Ziess axiocam digital camera

Following published procedures from our lab²⁸ , muscle complexes were divided at the midline along the axial plane and embedded in Tissue-Tek (Fisher Scientific). Three transverse sections per sample were cut for each histological assessment using a CM3050S cryostat (Lecia, Wezlar, Germany). Sections were placed on frozen microscope slides and stored at − 80 °C before staining with rat monoclonal anti-CD31 (BD, San Diego, CA), a marker for endothelial cells. Samples were co-stained with mouse monoclonal anti-dystrophin (MANDRA1) (Sigma-Aldrich, St. Louis, MO) to outline myofibers and measure skeletal muscle area, necessary for calculation of capillary density. For details on immunohistochemistry, see Supplementary Methods. Adobe Photoshop CS5 was used to analyze 5 digital images (randomly selected) per individual, acquired at 200 × total magnification with a Ziess AxioCam digital camera and Axiovision software (Zeiss, Thornwood, NY). All punctate CD31⁺ capillaries were manually counted per image and normalized to image area (capillary density).

The paraffin-embedded samples were deparaffinized,
rehydrated and 5 μm thick sectioned samples were cut.
Representative sections from all cartilage subtype samples
were stained with hematoxylin and eosin (H&E) for
the descriptive analysis of morphological details. Light
microscope at ×10 magnification (Ziess AxioCam digital
camera, Carl Zeiss, Germany) was used to visualize and
photograph the stained sections.