Bs 1 lectin

After microCT scanning, decalcified hind limbs or single muscles were further processed for histology. 5 μm-thick paraffin-sections were prepared and stained either with Azan trichrome, Masson trichrome, BS-1 Lectin (L3759, Sigma Aldrich Co., St. Louis, MO, USA) or with a monoclonal anti-slow skeletal myosin heavy chain antibody [NOQ7.5.4D] (Abcam, Cambridge, UK). Azan and Masson trichrome were chosen to facilitate the comparability between histology and microCT: both stainings highlight the connective tissue (appearing in blue), which also appears lighter colored in x-ray projections (higher x-ray attenuation than muscle tissue).

The oxygen exposure protocol placed oxygen-exposed mouse pups with their nursing mothers in the same covered plastic box with 75% oxygen from postnatal day 7 through postnatal day 12 as previously described42 (link). The oxygen was delivered at 75 ± 2%, and it was monitored at least three times daily during the oxygen exposure period. Oxygen concentrations were measured with an oxygen monitor (Teledyne Electronic Technologies, Thousand Oaks, CA, USA). On postnatal day 12, the animals were returned to room air and were subsequently sacrificed by a lethal intra-peritoneal injection of chloral hydrate (360 mg/kg) on postnatal day 17. DPP4-inhibitor injection (DipA; 70 μg/kg, twice daily) was administered from postnatal day 12 to 17. The control mice were injected with PBS in the same manner as DPP4-inhibitor. CXCR4-blocker (AMD3100; 7.5 mg/kg, once per day) was also injected in the same manner as that for the DPP4-inhibitor. BS-1 Lectin (Sigma-Aldrich, St. Louis, MO, USA) was infused systemically for vascularity examination and FITC-dextran (70 kDa; Sigma-Aldrich, St. Louis, MO, USA) for permeability examination. Both eyes of each animal were used for examination of the retinal vascular pattern after flat mounting of the retina.