Mouse anti human mmp 9 antibody

Serum samples from the median cubital vein of 40 patients and 20 healthy people were collected in Fujian Provincial Hospital and diluted with PBS (pH 7.4). The serum MMP-9 level was analyzed using the ELISA method. In brief, 100 μL serum specimens were applied to the 96-well plates and incubated overnight at 4°C. After three washes with PBS, each well was blocked with 0.5% skimmed milk at room temperature for 1 hour. After another three washes with PBS containing 0.05% Tween 20, mouse anti-human MMP-9 antibody (1:200; Abcam, Cambridge, UK) was applied to wells and incubated at room temperature for 2 hours. Then horseradish peroxidase-conjugated goat anti-mouse IgG (1:1,000; Dako, Glostrup, Denmark) was added, followed by incubation with SureBlue™ TMB Microwell Peroxidase Substrate (KPL, Gaithersburg, MD, USA). After color development, the reaction was stopped with 1 M HCl and the optical density of the solution was read at a wavelength of 450 nm using a microplate reader (iMark™ Microplate Reader; Bio-Rad, Hercules, CA, USA). The serum hypersensitive C reactive protein level was analyzed using an automatic biochemical analyzer (AU5800, BECKMAN, Brea, CA, USA).

Cells were solubilized in cold RIPA lysis buffer (Beyotime). Proteins were separated with 10% SDS-PAGE (Beyotime) and transferred onto a nitrocellulose membrane (Life Technologies), and then incubated with TBST (Beyotime) containing 5% nonfat milk (Yili, Beijing, China) at room temperature for 3 h. The membrane was then incubated with mouse anti-human TGIF2 antibody (Abcam, Cambridge, MA, USA), mouse anti-human MMP9 antibody (Abcam), and mouse anti-human GAPDH antibody (Abcam), respectively, at room temperature for 3 h. After washing three times with PBST, the membrane was incubated with the rabbit anti-mouse secondary antibody (Abcam) at room temperature for 1 h. Detection of immune complex was performed with an Pierce ECL Western Blotting KIT (Pierce, Thermo Fisher, USA), according to the manufacturer’s instruction. Image-Pro plus software 6.0 was used to analyze the expression of the protein.