2.10. Immunohistochemistry Analysis of Bcl-2-Associated X Protein (BAX) and B-Cell Lymphoma/Leukemia-2 (BCL-2)

The fresh tumor tissue samples were fixed in 4% paraformaldehyde and embedded in paraffin. 5 μm thick paraffin sections were mounted on glass slides and treated with 3% hydrogen peroxide for 10 min at room temperature. After washing with phosphate-buffered saline (PBS), the sections were blocked with sheep serum for 10 min at room temperature, followed by incubation with anti-BAX or anti-BCL-2 primary antibodies at 4°C overnight. The secondary antibodies were applied for 30 min at room temperature, and, then, the slides were stained with diaminobenzidine (DAB) and hematoxylin. The immunohistochemistry staining was observed under a microscope (BX-53; Olympus).