Diaphragm was fixed in formaldehyde and embedded in paraffin, and 8 µm transversal and longitudinal sections were stained with hematoxylin and eosin (H&E).
TA muscles were frozen in liquid nitrogen-cooled isopentane and stored at −80 °C. 8 µm transversal sections were stained with H&E, succinate dehydrogenase (SDH), reduced nicotinamide adenine dinucleotide (NADH) and Periodic Acid-Schiff (PAS) to assess muscle fiber morphology, nuclear position, and distribution of mitochondria, reticulum, and glycogen. The images were recorded using the Nanozoomer 2HT slide scanner (Hamamatsu). Myofiber segmentation was performed using Cellpose 1.0 algorithm60 (link) and LabelsToROIS Fiji plugin61 (link) minimum Feret diameter (MinFeret) calculated using ImageJ. Fibers with mislocalized nuclei (centralized or internalized) and fibers with abnormal SDH distribution were counted using Cell Counter ImageJ plugin. Minimum of 800 fibers were measured for each muscle section and a percentage was calculated for each mouse.
TA muscles were frozen in liquid nitrogen-cooled isopentane and stored at −80 °C. 8 µm transversal sections were stained with H&E, succinate dehydrogenase (SDH), reduced nicotinamide adenine dinucleotide (NADH) and Periodic Acid-Schiff (PAS) to assess muscle fiber morphology, nuclear position, and distribution of mitochondria, reticulum, and glycogen. The images were recorded using the Nanozoomer 2HT slide scanner (Hamamatsu). Myofiber segmentation was performed using Cellpose 1.0 algorithm60 (link) and LabelsToROIS Fiji plugin61 (link) minimum Feret diameter (MinFeret) calculated using ImageJ. Fibers with mislocalized nuclei (centralized or internalized) and fibers with abnormal SDH distribution were counted using Cell Counter ImageJ plugin. Minimum of 800 fibers were measured for each muscle section and a percentage was calculated for each mouse.
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