Benzoyl peroxide

Femurs were stripped of soft tissue and fixed in 4% PFA for 48 hours before proceeding to dehydration and embedding steps as previously described Qing et al. (2012) (link). Briefly, femurs were dehydrated in graded ethanol and placed into acetone. Subsequently, the femurs were immersed in infiltration solution made of 85% destabilized methyl methacrylate (MMA, Sigma), 15% dibutyl phthalate (Sigma), 1% PEG400 (Sigma), and 0.7% benzoyl peroxide (Polysciences, Inc., Warrington, PA)/acetone until infiltration was complete. The femurs were then placed on pre-polymerized base layers, covered with freshly catalyzed MMA embedding solution (for 100 mL, 85mL MMA, 14mL dibutyl phthalate, 1mL PEG400, 0.33uL DMT, and 0.8g BPO), and incubated under vacuum until the MMA was polymerized. The polymerized blocks were trimmed, sequentially polished to a completely smooth surface, and coated with gold using a sputter coater (Desk V, Denton Vacuum, NJ, USA). Then BSEM (JEOL: JSM-7800F) was performed to image the osteocyte lacunae on the sectioned bone surface at 450X magnification starting 2 mm distal from the growth plate. Six fields from the endosteal and periosteal sides of the cortical bone were taken as described previously Qing and Bonewald (2009) . Using ImageJ (NIH), the images were thresholded for background removal, binarized, and the lacunar area from each sample quantitated.