Succinate Dehydrogenase Stain

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

Protocol

  • 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.

10 thoughts on “Succinate Dehydrogenase Stain

  1. Anthony.Sinadinos@port.ac.uk

    Hello,
    I’ve just stumbled upon this website. I am finishing a phd looking into the role of a purinergic receptor, P2RX7, in the pathogenesis of DMD at the University of Portsmouth, UK. I think your images are great and I appreciate your philosophical stance on sharing information within science.
    I’m mostly commenting to inform you that I may try some of the innovations you mention in the methods sections of your website. If I end up adopting any of your innovations, I’ll acknowledge you.

    Best regards,
    Anthony Sinadinos

    1. Hi Anthony,

      Thanks for your message. I hope that you have (or are about to) submitted your thesis. Always glad to hear that people find some of these methods useful.

      James

      1. Irne

        Hi,
        did you ever tried or know someone who tried this staining in fixed primary cultured cells?
        Thanks!
        Irene

      2. Hi Irene,
        SDH is actually an enzymatic reaction which leads to the buildup of the blue ‘stain’. For the reaction to work the enzyme must still be active, which means this will only work on fresh-frozen sections.
        You won’t be able to measure oxidative activity on cells that have been fixed, but you could do an immunofluorescent stain for OXPHOS proteins instead.

        Hope this helps,

        James

  2. Louise

    Just a question on how long one can maintain the sections at -20C before staining with SDH? I have samples of hindlimbs stored at -80C that I wish to section and wonder how soon after the sectioning I should do the stain?
    Also, will storing the limbs at -80C impact the enzyme activity?
    Many thanks for a really nice clear protocol!
    Louise

    1. Hi Louise,

      Glad you like the protocol! In regards to your question, the sooner you stain the sections the better (ideal if you can cut all sections in one day, or even cut n=1-2 per group and then stain the sections fresh). It is possible to store the sections at -20 (remember to keep the sections at -20 at all times), but the enzymatic activity will degrade over time. You can keep the whole muscle frozen at -80 for an extended period (years), but remember that the enzymatic activity will degrade. The best option is to dissect out the muscles, freeze in thawing isopentane immediately, and cut/stain within a week. As with all research, the higher the quality of your starting material, the higher the quality of the results (and vice-versa, garbage in = garbage out).

      Hope this helps, anything else, just ask.

      James

      1. Louise

        Thanks so much for that James, that’s really helpful. I also had a question regarding immunostaining cryosections that had already been stained with sdh – can this be done? I’d like to immunostain the basment membrane using nidogen, and wonder if the affinity of the antibody will be altered by the sdh. Any comments/suggestions would be greatly appreciated!

        Many thanks for such a great resource,

        Louise

      2. Absolutely, we occasionally link the SDH stain to either laminin or specific myosin heavy chain immunofluorescence (or both). Just try one slide first to make sure it all works as expected.

  3. Wayne Hancock

    You mention the interesting point that one can detect the actual enzyme, SDH, in sections using immunohistology. Had you tried that and how well do the results compare with the enzyme histochemical reaction (better, worse, same)? Likewise, can the immunohistologic approach be applied to paraffin sections?

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