Immunohistochemistry and Scoring

HCC tumor tissues were resected from the patients, and then were fixed with 10% formalin and embedded in paraffin before prepared for tissue microarray. Briefly, 1-mm cores were taken from intratumoral tissue and serial sections (4-μm thick) were placed on slides coated with 3-aminopropyltriethoxysilane. The immunohistochemistry analysis was conducted as described previously³⁰. Immunostaining scores were independently evaluated by two pathologists who were blinded to the clinical outcome. The sections were incubated with primary goat anti-human LASS2 polyclonal antibody (clone sc-65102, 1:80, Santa Cruz Biotechnology)³⁸ and rabbit anti-human TGF-β1 polyclonal antibody (18978-1-AP, 1:100, Proteintech)³⁹ at 4 °C overnight, and then secondary antibody within 30 minutes. The average sum of integrated optical density (IOD) of each sample was calculated using ImageJ software.