In order to assess expression of proliferating cell nuclear antigen (PCNA) in vivo, decalcified, paraffin-embedded femur sections were subjected to immunohistochemical analysis, as previously described [38 (link)]. Briefly, slides were deparrafinized in xylenes, rehydrated in sequential alcohol baths, and incubated in 10 mM sodium citrate buffer for 10 minutes at 37°C and 30 minutes at room temperature to unmask antigens. Sections were blocked with SuperBlock (Thermo Scientific) for 1 hour and incubated overnight at 4°C in rabbit anti-PCNA antibody (1:1000) in SignalStain Ab Diluent (Cell Signaling Technology). The following day, slides were incubated with biotinylated secondary antibody and the Vectastain ABC reagent (Vector Laboratories) according to manufacturer’s instructions. Slides were stained with Vector NovaRED substrate (Vector Laboratories) and counterstained with Fast Green (Electron Microscopy Sciences). Slides were rapidly dehydrated through alcohols and xylenes prior to mounting with Permount and coverslips.