Mouse anti mmp 3

Immunohistochemistry was carried out using antibodies against type I collagen, type III collagen, MMP-1, and MMP-3 to investigate changes of collagen and interstitial collagenase expressions in the sections. The sections were incubated with 0.3% hydrogen peroxide in PBS for 30 min, followed by 10% normal donkey or goat serum (Vector Laboratories, Inc., Burlingame, CA, USA) in 0.05 M PBS for 30 min. The sections were incubated with rabbit anti-type I collagen (diluted 1:400; Abcam, Cambridge, UK), mouse anti-type III collagen (diluted 1:300; Abcam), rabbit anti-MMP-1 (diluted 1:300; Abcam), and mouse anti-MMP-3 (diluted 1:300; Abcam). Thereafter, the tissues were exposed to biotinylated donkey anti-rabbit IgG, goat anti-mouse IgG, and streptavidin peroxidase complex (Vector Laboratories) and visualized with 3,3′-diaminobenzidine tetrachloride (Sigma-Aldrich, Darmstadt, Germany). After dehydration, the sections were mounted with Canada balsam (Kanto, Tokyo, Japan).
We performed quantitative analyses of the immunoreactivities against type I collagen, type III collagen, MMP-1, and MMP-3 using an AxioM1 light microscope (200× magnification; Carl Zeiss, Oberkochen, Germany) equipped with a digital camera (Axiocam) and connected to a PC monitor.

Total proteins were extracted with lysis buffer (150 mmol/L NaCl, 1% NP‐40, 50 mmol/L Tris, 0.2% sodium dodecyl sulphate and 5 mmol/L NaF) supplemented with a protease inhibitor and phosphatase inhibitor cocktail (Roche, Madison, WI, USA). MMP3 and MMP13 were detected after trichloroacetic acid precipitation as described previously.23 Each protein was visualized using the SuperSignal West Dura kit (Thermo Scientific, Waltham, MA, USA); total extracellular signal‐regulated kinase (ERK) was used as a loading control. Western blot analysis was performed to detect protein levels using the following antibodies: mouse anti‐Hif‐2α (sc‐13596; Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti‐Mmp3 (ab52915; Abcam, Cambridge, UK), mouse anti‐Mmp13 (ab51072; Abcam), goat anti‐COX‐2 (sc‐1745; Santa Cruz), mouse anti‐Erk1/2 (610408; Becton Dickinson, NJ, USA), mouse anti‐pErk1/2 (9101; Cell Signaling Technology, Boston, MA, USA) and mouse anti‐IκB (9242; Cell Signaling Technology). Anti‐Hif‐2α antibody (ab8365; Abcam) was used for immunostaining as previously described.14

According to our previously published method, we performed immunohistochemistry for collagens and MMPs. In short, sections were reacted with 0.3% hydrogen peroxide (H₂O₂) solution in 0.01 M PBS, pH 7.4 and, subsequently, immersed into 5% normal donkey or goat serum (in 0.01 M PBS, pH 7.4; Vector Laboratories, Inc., Burlingame, CA, USA) at room temperature for 30 min, respectively. Next, the sections were immunoreacted with each primary antibody overnight at room temperature: rabbit anti-collagen I (diluted 1:500; Abcam, Cambridge, UK), mouse anti-collagen III (1:500; Abcam, Cambridge, UK), rabbit anti-MMP-1 (diluted 1:250; Abcam, Cambridge, UK) and mouse anti-MMP-3 (diluted 1:250; Abcam, Cambridge, UK). Thereafter, the sections were reacted with corresponding secondary antibodies, which were conjugated with biotin for 2 h at room temperature: donkey anti-rabbit immunoglobulin G (IgG) (diluted 1:250; Vector Laboratories, Inc., Burlingame, CA, USA) and goat anti-mouse IgG (diluted 1:250; Vector Laboratories, Inc., Burlingame, CA, USA). These immunoreacted tissues were exposed to avidin-biotin complex (ABC, 1:300; Vector Laboratories, Inc., Burlingame, CA, USA) for 1 h at room temperature. The sections were finally visualized via reacting with 3,3′-diaminobenzidine tetrahydrochloride (DAB, in 0.01 M PBS; Sigma-Aldrich, St. Louis, MO, USA).