Rabbit anti fibronectin

Dermal fibroblasts were cultured and treated as described above for 48 hours. The whole cell proteins were separated by running the samples on 10% SDS-polyacrylamide gel and then transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA). Membranes were blocked and probed for MMP1, type-I collagen and fibronectin using rabbit-anti MMP1 (1∶2000 dilution, Abcam, Cambridge, MA, USA), mouse-anti collagen-I (1∶100, Developmental Studies Hybridoma Bank), and rabbit-anti fibronectin (1∶1000, Santa Cruz Biotechnology, CA). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit Ab (1∶3000 dilution, Bio-Rad) and HRP-conjugated goat anti-mouse Ab (1∶3000 dilution, Bio-Rad) were used as the secondary antibodies. β-actin was used as the loading control.
Conditioned medium (30 μl) from the untreated and treated dermal fibroblasts were subjected to Western blotting for detection of secreted MMP3 protein. Mouse-anti MMP3 antibody (1∶2000, R&D Systems, Minneapolis, MN) and HRP-conjugated goat anti-mouse Ab were used as the primary and secondary antibodies, respectively.

Samples were fractioned by electrophoresis in 4–12% polyacrylamide gels (Thermo Scientific). Proteins were transferred electrically onto polyvinylidene fluoride (PVDF) membranes (Millipore) using a constant current of 400 mA for 80 min. PVDF membranes were saturated with 5% nonfat dried milk and 0.1% Tween-20 for 1 h and then probed with the following primary antibodies overnight at 4 °C: mouse anti-type I collagen (dilution 1:200, Santa Cruz), rabbit anti-type IV collagen (dilution 1:400, AbD Serotec), rabbit anti-type VI collagen (dilution 1:2000, Abcam), rabbit anti-fibronectin (dilution 1:1000, Santa Cruz), rabbit anti-laminin (dilution 1:500, Sigma Aldrich), and mouse anti-β-actin (dilution 1:10000, Sigma Aldrich). After 60 min of incubation at room temperature with appropriate horseradish peroxidase-conjugated secondary antibodies (diluted 1:2000, Bio-Rad Laboratories), proteins were visualized by enhanced chemiluminescence on ChemiDoc camera (Bio-Rad).

Immunohistochemistry was performed to measure the amount of type I collagen. Slides were incubated overnight at 4 °C with rabbit anti-collagen I antibody (Abcam) and rabbit anti-fibronectin (Santa Cruz Biotechnology) diluted in blocking serum. After several stages of washing with PBS, the tissue sections were incubated with peroxidase-conjugated goat anti-rabbit polyclonal antibody (DAKO, Carpinteria, CA, USA) at a dilution of 1:200 in PBS for 30 min at 37 °C. After several washes in PBS, the signals on the tissues were revealed by incubating the tissues with diaminobenzidine in PBS for 10 min.
Semi-quantitative analysis of the synthesis of type I collagen was executed using MetaMorph image analysis software (Universal Image Corporation, Buckinghamshire, UK). Results are expressed as the average optical density (OD) of five different digital images.