Ab80673

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).

NPC cells (1 × 10⁵ cells/well) were plated on 8-well chamber slides (SPL Life Science, South Korea) for 24 h. The cells were treated with 10 µM baicalein for 24 h. Cells in 1% DMSO were used as a control. They were washed, fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, blocked with 10% FBS, and probed with 4.5 µg/mL rabbit polyclonal anti-integrin β8 antibody overnight (ab80673; Abcam, Cambridge, MA, USA) (1:100). The cells were incubated with 5 µg/mL Alexa-488-conjugated goat anti-rabbit IgG secondary antibody (A-11034; Thermo Scientific™) (1:100) for 1 h. The cells were then counterstained with 1 µg/mL Tetramethylrhodamine (TRITC)-conjugated phalloidin and 5 µg/mL DAPI (AppliChem, Germany) before imaging using an FV1000 confocal microscope (Olympus, USA).

NPC cells (1 × 10⁶ cells/10 mL medium) were plated onto a 10 cm Petri dish and cultured for 24 h before exposure to 10 µM baicalein for 24 h. They were washed with PBS and lysed with ice-cold RIPA lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, 0.1% SDS, 0.5% sodium deoxycholate and 1% NP-40 with 1 mM PMSF). To remove cell debris, the samples were centrifuged at 9000× g for 20 min at 4 °C. The protein concentrations were determined by Bradford assay (Bio-Rad, CA, USA). Total proteins were then separated by SDS-PAGE and transferred onto nitrocellulose membranes. Nonspecific binding was blocked with 10% BSA for 2 h at room temperature. The primary antibodies against ITGB8 (ab80673; Abcam, MA, USA) (1:1000), β-actin (A1978; Sigma-Aldrich, MO, USA) or and GAPDH (ab128915; Abcam, MA, USA) (1:20,000) were incubated with the membrane overnight at 4 °C. The secondary anti-rabbit antibody conjugated with horseradish peroxidase (HRP) (1:1000) (Cell Signaling Technologies, MA, USA) was added and further incubated for 30 min. The immunoreactivities were detected by SignalFire™ ECL Reagent (Cell Signaling Technologies). The experiments were independently performed in triplicate. The intensities of bands were evaluated using the ImageJ program (Version 1.52r) [59 ].