Quantifying Bone Sialoprotein Expression

Samples were completely decalcified in 15% EDTA pH 7.0 in ddH₂O and embedded in paraffin. Histological tissue sections (7 μm) were stained with Masson’s trichrome (Réactifs RAL, Martillac, France) as previously described [34 (link),35 (link)]. Expression of bone sialoprotein (BSP) was determined using immunohistochemistry. Binding of BSP primary antibody (ab52128, Abcam, Cambridge, UK) was visualized using Vectastain Fast Red kit (Vector Laboratories, Glostrup, Denmark) according to the manufacturer’s instructions. Images were taken with a BX61 microscope (Olympus, Tokyo, Japan). Quantification of BSP staining area per scaffold area was performed using ImageJ (National Institutes of Health, Bethesda, MD, USA).