PMC:7432599 / 41564-43273 JSONTXT

{"project":"2_test","denotations":[{"id":"32752280-22078881-52426040","span":{"begin":391,"end":393},"obj":"22078881"},{"id":"32752280-26069013-52426041","span":{"begin":414,"end":416},"obj":"26069013"},{"id":"32752280-27678062-52426042","span":{"begin":1071,"end":1073},"obj":"27678062"},{"id":"T52074","span":{"begin":391,"end":393},"obj":"22078881"},{"id":"T15462","span":{"begin":414,"end":416},"obj":"26069013"},{"id":"T87284","span":{"begin":1071,"end":1073},"obj":"27678062"}],"text":"4.9. Mitochondria Abundance and Lipid Content in Skeletal Muscle\nTo visualize mitochondrial abundance and lipid content in skeletal muscle; gastrocnemius and soleus muscles were embedded in optimal cutting temperature compound and rapidly frozen by immersion into liquid nitrogen and kept at −80 °C. Muscle succinate dehydrogenase (SDH) activity was determined according to Yamamoto et al. [61] and Kalmar et al. [62]. Briefly, frozen sections (12 μm) were mounted in positively charged slides (Kling-On SFH1103, BIOCARE Medical, Concord, CA, USA) and incubated in SDH staining solution (0.55 mM nitro-blue tetrazolium and 0.05 mM sodium succinate) and incubated at 37 °C for 60 min. Afterwards, the slides were washed with deionized water and sequentially dehydrated (2 min) in 30%, 60% and 90% acetone. Then, the slides were rehydrated (2 min) with 60% and 30% acetone in deionized water. Digital photographs were taken from each section at 20× magnification as described above, and positive fibers (blue) were quantified with Image J software as described previously [63].\nTo evaluate lipid content in muscle fibers, frozen slides were rapidly fixed by immersion in ice-cold PBS-buffered 4% paraformaldehyde for 10 min, washed in deionized water and incubated for 30 min with the lipophilic dye BODIPY 493/503 (790389, Sigma-Aldrich, 20 µg/mL in PBS). Slides were washed in PBS and mounted with an aqueous mounting medium with DAPI (ProLong Gold Antifade Mountant P36931, Invitrogen, Carlsbad, CA, USA). Digital photographs were taken from each section at 20X magnification under fluorescence illumination. DAPI (nuclei) and BODIPY (fat) images were merged and fat content quantified using ImageJ software."}