Immunohistochemistry staining
Immunohistochemical analysis for Ki67 was performed on 4-μm sections. The Envision Plus detection system (Dako, USA) was used for the detection of immunostaining. Tissue sections were pretreated with 10 mM sodium citrate buffer for antigen unmasking (pH 6.0) after deparaffinized in xylene. Endogenous peroxidase activity was blocked by incubation with 0.03% hydrogen peroxide in methanol for 15 min. Then sections were incubated with Ki67 primary antibody (MA5-14520, Thermo Scientific, USA) at 4°C overnight after blocked in normal serum for 30min. Next, Sections were incubated with secondary antibody at room temperature for 60 min before staining for 5 min with 3’3-diaminobenzidine tetrahydrochloride, counterstained by hematoxylin, and observed by microscope (200×).