The study was approved by the Research Ethics Committee of Xi'an Jiaotong University. HCC samples from mice xenograft were examined immunohistochemically by treptavidin-peroxidase (SP) method, as described previously [56 (link)]. Briefly, paraformaldehyde-fixed paraffin embedded tissues were cut into 4-μm sections. The sections were dewaxed, dehydrated, following antigen retrieval in a pressure cooking for 5 min then incubated with 0.3% (v/v) hydrogen peroxide for 10 min to block non-specific antigens and any endogenous enzyme. The sections were incubated with normal goat serum for 10 min then with the primary antibody against NICD1, P21 or Bcl-2 at 4°C overnight. The slides were washed with PBS and incubated with biotinylated goat anti-rabbit IgG (1:100 dilution; Zhongshan Golden Bridge Biotechnology, Beijing, China) for 20 min as the secondary antibodies. Each slide was colored with DAB (Sigma, St. Louis, MO, USA) in a dark room before incubated with peroxidase-conjugated streptavidin (Zhongshan Golden Bridge Biotechnology) for 10 min. At last, all the sections were rinsed with running water, counterstained by hematoxylin and dehydrated in graded ethanol. The slides were read and marked by two independent pathology experts under a microscope (Olympus Optical Co, Tokyo, Japan). Haematoxylin and eosin (H&E) staining was performed according to regular procedures.
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