Hrp conjugated donkey anti rabbit or mouse secondary antibody

For paraffin slides from the tissue of patients, after deparaffinization and hydration, slides were repaired by boiling in Tris-EDTA buffer (PH = 8.0) for 10 min and then washed with PBS. Frozen tissue slides of xenograft were washed with PBS three times. Next, sections were treated with 3% H₂O₂ for 20 min to bleach endogenous peroxidase. After blocking with donkey serum in PBS for 30 min, slides were incubated overnight at 4 °C overnight with primary antibodies. Following washes with PBS, slides were then incubated with HRP-conjugated donkey anti-rabbit or mouse secondary antibody (DAKO) for 45 min at room temperature. Sections were then stained by DAB with Gill hematoxylin counterstaining. Samples incubated without primary antibodies were used as negative controls.

For paraffin slides from tissue of patients, after deparaffinization and hydration, slides were boiled in Tris-EDTA buffer (PH = 8.0) for 10 min for antigen retrieval, then washed with PBS; while frozen tissue slides of xenograft were washed with PBS for three times. Next, sections were treated with 3% H₂O₂ for 20 min to bleach endogenous peroxidase. After blocking with donkey serum in PBS for 30 min, slides were incubated at 4 °C overnight with primary antibodies (Supplementary Table 1). After wash with PBS, slides were incubated with HRP-conjugated donkey anti-rabbit or mouse secondary antibody (DAKO) for 45 min at room temperature, followed by staining by DAB with Gill hematoxylin counterstaining. Samples incubated without primary antibodies were used as negative controls. The expression of PPFIBP1 in human glioma specimens was scored as 0 (absent), 1 (weak), 2 (moderate), and 3 (strong) in a double-blinded manner. Patients were accordingly stratified into PPFIBP1^Low (score of 0–1) versus PPFIBP1^High (score of 2–3) groups.