Aqueous osmium tetroxide

Long bones were dissected free of soft tissues and fixed in 10% neutral buffered formalin (Fisher #SF100-4) overnight at 4°C with gentle agitation. The next day, bones were washed in cool running tap water. The bones were decalcified in 4% EDTA for 15 days at 4°C, changing the EDTA every 3–4 days. The bones were then stained for lipid using a 1:1 mixture of 2% aqueous osmium tetroxide (Polysciences Inc, Warrington, PA) and 5% potassium dichromate for 48 hrs (34 (link)). The bones were then washed in cool running tap water for 2 hrs. Whole bones were imaged using micro-CT performed in water with energy of 55kVp, an integration time of 500 ms, and a maximum isometric voxel size of 10 μm (the “high” resolution setting with a 20mm sample holder) using a Scanco microCT-35. When creating volumes of interest (VOI), the interface between the decalcified bone and the marrow adipose tissue (MAT) is usually apparent, facilitating placement of graphical objects. When segmentation is applied, MAT can be visualized unencumbered by the surrounding bone. The data is a volumetric measurement analogous to the volumetric bone measurement, bone volume/total volume (BV/TV). As such, it is more sensitive and provides a better representation of the physical distribution of MAT than 2-dimentional data. The Yale micro-CT facility at Yale Medical School was used to perform the micro-CT analysis.

Quantification and visualization of marrow adipose tissue was performed as described in (Scheller et al. 2014 (link)). Briefly, long bones were dissected free of soft tissues and fixed in 10% neutral buffered formalin (cat# SF100-4, Fisher Scientific, Pittsburgh, PA, USA) overnight at 4°C with gentle agitation. The following day, bones were washed in cool running tap water. The bones were then decalcified in 4% EDTA for 14 days at 4°C, with EDTA changes every 3-4 days. The bones were then stained for lipid using a 1:1 mixture of 2% aqueous osmium tetroxide (cat# 23310-10, Polysciences Inc., Warrington, PA) and 5% potassium dichromate for 48 hours. The bones were then washed in cool running tap water for 2 hours. Whole bones were imaged using micro-computed tomography (μCT) performed in water with energy of 55kVp, an integration time of 500 ms, and a maximum isometric voxel size of 10μm using a μCT-35 (Scanco Medical, Bruttisellen, Switzerland).