Hdac3 Regulates Bone Density Figure 8. Hdac3-depletion in osterix-expressing cells increases adipocyte volume within bone marrow. A. Goldner’s Trichrome stain of femurs from 5.5 week-old male wildtype and Hdac3-CKO mice. B–D. Adipocyte density (Adipocyte volume/tissue volume (B), perimeter (C), and number (D) are increased in Hdac3 CKO mice. n = 426, *p,0.05, **p,0.01. doi:10.1371/journal.pone.0011492.g008 destained in 20% glycerol, 1% KOH for 2 weeks, with daily solution changes. Skeletons were transferred to a 20% glycerol, 20% ethanol solution overnight and then stored in a 50% glycerol, 50% ethanol solution indefinitely. Microcomputed Tomography Bones were dissected from euthanized mice and fixed in 70% ethanol. Samples were scanned and measured in a Scanco 35 System (Scanco Medical, Switzerland), which was calibrated monthly with an aluminum rod phantom and weekly with a hydroxyapatite phantom. Images were reconstructed with cubic voxels 5.7 mm on a side using a modified Feldkamp cone beam tomographic reconstruction algorithm. Trabecular bone proper- ties were calculated with the manufacturer’s software. Cortical bone geometric properties were measured from femoral mid-shaft cross-sectional images as previously described [58]. Data from Hdac3 mutant mice were compared to data from wildtype mice with student t-tests. Histomorphometry After microcomputed tomography scanning, two transverse sections (,50 microns thick) were cut from the diaphysis of long PLoS ONE | www.plosone.org 11 July 2010 | Volume 5 | Issue 7 | e11492