Gb11021

Cells were lysed on ice for 30 min in lysis buffer containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, and 0.1% SDS supplemented with protease inhibitors (10 mg/ml leupeptin, 10 mg/ml pepstatin A, and 10 mg/ml aprotinin). Protein fractions were collected by centrifugation at 10,000 × g for 10 min, separated on 10% SDS-PAGE gels, and then electrotransferred onto nitrocellulose membranes (Whatman, Piscataway, NJ, USA). The membranes were blocked with 5% BSA and then incubated with specific antibodies overnight at 4 °C. The primary antibodies and their sources were as follows: COL2A1 (1:1000, GB11021, Servicebio), ADAMTS4 (1:1000, ab185722, Abcam), p65 (1:5000, ab32536, Abcam), MMP3 (1:1000, ab52915, Abcam), MMP13 (1:1000, ab84594, Abcam), Caspase-7 (1:1000, DF6441, Affinity), cleaved Caspase-3 (1:1000, AF7022, Affinity), Caspase-3 (1:1000, AF6311, Affinity), Bcl-2 (1:2000, ab182858, Abcam), Bax (1:1000, ab32503, Abcam), TNF alpha (1:1000, ab1793, Abcam), β-actin (1:1000, ab6276, Abcam), and GAPDH (1:2000, AF7021, Affinity). HRP-conjugated secondary antibodies (ab205719 and ab6721, Abcam) were used at a 1:2000 dilution. The antigen–antibody complexes were visualized using an enhanced chemiluminescence detection system (Millipore, Darmstadt, Germany) according to the manufacturer’s instructions.

Antigen retrieval was performed by incubating sections with 0.05% trypsin (pH 7.8) at 37 °C for immunohistochemistry (IHC). After being blocked with 1% BSA, the sections were incubated with primary antibodies against COL2A1 (1:200, GB11021, Servicebio), MMP3 (1:100, ab52915, Abcam), and MMP13 (1:100, ab84594, Abcam) at 4 °C overnight and then for 1 h at 37 °C with HRP-labeled secondary antibodies (Beyotime Biotechnology, Jiangsu, China). Then use DAB stain for 10 min until the brown color appeared. After hematoxylin counterstaining, 1% alcohol differentiation, and dehydration, use neutral gum to cover the slides. The positively stained cells were counted, and the percentage was calculated by Image-J 1.52t^{25 (link)}.

Immunohistochemistry was performed to evaluate the expression and location of specific protein. Sections were incubated with primary antibodies against Bax (1:200, GB114122, Servicebio), IL-1β (1:100, GB11113, Servicebio), COL2 (1:200, GB11021, Servicebio), ADAMTS5 (1:100, TD13268M, Abcam), MMP13 (1:100, 18164-1-AP, Proteintech), Bcl-2 (1:100, 26593-AP, Proteintech), IL-18 (1:100, 10663-1-AP, Proteintech), and a secondary antibody. IHC was performed using the DAB solution and hematoxylin. Safranin-O/Fast Green staining is an approach used to assess the severity of cartilage injury. The Safranin-O/Fast Green staining kit (Servicebio, China) was utilized to stain and evaluate the distribution and composition of the extracellular cartilage matrix in the intervertebral disc.