Muscle Fiber Composition and Morphology Analysis

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)