Primary antibody against sclerostin

Decalcified slices were probed with primary antibody against sclerostin (10 µg/ml; R&D Systems, USA) overnight at 4 ºC. Detection was achieved by using DAB kit (ZSGB-BIO) followed by counterstaining with hematoxylin.
Longitudinal bone growth results in cells with different age in tibia, which lead to differential sensitivity to disuse 12 (link), 13 . To investigate the response of osteocyte to the mechanical unloading in different anatomic sites, the analysis of positive-staining osteocytes was limited at three regions of tibia: the proximal region (0.5 - 1.5 mm to the growth plate), the distal region (2.5 - 3.5 mm to the growth plate), and the diaphysis region (5 - 6 mm to the growth plate). The numbers of sclerostin-positive (sclerostin+) osteocytes, exhibiting brown staining, and sclerostin-negative (sclerostin-) osteocytes, exhibiting blue staining, were separately counted. The percentage of sclerostin-positive osteocytes was calculated out of the total number of osteocytes (sclerostin+ plus sclerostin-) at the proximal, distal and diaphysis sites, respectively.

Decalcified sections of the proximal tibia were probed with primary antibody against sclerostin (10 µg/ml; R&D Systems) overnight at 4 ºC. Detection was achieved by using DAB kit (ZSGB-BIO, Beijing, China) followed by counterstaining with hematoxylin. The sections were photographed using 20× objective within cortical bone. The numbers of sclerostin-positive (sclerostin+) (stained brown) and -negative (sclerostin-) (stained blue) osteocytes were counted. The percentage of sclerostin-positive osteocytes out of the total number of osteocytes (sclerostin+ plus sclerostin-) was calculated.