Immunohistochemistry staining and quantification of type X collagen

Cryosections were rehydrated in 1× PBS and fixed with 4% PFA for 10 min at room temperature. Sections were pretreated with 0.2 mg/ml proteinase K stock concentration (1:100 dilution) for 5 min at room temperature. Sections were then blocked for 1 h with 5% BSA and 5% normal goat serum in 1× Tris-buffered saline/Tween 20 (TBST). Primary antibody to collagen X (1:1000 dilution; ab58632; Cell Signaling) in 1% BSA in 1× TBST was incubated on the sections at 4° overnight. Goat anti–rabbit immunoglobulin horseradish peroxidase–linked secondary (1:1000 dilution, sc-2004; Santa Cruz Biotechnology) was placed on sections for 1 h at room temperature. Sections were washed and then stained using the DAB substrate following the instructions in the ImmPACT DAB Kit (SK-4105; Vector Laboratories). Sections were mounted with Cytoseal 60.

The hypertrophic zone was measured by the chondrocytes expressing collagen X using FIJI. We cropped 20× images and centered them to include the entire growth plate. First, a line was drawn from the top of the resting zone to the bottom of the hypertrophic zone, and the length was measured using the Measure plug-in. Second, a line was drawn to included the collagen X–expressing cells, and the length was measured using the Measure plug-in. Eight measurements were averaged for each sample, and the results are presented as the percentage of the total growth plate that expresses collagen X. The 1- and 2-wk surgery limbs (N = 3) and contralateral control limbs (N = 3) were compared using the paired t test for significance.