Anti steap3

Sections (4 μm) were cut from formalin-fixed, paraffin-embedded tissues of WHO grade II – IV gliomas, deparaffinized, rehydrated in an alcohol series, and pretreated with high-temperature antigen retrieval to 98 °C in 0.01 M citrate buffer for 20 min. Endogenous HRP activity was quenched with 3% H₂O₂ diluted in ethanol for 30 min at room temperature. Sections were blocked with 10% normal goat serum (Beyotime, China), incubated with anti-STEAP3 (Abcam, UK; dilution 1: 500) and anti-TfR antibodies (Abcam, UK; dilution 1: 800) at 4 °C overnight, and rinsed with physiological phosphate buffered saline (PBS) three times for five minutes each. Detection was performed at 37 °C for 30 minutes with a goat anti-rabbit secondary antibody and HRP conjugated to a polymer according to the manufacturer's instructions. Signal was developed with 3,3′-diaminobenzidine (DAB; Beyotime, China), and slides were counterstained with hematoxylin. For negative controls, sections were incubated with normal goat serum instead of the primary antibody. Staining of cancer cells was scored as follows: 0, no staining; 1, weak staining in <50% of cells; 2, weak staining in ≥50% of cells; 3, strong staining in <50% of cells; and 4, strong staining in ≥50% of cells.