IHC staining for ki67 and cleaved caspase-3

Paraffin sections were deparaffinized and rehydrated, then antigen retrieval was performed using EZ antigen retrieval 3 solution (BioGenex Laboratories, San Ramon, CA, USA). Sections were then blocked with 3% hydrogen peroxide in methanol and 4% fish gelatin at room temperature for 30 min. Sections were then incubated with rabbit anti-ki67 (1:200) or rabbit anti-cleaved caspase-3 (1:100) overnight at 4°C. Following washing with PBS, sections were incubated with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (cat. no. 111-005-045; dilution, 1:200; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) for 60 min at room temperature. Sections were visualized with a 3,3′-diaminobenzidine kit (Vector Laboratories, Inc.), counterstained with hematoxylin at room temperature for 10 sec, dehydrated and mounted in Richard-Allan Scientific^™ Cytoseal^™ XYL (Thermo Fisher Scientific, Inc.). Antibody staining in the tissue sections was observed using a light microscope (×40 magnification).

The count of ki67- or cleaved caspase-3-positive cells was independently performed by two experienced pathologists. Briefly, each entire slide was evaluated and five fields were randomly visualized at a magnification of ×200. Subsequently, the average proportion of positively stained tumor cells was calculated based on the results from the five fields using ImageJ software (version 1.31; National Institutes of Health, Bethesda, MD, USA).