Alp kit

The measurement of ALP activity was conducted using an ALP Kit (Solarbio). After the treatment, the cells were immobilized using a 4% paraformaldehyde solution and then stained using a working solution of ALP. In addition, the specimens were stained with 2% ARS (pH 4.2) to visualize calcification. Specimens were fixed in 4% paraformaldehyde, followed by staining with ARS for 15‒30 min. To quantify the mineral deposits, the samples were eluted with 10% cetylpyridinium and determined at the absorbance of 562 nm.

The measurement of ALP activity was conducted using an ALP Kit (Solarbio). After the treatment, the cells were immobilized using a 4% paraformaldehyde solution and then stained using a working solution of ALP. In addition, the specimens were stained with 2% ARS (pH 4.2) to visualize calcification. Specimens were fixed in 4% paraformaldehyde, followed by staining with ARS for 15‒30 min. To quantify the mineral deposits, the samples were eluted with 10% cetylpyridinium and determined at the absorbance of 562 nm.

Primary osteoblasts harvested from one or two-day-old newborn mice calvaria, were washed with 75% ethanol for 5 min and PBS repeatedly to remove residual blood and adipose tissue. The calvaria were cut into 2-5 mm² fragments and digested with 0.25% trypsin for 15 min to remove the fibrous tissue cells. The digestions were discarded, and then 0.1% of type II collagenase was added to 10 ml at 37° C for 20 minutes to digest. The samples were centrifuged at 3000 rpm for 3 minutes at room temperature. Discarding the supernatant, osteoblast cells were collected and incubated for 7 days with alpha-MEM. 10% fetal bovine serum (FBS) in alpha-MEM was changed with mice serum which was obtained from 8 weeks OVX or OVX + HAMP mice before and osteoblasts were cultured for further 14 or 21 days. To visualize osteoblasts ALP and mineralization activity, these cells were fixed in 3.7% formaldehyde for 10 min, and analyzed using alkaline phosphatase and alizarin red staining with ALP kit (Solarbio, G1480) and alizarin red kit (Solarbio, G3280) following the manufacturer’s instructions.