Immunohistochemical Evaluation of Osteoblasts

To evaluate osteoblasts, immunostaining was performed using a standard protocol [17 (link)]. We incubated sections with primary antibodies to mouse Alkaline phosphatase (ALP, PA1004, Boster, Pleasanton, USA) and Osteocalcin (OCN, ab93876, Abcam, Cambridge, UK) overnight at 4°C. A biotinylated horseradish peroxidase detection system (Vectastain, PK-6200, Vector Laboratories, Burlingame, USA) was subsequently used to detect the immunoactivity, followed by incubation in 3,3'-diaminobenzidine (DAB, SK-4100, Vector Laboratories, Burlingame, USA) and counterstaining with hematoxylin. Also, tartrate-resistant acid phosphatase (TRAP) staining was performed for osteoclasts. Descriptive analysis to the immunostaining was performed by comparing the number of cells in the view field that are positive with the markers mentioned above. At least three mice per group were examined. Three equidistant sections spaced at 200 μm apart throughout the middle 1/3 coronal section of the vertebra were evaluated.