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Representative areas of the tumor were selected based on hematoxylin-eosin (HE) staining. Tissue sections were incubated for 60 min at 65°C and rehydrated by using xylene and ethanol series. The tissues were then dipped thrice in phosphate buffered saline (PBS). Then, microwave antigen retrieval was performed by the following method: sections were spaced in antigen retrieval buffer (pH 6.4) for microwaving, with high temperature for 5 min and 40°C for 15 min. After cooling to room temperature and washing in PBS, the tissues were quenched of endogenous peroxidase by 3% H₂O₂ for 20 min and blocked with goat serum at 37°C for 30 min, followed by incubation with anti-β8 antibodies (ab80673, 1:100, Abcam, US) or anti-CD8 antibodies (sc-1177, 1:100, Santa Cruz Biotechnology, US) overnight at 4°C. On the following day, tissues were incubated with the universal IgG antibody-Fab-HRP polymer for 30 min. Subsequently, diaminobenzidine and hematoxylin were stained and terminated sequentially. Normal mouse IgG was substituted for the primary antibody as the negative control. Finally, the samples were observed under a light microscope (Olympus Corp, Tokyo, Japan).

Mouse brains collected from the intracranial glioma model were fixed in 4% PFA for 24 h, followed by soaking in 30% sucrose in PBS at 4°C for 48 h. The samples were cut into 20-μm-thick brain slices by a freezing slicer (Leica). Sections were blocked with 10% normal goat serum in PBS plus 0.3% Triton-100 (PBST) for 1 h. Then, the sections were permeabilized with Triton X-100 (0.2%, in PBS containing 10% normal goat serum) for 40 min at RT. Mouse anti-CD8 monoclonal antibody (1:300, sc-1177; Santa Cruz) was added and incubated overnight at 4°C. Then, Alexa Fluor 594 goat anti-mouse secondary antibody (A20185; Invitrogen) was incubated for 1 h at RT in the dark. The nuclei were stained with DAPI. Fluorescence images were obtained by a confocal laser-scanning microscope (Zeiss LSM-710; Oberkochen).