Goat anti rabbit igg hrp

Total proteins were extracted using RIPA lysis buffer. Total proteins were quantified using BCA kit (Wanleibio, Shenyang, China). Proteins were separated by SDS-PAGE, and were transferred onto PVDF membrane (Millipore, Billerica, MA, US). The membrane was blocked in defatted milk powder, and was incubated at 4 °C overnight in primary antibody (mouse anti-E-cadherin at 1:500; rabbit anti-α-SMA at 1:500; goat anti-Snail 1:400 and goat anti-MGAT3 1:200; Wanleibio, Shenyang, China). On the following day, the membrane was rinsed in PBS, and was incubated in secondary antibody (goat anti-mouse IgG-HRP 1:5000; goat anti-rabbit IgG-HRP 1:5000; donkey anti-goat IgG-HRP 1:5000; Wanleibio, Shenyang, China) for 1 h at room temperature. ECL chromogenic substrate (BD Bioscience, US) was added for development, and was imaged using a gel imaging system. Relative protein expression was quantified against the beta-actin band.

The expression levels of bFGF, TGFβ1, Collagen I and Collagen II in degenerative disc tissue were measured using western blotting. Degenerated intervertebral disc tissue was frozen at −80°C, homogenised and lysed and then placed on ice for 5 min. The samples were centrifuged at 23,188 × g. and 4°C for 10 min, and the protein was extracted using RIPA lysis buffer (WLA019, Wanleibio Co.). and separated by electrophoresis. The protein concentration in the supernatant was determined using the BCA method, and each sample was boiled in water for 5 min at 100°C. The samples (20 µl per lane, containing 40 µg of protein) were examined using 7.5–15% SDS-PAGE and transferred to PVDF membranes. The PVDF membrane was blocked with 5% (M/V) non-fat milk powder for 1 h at 4°C. The blots were incubated first at 4°C with primary antibodies against bFGF (1:2,000), TGFβ1 (1:500), Collagen I (1:500) and Collagen II (1:500) overnight. The samples were then incubated with secondary antibodies (Goat Anti-rabbit IgG-HRP, 1:5000, WLA023, Wanleibio Co.) for 45 min at 37°C. The reaction was visualised by chemiluminescence, and the optical density of the target band was analysed by a gel image processing system (Gel-Pro-Analyzer 6.0; Media Cybernetics, Inc.). The protein levels were normalised to that of β-actin (1:1,000, WL01845, Wanleibio Co.).