Quantifying Collagen and MMP-1 in Fibroblasts

Collagen type I and III (COL-I and COL-III) and matrix metalloproteinase (MMP)-1 protein levels secreted by muscle fibroblasts were analysed by Slot Blot in serum-free cell supernatants as previously described [67 (link)]. After blocking, membranes were incubated for 1 h at room temperature with primary polyclonal antibodies to COL-I (1:1000 in 1X Tris-Buffered Saline plus 0.1% Tween 20, TBST) (Sigma-Aldrich, Milan, Italy), COL-III (1:1000 in TBST) (Sigma-Aldrich), or MMP-1 (1 μg/mL in TBST) (Millipore, Milan, Italy). After 1 h incubation with HRP-conjugated antibody (1:20,000 in TBST), immunoreactive bands were revealed by the Amplified Opti-4CN substrate (Amplified Opti-4CN, Bio Rad, Segrate, Milan, Italy) and quantified by densitometric scanning (UVBand, Eppendorf, Milan, Italy).