A1195

Slices were dewaxed in xylene twice, and then rehydrated in gradient ethanol solution. After antigen recovery using the microwave method in citric acid solution, the sections were blocked in 5% BSA in TBST for 1 h at room temperature, then incubated with the primary antibodies against CD31 (1:250, A19014), VEGFA (1:200), PDGFRB (1:200), VWF (1:200), PDGFB (1:200, A1195), and VEGF (1:200, A16703) purchased from ABclonal (Wuhan, China) and Ki67 (ready-to-use, MAB-0672, Maixinbio, China) at 4°C overnight. The secondary antibody in the streptavidin-biotin kit (#KIT-9720; Maixin-Bio, China) was added to the slides and incubated for 1 h. DAB substrate solution was used to show the positive stain, and the sections were counterstained with hematoxylin. The sections were photographed, and two individuals (G. Y. and Y. C.) independently evaluated all these slides and analyzed the final results using a semiquantitative scoring system as previously described (Gao et al., 2021 (link)).

Paraffin sections were dewaxed with a dewaxing solution and rehydrated with anhydrous ethanol before immunohistochemistry. Following high-temperature and high-pressure antigen retrieval, the sections were treated with 3% H₂O₂ at room temperature in the dark for 25 min to inhibit endogenous peroxidase activity, and then washed with PBS for 3 times. Subsequently, the sections were incubated with 3% BSA at room temperature for 30 min. After the serum blocking buffer was shaken off gently, the sections were incubated with the primary antibodies (TGF-β1 Rabbit pAb, A15103, ABclonal, China; PDGFβ Rabbit pAb, A1195, ABclonal) at a dilution of 1:200 at 4°C overnight. On the next day, all sections were washed with PBS and incubated with the secondary antibody (GB21303, Servicebio) for 50 min at room temperature, and then stained using 3, 3′-diaminobenzidine (DAB). Finally, the sections were counterstained with hematoxylin for 3 min and washed with PBS. The positive signals were observed and evaluated by an optical microscope (E100, Nikon), and the IOD/area (Integrated Optical Density) and percent positive rate were analyzed by Image Pro Plus 6.0 software.

Anti‐HIF‐1α (WB 1:500; IHC 1:100; ab51608), anti‐METTL3 (WB 1:1,000; IHC 1:500; ab195352), anti‐FOXO3 (WB 1:1,000; ab53287), goat anti‐rabbit IgG (WB 1:2,000; ab6721), and goat anti‐rabbit IgG‐FITC (IF 1:200, ab6717) antibodies were purchased from Abcam, USA. Anti‐MAP1LC3‐B (WB 1:1,000; IHC 1:100; IF 1:100; A7198), anti‐PDGF‐B (WB 1:1,000; A1195), anti‐VEGF‐A (WB 1:1,000; IHC 1:100; A5708), anti‐Ki67 (IHC 1:100; A11390), anti‐GAPDH (WB 1:1,000; AC027), anti‐tubulin (WB 1:5,000; AC021), anti‐β‐actin (WB 1:50,000; AC026), anti‐YTHDF1 (IHC 1:1,000; A18126), and anti‐YTHDF2 (IHC 1:1,000; A15616) antibodies were purchased from Abclonal, China. Anti‐m⁶A (MeRIP 1:1,000; ABE572) antibody was purchased from Merck Millipore (Massachusetts, USA). Western blot analyses were performed by standard methods described previously (Niu et al, 2019).