Histofine mouse staining kit

All tumors resected from mouse primary lesions at the right knee joint were fixed with 10% buffered formalin and embedded in paraffin. Thick sections of 3 μm were examined using immunohistochemistry. The sections were deparaffinized, and antigens were retrieved by autoclaving in 10 mmol/L citrate buffer (pH 6.0) at 121°C for 10 min. Endogenous peroxidase activity was blocked by immersing the slides in 0.6% hydrogen peroxide in methanol for 30 min. The sections were immunostained using a Histofine mouse staining kit (Nichirei, Tokyo, Japan). The primary antibodies used in this study were a mouse monoclonal antibody against human Ki‐67 (1:50; DAKO, Glostrup, Denmark), a goat polyclonal antibody against human PAI‐1 antigen (1:100; SEKISUI, Tokyo, Japan) and a rabbit polyclonal antibody against human MMP‐13 antigen (1:400; Abcam). Immunoreactions were visualized with diaminobenzidine and the sections were counterstained with hematoxylin.

Paraffin‐embedded tissue sections were deparaffinized, and antigens were retrieved for 5 min in a pressure cooker at 121°C in pH 9.0 antigen retrieval solution (Nichirei Bioscience). For MMP‐12 staining, the sections were incubated with a rabbit monoclonal antibody (1:100 dilution, BS9869M, Bioworld Technology) at room temperature for 60 min. Sites of antibody binding were visualized with the Histofine simple stain MAX‐PO(R) kit (Nichirei Bioscience). 3,3′‐Diaminobenzidine tetrahydrochloride was used as a chromogen, and the sections were counterstained with hematoxylin. For TIMP‐2 staining, a mouse monoclonal antibody (1:100 dilution, 3A4, Santa Cruz Biotechnology) was applied at room temperature for 60 min and visualized with the Histofine mouse staining kit (Nichirei Bioscience). For immunofluorescence, a rabbit polyclonal antibody against p19^ARF (1:300 dilution, ab80, Abcam) and a rat monoclonal antibody against fibroblasts (1:50 dilution, ER‐TR7, Santa Cruz Biotechnology) were used. The sections were visualized with Alexa Fluor 488‐conjugated anti‐rat IgG and Alexa594‐conjugated anti‐rabbit IgG and were counterstained with DAPI.