Histological Analysis of Mouse Skeletal Tissues

Postnatal and adult mouse femurs were dissected and fixed in 4% PFA/PBS for 2 days at 4 °C. Bone tissues were decalcified in 20%EDTA for 5days. Embryonic and neonatal tissues were fixed overnight in 4%PFA/PBS, but not decalcified. Fixed skeletal tissues were dehydrated through ethanol and embedded in paraffin; 8-μm serial sections were then prepared for histological analysis49 (link). Hematoxylin and Eosin (HE) staining and immunohistochemical analyses were carried out by standard procedures using the following antibodies (Ab)50 (link): anti-Cathepsin K (1:300, cat. no. ab19027, abcam)51 (link), anti-Osterix (1:1000, cat. no. ab22552, abcam)52 (link), anti-Axin2 (1:300, cat. no. ab32197, abcam)53 (link), anti-Osteocalcin (1:1000, cat. no. M173, Takara)54 (link), anti-Alkaline Phosphatase (1:500, cat. no. M190, Takara)55 (link). Bone-tissue cells with positive immunohistochemical signals representing equal areas of control and sFRP4 KO mice were counted and their average numbers were compared.