Immunohistochemical Analysis of Liver Tissue

Liver tissues were excised and fixed with 4% paraformaldehyde in PBS for 24 h and then dehydrated in PBS containing 30% sucrose for at least 16 h. Following paraffin embedding, the tissue sections were stained with hematoxylin and eosin (H&E). Immunohistochemistry was performed as described previously with modifications (Wu et al, 2011 (link)). Briefly, sections were deparaffinized with xylene and rehydrated. Heat‐induced epitope retrieval was performed by placing slides immersed in sodium citrate buffer (pH 6.0) at 100°C for 20 min followed by endogenous peroxidase blocking with 3% H₂O₂ for 30 min. Blocking was performed with 2% (w/v) BSA, and then the sections were incubated with Ki67 antibody (CST, 9449, 1:400) at 4°C overnight. Subsequently, the sections were incubated with Horseradish peroxidase (HRP)‐conjugated goat anti‐mouse antibody for 2 h at room temperature, and then stained by diaminobenzidine tetrahydrochloride (Gene Tech, GK500710). The sections were counterstained with hematoxylin.