4.6. Histology and Succinate DeHydrogenase (SDH) Staining and Enzymatic Activity

During necropsy, one tibialis anterior muscle was mounted in OCT and frozen in melting isopentane for histology. Transverse sections (10 μM) were cut on a cryostat and stained for SDH incubating for 30 min at 37 °C with 1 mg/mL NTB (nitrotetrazolium blue chloride) and 27 mg/mL Na-succinate in PBS. Several pictures were taken and the whole TA section was digitally reconstructed. Fiber cross-sectional area (CSA) was determined on the whole muscle using the Image J software (freely available at https://imagej.nih.gov/ij/, accessed on 20 January 2021). The images were analyzed by three different researchers. The reliability among individuals was verified by analyzing one muscle of each group by all three researchers. The researchers were not blinded to groups when performing the analysis. As for the total SDH activity, the muscles were homogenized (5% wt/vol) in ice-cold 150 mM NaCl, 10 mM KH2PO4, 0.1 mM EGTA and centrifuged 5 min at 800 × g collecting the supernatant. 50 µL protein homogenates were incubated with 200 μL reaction buffer containing 10 mM Na-succinate, 50 μg/mL DCPIP, 10 mM phosphate buffer (pH 7.4), 2 mM KCN, 10 mM CaCl2, 0.05% BSA. The rate of absorbance decrease at 600 nm between 3 and 20 min was corrected for the protein loading and used to calculate the relative SDH activity.