Diaminobenzidine substrate

Caspase 3 and epidermal growth factor receptor (EGFR) in paraffin sections were visualized with rabbit polyclonal Caspase 3 antibody (Abcam, Cambridge, MA, USA) and EGFR antibody (Zen BioScience, Chengdu, China) followed by staining with a horseradish peroxidase (HRP)-conjugated secondary antibody, and diaminobenzidine substrate (Maixin, Fuzhou, China). Five random fields from each section were photographed with a Zeiss digital camera, and analyzed using a semi-quantitative scoring system as follows (Table 3, [19 (link),20 (link)]).

Serial tissue sections were incubated with either mouse anti-human pAkt1/2/3 (1:100 dilution, Thr308/Thr309/Thr305; Santa Cruz Biotechnology, Dallas, TX, USA) or rabbit anti-human pERK1/2 (1:200 dilution, Thr202/Tyr204; Cell Signaling Technology, Danvers, MA, USA) monoclonal antibody at 4°C overnight, and then incubated with a horseradish peroxidase-conjugated secondary antibody (Maixin-Bio, Fuzhou, China) at room temperature for 45 min. The immunoreactivity was visualized using diaminobenzidine substrate (Maixin-Bio). The positivity was determined independently by three liver pathologists considering both intensity and subcellular localization as reported (9).

Tumor tissues were sectioned, mounted on to slides, deparaffinized with xylene, and dehydrated in a series of graded ethanol. For antigen retrieval, the sections were boiled in citric buffer (pH 6.0) for 10 min using a hot plate and then cooled for 1 h at room temperature. Endogenous peroxidase was blocked with 3% H₂O₂ for 15 min, and the sections were blocked with 10% normal goat serum for 20 min. Antibodies against p-Akt, p-ERK, E-cadherin, Vimentin, Snail, cyclin D1, PCNA, Bax, Bcl-2, HIF-1α, and VEGF were applied overnight at 4°C, followed by incubation with secondary antibodies for 1 h at 37°C. Sections were then stained with freshly prepared diaminobenzidine substrate (Maixin Biotech., Fuzhou, China), counterstained with hematoxylin, dehydrated, and mounted. Five nonoverlapping fields per slide were randomly selected, and images were captured with a light microscope attached to a digital camera (Olympus). The captured images were examined independently by two experienced pathologists in a blinded manner.