2.9. Osteocalcin
DECALCIFICATION
The detached femoral bone tissues were fixed with 10% neutralized formalin for 24 h. After decalcification and 1 M ethylenediaminetetraacetic acid for 2 weeks, the tissue paraffin block was made for immunofluorescence. The bone tissues were sliced into 7 μm thickness and incubated with primary anti-rabbit OCN overnight at 4 °C. After washing, the samples were incubated with the anti-fluorescence quencher conjugated goat anti-rabbit OC for 2 h and then Topro 3 for counterstaining of nucleus for 3 min, respectively. Sections were observed using a fluorescence microscope (LSM 5 PASCAL; Carl Zeiss, Oberkochen, Germany).