Rabbit anti tgf β1

The specimens were lysed with lysis buffer (100 mmol/L dithiothreitol, 50 mmol/L Tris-HCl, pH 6.8, 2% SDS, and 10% glycerol) containing protease inhibitors. The BCA method was used to determine the total protein concentration. Lysates with equal amounts of protein were resolved on SDS-PAGE, and then transferred to a PVDF membrane. The membrane was blocked with 5% non-fat dry milk in TBST buffer (100 mmol/L NaCl, 50 mmol/L Tris-HCl, pH 7.4 and 0.1% Tween-20) at 4°C overnight. The membranes were washed with TBST buffer and then incubated with monoclonal antibodies against rabbit anti-substance P(dilution: 1∶500, GeneTex, Inc., USA), mouse anti-c-fos (dilution: 1∶200, Santa Cruz Biotechnology, USA), mouse anti-NGF (dilution: 1∶200, Santa Cruz Biotechnology, USA), rabbit anti- TGF-β1 (dilution: 1∶300, Santa Cruz Biotechnology, USA), mouse anti-collagen I (dilution: 1∶300, Santa Cruz Biotechnology, USA) and III (dilution: 1∶200, Boster, Wuhan, China) at room temperature for 1.5 h. After washing, the samples were incubated for 2 hr at room temperature with HRP-conjugated goat anti-mouse IgG (1∶8 000) or goat anti-rabbit IgG(1∶5000). The membrane was then washed with buffer and the image scanned with a GS800 Densitometer Scanner. Optical density data were analyzed using PD Quest 7.2.0 software. Beta-actin (dilution: 1∶1000, Santa Cruz Biotechnology, USA) was used as a control.