Sections for immunohistochemistry were fixed in 4% PFA, then digested with 942–2,000 U/ml type IV-S bovine hyaluronindase (Sigma, St. Louis, Missouri, United States) in PBS (pH 5) at 37 °C for 30 min to 2 h depending on the stage. Slides were then washed in PBS, treated with 0.3% hydrogen peroxide in 100% methanol for 30 min, washed, blocked with PBS + 0.05% Tween20 + 5% goat or fetal bovine serum, washed again, and incubated with primary antibodies in PBS + 0.05% Tween 20 + 1% goat or fetal bovine serum overnight at 4 °C. Biotin-labeled secondary antibodies (Vector Labs) were tagged with HRP using the Vectastain Elite ABC kit (Vector Labs) followed by detection with DAB (Vector Labs). Primary antibodies and dilutions used were: goat anti-mouse MMP13, 1:100 (Chemicon International, Temecula, California, United States); rabbit anti-human SOX9, 1:500 (Morais da Silva et al. 1996); rabbit anti-phosphorylated-SOX9 (SOX9.P), 1:10–1:250 (Huang et al. 2000).