Succinate dehydrogenase (SDH, also known as complex II in the mitochondrial respiratory chain) is responsible for the oxidation of succinate to fumerate. A simple enzymatic reaction can be used to determine the relative SDH activity of individual muscle fibers in a skeletal muscle that has been fresh-frozen, and cryosectioned (with the cryosections maintained at -20C). This can be used to demonstrate changes in the overall oxidative capacity of a skeletal muscle, at the individual fiber level. The protocol I describe here is based upon a modified version of the methodology of Blanco et al. (1988).
Stock solutions (can be stored <12months at -20C)
Disodium succinate stock (0.24M)
- disodium succinate hexahydrate [3.24g]
- ddH2O up to 50ml
- store in 5ml aliquots
Azide/EDTA/PO4 Buffer stock
- Sodium Azide [12.2mg]
- disodium EDTA [465.3mg]
- NAH2PO4.H2O [448.5mg]
- Na2HPO4.7H2O [5.83g]
- Adjust pH to 7.6
- ddH2O up to 100ml
- Store in 10ml aliquots
Working SDH solution (made fresh each day, immediately prior to use)
- 1-methoxyphenzine methosulphate (mPMS) [8.4mg]
- Nitroblue tetrazolium (NBT) [30.7mg]
- disodium succinate stock [5ml]
- Azide/EDTA/PO4 Buffer stock [10ml]
- ddH2O up to 25ml
- Take slides directly from -20C and place in coplin jar containing SDH solution (place at least one control and one experimental sample per jar for comparison)
- Incubate for 5-20minutes (I find that the time will depend on: the muscle used, EDL will be closer to 15-20mins, while soleus will be closer to 5-10mins; and the animal used, rat muscle takes less time to stain than mouse). It is important to determine the optimal time first with a number of control slides, and then use this time exactly for all experiments.
- Stop enzymatic reaction by dipping the slides ONCE quickly in ddH2O
- Air-dry for ~10mins in the dark
- Mount slides
- The NBT should form a blue colored nitroblue-diformozan precipitate as shown below, the darker the intensity the higher the SDH activity.
- SDH activity can be quantified by converting the image to greyscale and measuring the grey intensity of each fiber (this can be done using the free image analysis software ImageJ).
Figure. Rat EDLmuscle stained using the SDH protocol described above, note the small fibers with high oxidative capacity (likely type I or IIa fibers), and larger fibers with intermediate (likely IId/x fibers) or low (likely IIb fibers) oxidative capacity.