Anti tnfα

For immunohistochemical staining, after antigen retrieval, the slides were blocked with a 5% BSA solution for 1 ​hour and incubated with mouse anti-CD68 (1:100, Invitrogen, USA), anti-iNOS (1:100, Abcam, UK), anti-CD163 (1:1000, Proteintech, USA), anti-TNFα (1:100, ZenBio, China), anti-IL1β (1:100, Proteintech, USA), anti-IL10 (1:100, Proteintech, USA), anti-TGFβ1 (1:100, Proteintech, USA), anti-MMP9 (1:100, Proteintech, USA), anti-OCN (1:100, Affinity, China), anti-HIF1α (1:100, Proteintech, USA) and anti-SDHB (1:100, Proteintech, USA) at 4 ​°C overnight. Goat anti-rabbit IgG (1:100, Servicebio, China) was used as a secondary antibody and the nuclei were stained using 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI, Servicebio, China). Images were captured using Aperio AT2 (Leica, German).

Paraffin slices were deparaffinized and rehydrated before H&E staining (HE; BL700A; Biosharp). Immunohistochemical staining was conducted with VECTASTAIN® Elite® ABC‐HRP Kit (PK‐6101; Vector laboratories). Slides were incubated in antigenic repair fluid for 20 min at 100°C for antigen retrieval. Endogenous peroxidase activity was quenched via incubation with 3% H₂O₂ for 30 min. Following PBS rinse, sections were further treated with goat serum for 1 h. Afterwards, incubation with corresponding primary antibodies were applied at 4°C overnight, including anti‐AFF4 (14662‐1‐AP; Proteintech), anti‐Collagen I (R26615; ZEN‐BIO), anti‐Runx2 (860139; ZEN‐BIO), and anti‐TNFα (346654; ZEN‐BIO). Thereafter, sections were treated with biotinylated goat anti‐rabbit IgG for 2 h and visualized with DAB peroxidase substrate kit (SK‐4100, Vector laboratories). Images were captured with light microscopy (Olympus).