Quantification of Marrow Adipose Tissue

At the time of sacrifice, tibiae were isolated and placed into 10% neutral buffered formalin overnight at 4°C. Soft tissue was carefully removed to ensure that the fibula remained intact and the bones were washed under continuous cold PBS for 1 hr, then stored in PBS at 4°C. Quantification and visualization of marrow adipose tissue was performed as described previously (Scheller et al., 2014 (link)). Briefly, bones were decalcified in 14% EDTA (pH 7.4) for 14 days, with EDTA changes every 3–4 days. Bones were then washed for 10 min in PBS (three times) and stained with a 1:1 mixture of 2% aqueous osmium tetroxide (cat# 23310-10, Polysciences, Inc, Warrington, PA, USA) and 5% potassium dichromate for 48 hr. Stained bones were then washed with PBS (pH 7.4) for 5 hr (three times), and subsequently scanned by μCT. BMAT content was calculated by determining the whole volume of second osseous center of tibiae.