Dab reagent

The clinical glioma specimens were obtained from the Neurosurgery Department, the Second Affiliated Hospital of Kunming Medical University, China. Immunohistochemical staining was conducted according to standard procedures. Briefly, paraffin‐embedded sections were incubated with antibodies of rabbit anti‐human TNIP1 (Abcam, USA) overnight at 4°C. The sections were then further stained with goat anti‐human secondary antibodies conjugated with horseradish peroxidase (Santa Cruz Biotechnology, USA). The immunohistochemical signals were developed with DAB reagent (Boster Biological Technology Ltd., China) and further examined under a light microscope (Olympus, Japan).

Western blot analysis was performed using standard procedures, as described previously [61 (link)]. To obtain proteins, cells were lysed by 100 mg/mL RIPA lysis buffer containing 1% phenylmethylsulfonyl fluoride (Beyotime, Shanghai, China) on ice. Then, cell lysates were boiled in 5× SDS buffer for 5 min before being separated by 10% SDS-polyacrylamide gel, and transferred to a PVDF membrane (Bio-rad, Hercules, CA, USA). Subsequently, the membranes were blocked by TBST containing 5% non-fat dried milk for 2 h at 37 °C to avoid non-specific binding. After that, the membranes were incubated with primary antibodies overnight at 4 °C. Additionally, the immunoblot membranes were incubated with the secondary antibodies for 2 h at 37 °C after washing 3 times. The blots were visualized by DAB reagent (Boster, Wuhan, China) according to the manufacturer’s instructions. The antibodies included anti-β-actin (GB12001, Servicebio, Wuhan, China, dilution: 1:1000); anti-IL6 (GB11117, Servicebio, China, dilution: 1:1000); anti- IL 1 β (abs115412, Absin, Shanghai, China, dilution: 1:1000).

After intrinsic peroxidase activity, the articular cartilage sections were blocked with 3% hydrogen peroxide (H₂O₂) and then incubated with 1.5% BSA for 1 hour. The tissue sections were covered with the antibodies against SBP2 (12798‐1‐AP, 1:250 dilution), GPX1 (3120‐1, 1:250 dilution), GPX4 (14432‐1‐AP, 1:500 dilution) and SELS (15591‐1‐AP, 1:500 dilution), respectively, which were purchased from Proteintech (Wuhan, China). The samples were incubated at 4°C overnight on a wet box. The sections were rinsed with PBS. Sequentially, they were incubated with biotinylated secondary antibody for 1 hour and DAB reagent (Boster, Wuhan, China) for 5 minute at room temperature. Chromogenic reactions were terminated once claybank regions were observed under the microscope. Rabbit IgG was used as a negative control.