Na succinate

TA and TB muscles were dissected out and snap-frozen in liquid nitrogen. For the muscle fiber composition (SDH staining), 10 μm-thickness serial coronal cryosections from the mid-belly region of the TA muscle were air-dried and then incubated at 37°C for 30 min in phosphate buffer (0.2 M, pH 7.6) containing 13.5 mg/mL Na-succinate (Sigma-Aldrich) and 0.5 mg/mL nitro blue tetrazolium (Sigma-Aldrich, 0.29 mg/mL buffer solution). After staining, sections were fixed with 4% paraformaldehyde, dehydrated in 15% alcohol for 5 min, and finally mounted with DPX compound (Sigma-Aldrich).
For the muscle fiber cross-sectional area, 10-μm thickness serial coronal cryosections from the mid-belly region of the TA muscle were air-dried, fixed in 4% paraformaldehyde solution for 5 min, and stained with wheat germ agglutinin, Alexa Fluor 488 conjugate (1:500; W11261, Thermo Fisher, Pittsburgh, PA, USA) and Hoechst (1:1,000; Roche).
Images were acquired with an Olympus virtual slide system VS110 (Olympus, Center Valley, USA) at 20× magnification and analyzed through Fiji (ImageJ) on 3–5 serial sections per animal. For the SDH staining, a systematic random sampling procedure was applied as described above. For the muscle fiber cross-sectional area and centralized nuclei, the entire TA or TB muscle section was analyzed with the MuscleJ plug-in of Fiji software, as previously described.¹⁴¹ (link)

Experiments were conducted at room temperature (25°C), with mitochondria (0.5 mg protein/ml) suspended in experimental buffer (buffer B) that contained (in mM) 130 KCl (EMD Chemicals, Gibbs-town, NJ, USA), 5 K₂HPO₄, 20 MOPS, 0.001 Na₄P₂O₇, and 0.1% BSA. This assured that only 40 μM EGTA was carried over from the isolation buffer (buffer A) into the experimental buffer. Based on the experimental protocol and conditions, the buffer pH was specifically adjusted upward from 6.5 to 6.9 and 7.15 by adding KOH. The respiration buffer contained 0 or 150 μM CaCl₂; concentrations of CaCl₂ between 20 and 60 μM had no significant effects on H₂O₂ production (Figure S.3) and 100 μM CaCl₂ gave inconsistent data, so these data are not reported. From the residual EGTA concentration of 40 μM, we estimated 150 μM CaCl₂ to be equivalent to ≈ 220 nmol CaCl₂/mg protein. After adding CaCl₂ (or H₂O), 10 mM Na⁺ pyruvate or Na⁺ succinate (Sigma) was added. Then either complex I blocker ROT (10 μM; Sigma) or complex III blocker AA (5 μM; Sigma) was added.