Immunostaining for interleukin 6 (IL-6) was performed on 4 μm decalcified distal femoral sections. The procedures were performed as described previously7 (link) with an immunohistochemical kit (Boster, Wuhan, People’s Republic of China) by following the manufacturer’s instruction. The anti-IL-6 antibody was purchased from Abcam. The region of interest was the distal femur growth plate and the distal femur metaphysis located between 0 and 2 mm distal to the growth plate epiphyseal junction. The images in the region of interest of each sample were captured at 20× magnification. Six different images were randomly collected per sample and subjected to final analysis. The analysis was performed by the biomedical image analysis software (Image Pro plus 6.0, IPP 6.0; Media Cybernetics Inc., Silver Spring, MD, USA). The evaluation of IL-6 expression was determined by integral optical density values of the images. The integral optical density values of positive staining in the distal femur are presented as mean ± standard deviation. All image groupings analyzed using IPP 6.0 were blinded to the analysts.