Immunohistochemical Staining Protocol

Immunohistochemistry assay was performed as described by Xu. et al. [18 (link)]. In brief, serial cross sections of 4 μm thick were collected and stained immunohistochemically according to the manufacturer's instructions. The samples were deparaffinized, rehydrated, and incubated with fresh 0.3% hydrogen peroxide in methanol for 10 min at 37°C. The sections were then autoclaved for antigen retrieval in citrate buffer at 100°C for 5 min and incubated with indicated antibodies overnight at 4°C. The sections were washed with phosphate-buffered saline (PBS) and incubated with biotinylated anti-rabbit IgG as a secondary antibody for 15 min at 37°C and then with streptavidin-conjugated horseradish peroxidase for 15 min at 37°C (Zymed, Carlsbad, USA). The immune reaction was demonstrated using DAB, and the sections were counterstained with hematoxylin, dehydrated, and then mounted.