Immunohistochemical Analysis of Tumor Markers

Mice were killed on day 14 after treatments. Tumors were removed from mice and fixed in 4% paraformaldehyde (PFA) at 4°C. Paraffin-embedded tumor tissues were sectioned in 5 μm-thick slices by Bio-Check Laboratories Ltd (New Taipei City, Taiwan). Protein levels in tumor tissues were evaluated with IHC staining. The instructions provided with the kit were followed to perform IHC staining. The sectioned slices were immunohistochemically stained with MMP-9, VEGF, MCL-1, XIAP, C-FLIP, Cyclin-D1, anti-P-ERK, NF-κB p65 (Ser536), P-AKT (Ser473), Caspase-9, -8, and -3 antibodies respectively. The stained slides were scanned using the microscopy-based TissueFAXS platform (TissueGnostics, Vienna, Austria) and images were captured at 100× magnification [12 (link)]. ImageJ software version 1.50 (National Institutes of Health, Bethesda, MD, U.S.A.) was used to evaluate indices of positivity on IHC slides.