Immunohistochemical Localization of CEMIP

CEMIP localization was assessed by immunohistochemistry in knee joint sections using anti-KIAA1199 antibody (1:1000) as previously described [10 (link)]. Deparaffinized sections were pretreated with chondroitinase ABC (1 U/mL; Sigma-Aldrich) at 37 °C for 2 h. The endogenous peroxidase was blocked with 3% H₂O₂ in PBS at 20 °C for 15 min and incubated in normal goat serum at 20 °C for 60 min. Rabbit anti-aggrecan neo antibody (10 mg/mL) was used as the primary antibody at 4 °C for 16 h. Histofine simple stain rat MAX PO(R) (Nichirei, Tokyo, Japan) was used as the secondary antibody [40 (link)]. The reaction was visualized by diaminobenzidine (DAB; Histofine simple stain DAB, Nichirei), resulting in a brown color. Counterstaining was carried out with hematoxylin. Sections incubated with normal rabbit non-immune serum or incubated without primary antibody were used as negative controls.