4.7. Liver Pathology and Tumor Immunohistochemistry Staining

The liver and tumor tissue isolated from mice were fixed with 10% neutral buffered formalin and embedded by paraffin. The liver tissue section stained with hematoxylin and eosin (H&E) was used as an anatomical pathology diagnosis to compare untreated and DMC-treated mice [42]. Tumor immunohistochemistry staining of Bcl-2, XIAP, cleaved caspase-3, and BAX was performed as previously described [41].In brief, tumor sections from the individual group of mice were incubated with primary monoclonal anti-Bcl-2 (1:300 dilution; Cell Signaling, MA, USA), anti-XIAP (1:300 dilution; Elabscience Biotechnology Inc., Houston, TX, USA), anti-cleaved caspase-3 (1:300 dilution; Cell signaling, Danvers, MA, USA), and anti-BAX antibodies (1:300 dilution; Rosemont, IL, USA) at 4 °C overnight. Then, followed with secondary antibodies staining for 1 hr and wash twice by rinse buffer before Horseradish Peroxidase Streptavidin (HRP Streptavidin) inoculation. Finally, slides were dehydrated, stabilized with mounting medium, and scanned by Nikon ECLIPSE Ti-U microscope (×100 magnification, Nikon Instruments Inc., Melville, NY, USA). A total of five view images was quantified by Image J and used to represent the protein level alteration (version 1.50, National Institutes of Health, Bethesda, MD, USA) [43]. Image was quantified by Image J IHC tool box developed. The procedure of analysis was followed by the protocol described on the website (https://imagej.nih.gov/ij/plugins/ihc-toolbox/index.html, since 2014, accessed on 1 January 2021) [44].