Quantitative Analysis of Bone Cell ALP Expression

Bone samples were fixed with 4% paraformaldehyde, decalcified, dehydrated, embedded in paraffin, and cut into 5 μm slices, after which they were dewaxed by xylene and alcohol (in the following order: 100, 95, 75, and 50%). Slices were immersed in distilled water and HE staining was performed in accordance with standard protocols. Fields with positive staining were captured by microscopy. Additionally, primary antibody anti-ALP (abcam, UK) was incubated overnight at 4 °C and, subsequently, incubated with anti-mouse IgG-HRP (SV-0001; Boster) for 1 h. Then, sections were observed under the microscope (OPTEC CCD TP510) with a 100× objective and pictures were collected. The staining intensity was scored according to previous research (Lian et al. 2019 (link)) as follows: 0 (negative), 1 (weakly positive), 2 (moderately positive), and 3 (strongly positive). The percent of positivity was scored according to five categories: 0 (< 5%), 1 (5–25%), 2 (25–50%), 3 (50–75%), and 4 (> 75%). Finally, the percent positivity score was multiplied by the staining intensity score to generate final expression scores, which ranged from 0 to 12.