Nanozoomer rs c110730

For immunohistochemistry, whole slide images were acquired with a digital slide scanner (Nanozoomer-RS C110730, Hamamatsu) and then visualized using NDP.view2 software (Hamamatsu Photonics). For immunofluorescence, a confocal microscope (Nikon A1R, Nikon) was used to acquire a 20 × 5-z-stack tile-scan of the whole adductor muscle section every 1.5 µm. Fiji/Image J software was used to manually select superficial adductor muscle area, where the same software automatically selected and counted the remodeled arterioles (> 40 µm diameter)^{42 (link)} in both immunohistochemistry and immunofluorescence samples. Total number of VSMCs (DAPI⁺/SMA⁺) and proliferative VSMCs (DAPI⁺/SMA⁺/EdU⁺) were counted within the remodeled arterioles. For analysis of PGC1α in arterioles, a confocal microscope (Leica SP8) was used to acquire 63 × images of individual arterioles along a stack of 5z every 1 µm. The mean fluorescence intensity (MFI) of PGC1α was calculated for the brightest section of the z-stack to avoid the high background obtained when creating maximal projections. To analyze mitochondria in arterioles, a confocal microscope (Leica SP8) was used to acquire 63 x—2 × zoom images of individual arterioles in a stack of 5z every 0.5 µm. TOMM20⁺/LAMP1⁺ double positive events were considered as mitolysosomes and their number was normalized by VSMC area (SMA⁺ area) to obtain mitolysosome density.