Sf100 4

For cytospins, 1×10⁶ cells were collected in 100ȝL PBS/2% FBS from single cell preparations of BM, added to Shandon Single Cytofunnels (Thermo Scientific #1102548) clipped to glass slides (FisherScientific #12–550-20), and centrifuged using a Cytospin 3 centrifuge (Shandon) set at 500 rpm for 5 min at medium acceleration. After drying, cells were stained with the Hema 3 stat pack (Fisher Scientific #23–123869) and images captured with a Nikon Eclipse E200 microscope equipped with an Infinity 2 color camera (Lumenera) controlled by Infinity Capture software (Lumenera).
For histopathology, mouse tibia and spleens were fixed in 10% neutral buffered formalin (Fisher Scientific #SF100–4) overnight at 4^oC or 1 hr at room temperature, respectively. Spleens were inoculated in 35% sucrose (Fisher Scientific #525590A) overnight, whereas bones were decalcified (Calrite from Richard-allen Scientific #5501) for 12 days prior to overnight inoculation in 35% sucrose. Both tibias and spleens were rinsed with PBS and processed for paraffin embedding and sectioned at 5 μM. Both H&E and reticulin staining was performed by the Histology Lab of Washington University. Images were captured using an inverted microscope and analyzed with NIS-Elements software from Nikon.

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.