Amplified opti 4cn substrate

Collagen type I (COL-I) and matrix metalloproteinase (MMP)-1 protein levels secreted by tenocytes in serum-free cell supernatants were analyzed by slot blot analysis, as previously detailed [18 (link)]. Membranes were incubated for 1 h at room temperature with primary monoclonal antibodies to COL-I (1:1000 in TBST) (Sigma-Aldrich, Milan, Italy) or MMP-1 (1 µg/mL in TBST) (Millipore, Milan, Italy). Immunoreactive bands were revealed by the Amplified Opti-4CN substrate (Amplified Opti-4CN, Bio Rad, Segrate, Milan, Italy) and quantification was obtained after densitometric scanning of immunoreactive bands (UVBand, Eppendorf, Italy).

Collagen type I and III (COL-I, COL-III), matrix metalloproteinase (MMP)-1 protein levels secreted by tenocytes were assessed in duplicate samples by Slot blot in serum free cell culture medium, as previously detailed [21 ]. Membranes were incubated for 1 h at room temperature in monoclonal antibody to COL-I (1:1000 in TBST) (Sigma-Aldrich, Milan, Italy), COL-III (1:2000 in TBST) (Sigma-Aldrich, Milan, Italy), MMP-1 (1 µg/mL in TBST) (Millipore, Milan, Italy). Immunoreactive bands, revealed by the Amplified Opti-4CN substrate (Amplified Opti-4CN, Bio Rad, Italy), were scanned densitometrically (UVBand, Eppendorf, Italy).