Publication protocol
Endogenous peroxidase was devitalized by 3% H2O2 for 20 min. After blocking with normal bovine serum for 60 min at room temperature, the sections were incubated overnight with primary antibodies (15 h at 4°C). Sections for control staining were incubated with normal rabbit serum and normal goat serum (15 h at 4°C) instead of primary antibodies. The primary antibodies were as follows: anti-AT1R goat polyclonal antibody (1:200 in PBS; Santa Cruz Biotechnology, Santa Cruz, CA, #31181), anti-AT2R rabbit polyclonal antibody (1:200 in PBS; Santa Cruz Biotechnology, #9040), anti-Col X rabbit monoclonal antibody (1:100 in PBS; LSL, Tokyo, Japan, #0092), and anti-MMP-13 rabbit polyclonal antibody (1:100 in PBS; Abcam, Cambridge, UK, #39012). The sections were incubated with the secondary antibody for 1 h at room temperature. Horseradish peroxidase-conjugated bovine anti-goat IgG antibody (1:1000 in PBS; Santa Cruz Biotechnology, #2350) was used as the secondary antibody for AT1R staining. Horseradish peroxidase-conjugated bovine anti-rabbit IgG antibody (1:1000 in PBS; Santa Cruz Biotechnology, #2370) was used as the secondary antibody for AT2R, Col X, and MMP-13 staining. The staining was visualized with a diaminobenzidine chromogen kit (DAB Chromogen; Dako, Glostrup, Denmark) and counterstained lightly with Mayer’s hemalum solution. These stained samples were observed using a light microscope (BZ-9000; Keyence, Osaka, Japan). To investigate whether the expression of AT1R and Col X increased with the development and progression of articular degeneration, we examined the correlations between the modified Mankin score and the immunological positive cell rates of AT1R and Col X. Correlations between the immunological positive cell rate of AT1R and Col X, and between the rates of AT1R and AT2R were also investigated in the running THM group.
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