Immunohistochemical Analysis of Callus Formation

To examine the localization of Cnmd, type II collagen and type X collagen, and CD31 in the lengthened callus, immunohistochemistry was performed using an avidin-biotin peroxidase detection system (Vector Lab, Burlingame, CA, USA). After blocking in 5% skim milk in PBS-T (phosphate-buffered saline containing 1% Tween 20) at 4°C for 1 hour, the sections were incubated overnight at 4°C with a primary antibody, anti-Cnmd rabbit polyclonal antibody (1:1000), anti-type II collagen rabbit polyclonal antibody (1:400: Rockland Immunochemicals, Philadelphia, PA, USA), anti-type X collagen polyclonal antibody (1:400: LSL, Japan), and anti-CD31 rabbit polyclonal antibody (1:50: Abcam, Cambridge, MA, USA). After washing with PBS-T, the sections were incubated with secondary antibodies, a biotinylated anti-rabbit IgG (Vector Lab). Reactions were visualized with diaminobenzidine as substrate (Vector Lab). The sections for Cnmd, type II collagen, type X collagen, and CD31 were counterstained with hematoxylin. Five random fields (60000 μm²) per lengthened segment were measured, on CD31 stained slides, to determine the number of newly formed vascular vessels for samples 3 and 4 weeks after osteotomy. Three specimens were included for each genotype at each time point.