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×).