Anti mmp 3

To detect the activity and location of gelatinase and collagenase, in situ zymography was performed. Dorsal skin tissues were rapidly frozen using 2-methylbutane and liquid nitrogen, and 20-μm cryosections were prepared. Sections were reacted at 37 °C for approximately 8 h in dark humidified chambers with a Molecular Probes EnzChek Gelatinase/Collagenase Assay Kit (Life Technologies, Eugene, OR, USA). After washing, the skins were fixed in 10% neutral-buffered formalin in the dark. To evaluate MMP-9 and MMP-3 co-localization, sections were incubated with primary anti-MMP-9 (Millipore, Billerica, MA, USA) or anti-MMP-3 (Santa Cruz Biotechnology Inc., CA, USA) in antibody dilution buffer (1 × PBS, 1% bovine serum albumin [BSA], and 0.3% Triton X-100) overnight in the dark, followed by washing with PBS. The sections were incubated with the corresponding secondary goat anti-rabbit IgG-TR (Vector Laboratories Inc.) or rabbit anti-goat IgG-TR (Vector Laboratories Inc.) antibody for 2 h at room temperature in the dark, followed by washing with PBS. Subsequently, the slides were mounted using mounting medium (Vector Laboratories Inc.), and images were acquired using a fluorescence microscope (Carl Zeiss Imager M1, Carl Zeiss Inc.). IMT i-Solution (IMT i-Solution Inc.) was used for automatic measurement of the integrated optical density (IOD).

DSF was purchased from Sigma (USA) and dissolved in DMSO. TGF-β was purchased from Peprotech (USA) and dissolved in 10 mM citric acid, PH3.0 to a concentration of 0.1–1.0 mg/ml. U0126 was purchased from Selleck Chemicals (USA) and dissolved in 10% DMSO, 30% Cremophor EL and 60% PBS to a concentration of 10 mM.
The following primary antibodies were used: anti-E-cadherin (WB: 1:1000, IF:1:200, IHC:1:400 dilution, CST), anti-vimentin (WB: 1:1000, IF:1:100, IHC:1:100 dilution, CST), anti-N-cadherin (WB: 1:1000 dilution, CST), anti-Snail (WB: 5 ng/ml, IF:10 μg/ml, IHC: 10 μg/ml, R&D Systems), anti-NF-κB/p65 (WB: 1:1000, IF:1:50, IHC:1:800 dilution, CST), anti-ERK (WB: 1:500, IF:1:50, IHC:1:50, dilution, Boster, Wuhan, China), anti-p-ERK (WB: 1:1000, IF:1:200, IHC:1:400 dilution, CST), anti-IκB-α (WB:1:500, IF: 1:50, IHC:1:50 dilution, Anbo, USA), anti-CD24 (WB: 1:200, IF:1:50 dilution, Santa Cruz), anti-CD24 (IHC:1:50 dilution, Abcam), anti-CD44 (WB:1:1000, IF:1:200, IHC:1:50 dilution, CST), anti-MMP-1 (WB:1:200 dilution, Santa Cruz), anti-MMP-3 (WB:1:200 dilution, Santa Cruz).

Western blot assays were carried out as previously described [25 (link)]. The protein from primary human chondrocytes was isolated after 24 h of treatment. The primary antibodies used were rabbit polyclonal anti-MMP1 (1:500), anti-MMP2 (1:500), anti-MMP13 (1:500), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:1000), goat polyclonal anti-MMP3 (1:500), mouse monoclonal anti-MMP9 (1:1000; Santa Cruz Biotechnology, Inc.), and anti-SRY-related high mobility group-box gene9 (SOX9) (1:2000; Millipore).