 10-14μm cryostat sections. Thaw sections onto slides at room temperature for 2-5 minutes, and store slides without cover-slipping at -20 °C until ready to use.
- Prepare COX incubation solution: Defrost one COX A Solution (VB-3022-1) and one COX B Solution (VB-3022-2) vial. One vial of COX A solution are mixed with 1 vial of COX B solution.
- Immediately put 80~200ul of COX incubation solution onto frozen sectioned slides in humidity chamber and incubate in dark for two hours at room temperature.
- Check staining and replace for longer if required
- Rinse slides in distilled water
- Freshly prepare SDH incubation medium: Defrost a vial (0.3ml) of Succinate Solution (VB-3022-3) and a vial of Yellow SDH Incubation Medium (1.5 ml) (VB-3022-4); Add solution succinate solution to Yellow SDH Incubation Medium prior to use and mix well.
- Add 80~200ul of SDH incubation medium onto slide and incubate at 37 ͦ C for 1-2 hour.
- Rinse slides in distilled water.
- Rinse in water, dehydrate, clear and mount.
Appropriate specificity controls
- For specificity controls for COX activity, repeat “COX histochemistry” steps, and add 100µl of COX Inhibitor Solution (VB-2022-5), a terminal respiratory chain inhibitor.
- For specificity controls for SDH activity, repeat “SDH histochemistry” steps with the removal of sodium succinate and the addition of 100µl of SDH Inhibitor Solution (VB-2022-6), a competitive inhibitor of SDH.
Results
| Cytochrome Oxidase positive mitochondria——- | Brown |
| Cytochrome Oxidase negative mitochondria——- | Blue |
References
- Ross, J.M. Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase (COX/SDH) Double-labeling Histochemistry. J. Vis. Exp. (57), e3266, DOI : 10.3791/3266 (2011).
- Seligman etal (1968) J Cell Biol 38:1-14.
- Loughlin M. (1993). Muscle biopsy. A laboratory investigation. Butterworth-Heinemann p.38-39.
- Sheehan D, Hrapchak B. (1987). Histotechnology, 2nd Ed. Batelle Press, Columbus p306-307
Recent Publications Using VB-3022
- Dombrecht D, Daele UV, Asbroeck BV, David R. Schieffelers DR, Guns PJ,Breda EV (2023). Skeletal muscle wasting after burn is regulated by a decrease in anabolic signaling in the early flow phase. BurnsVolume; 49 ( 7): 1574-1584
- Dang X, Zhang L, Franco S, Dorn II GW (2023). Activating mitofusins interrupts mitochondrial degeneration and delays disease progression in SOD1 mutant amyotrophic lateral sclerosis. Human Molecular Genetics; 32 (7): 1208–1222
- Mosteiro L, Nguyen TTT, Hankeova S, Alvarez-Sierra D, Reichelt M, Vandriel SM, Lai Z, Choudhury FK, Sangaraju D, Kamath DM, Scherl A, Pujol-Borrell R, Robert Piskol R & Christian W. Siebel (2023). Notch signaling in thyrocytes is essential for adult thyroid function and mammalian homeostasis. Nature Metabolism; 5: 2094–2110
Note
This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.
Precautions
Handle with care. Avoid contact with eyes, skin and clothing. Do not ingest. Wear gloves.
