Application suite las x imaging software

Histochemical succinate dehydrogenase (SDH) staining was performed on fresh-frozen tissue (FFT). Appropriate cross-sections of muscle were selected and submerged in liquid nitrogen. The tissue was then stored in a −80°C freezer until used. All samples were sectioned on a cryostat at 8–12 μm. Enzymatic activity of SDH was assayed by placing the slides in SDH incubating solution, containing 100.0 mM of sodium succinate salt as a substrate and Nitro-Blue Tetrazolium (NBT) for visualization of reaction, and 1.2 mM of NBT in 0.2 M phosphate buffer for 1 h at 37 °C. Reduced NBT forms a highly colored formazan dye that is finely granular blue. Samples were dehydrated and mounted with Eukitt^® mounting medium (Bio-Optica Improving Pathology, Milan, Italy). Images were captured using the Leica Application Suite LAS X Imaging Software with a fluorescent Leica DMi8 microscope and the quantification of SDH activity in muscle was performed using ImageJ software (NIH Image, Bethesda, MD, USA).

For immunofluorescence analysis, pOBs were induced to mineralization and treated on glass in the conditions described above. Then, cells were fixed in 4% PFA for 15 min at RT, permeabilized with 0.2% Triton™ X-100 (Sigma-Aldrich) in PBS, and blocked with PBS 5% FBS for 1 h at RT. The primary antibodies anti-p-DRP1[Ser616] (Cell Signaling Technologies, Danvers, MA, USA) were diluted 1:250 in blocking buffer and incubated for 2 h at RT. The secondary antibody conjugated with Alexa Fluor 488 (donkey anti-rabbit IgG, Invitrogen Thermo Fisher Scientific) was diluted in blocking buffer 1:300 at a concentration of 0.0067 mg/mL, and incubated for 45 min at RT. Nuclei were visualized with DAPI (Sigma Aldrich). Images were acquired with fluorescence microscope LEICA DMi8 equipped with Leica Application Suite LAS X Imaging Software. ImageJ software (NIH Image, Bethesda, MD, USA) was used to analyze fluorescence intensity and data were represented as fluorescence intensity/cell area versus G condition.

Muscles were dissected and frozen in liquid nitrogen-cooled isopentane, and 7 μm muscle cryosections were used for histology analyses. According to classical methods [62 (link)], cryostat sections were stained with Hematoxylin/Eosin (H&E). Briefly, for histology analysis, cross-sections were fixed in 4% formaldehyde at room temperature for 15 min and stained with H&E. Fiber size distribution was quantified by Image-J software (NIH Image, Bethesda, MD, USA). Up to 6 fields of view were captured from the same location within each muscle, and then 600 myofibers/muscle were measured. Images were captured using the Leica Application Suite LAS X Imaging Software with a fluorescent Leica DMi8 microscope.