Anti ki 67 antibody

Formalin-fixed paraffin-embedded sections (4 μm) of resected tumor tissue were used for histologic analysis. Hematoxylin and eosin staining was conducted according to standard protocol. Ki-67 staining was performed with anti-Ki-67 antibody (Nichirei Biosciences, Tokyo, Japan) and hematoxylin background. The Ki-67 index (%) was estimated by counting the number of Ki-67-positive cell nuclei per 1200–1800 tumor cells in the three regions of the tumor with the greatest staining density.

Preparation of sections from paraffin-embedded samples and antigen retrieval were
performed in the same process above-mentioned. To inactivate endogenous peroxidase, the
section was incubated with 3% hydrogen peroxide (H₂O₂) for 30 min.
After blocking for 30 min with 10% normal goat serum, the section was incubated with
rabbit polyclonal anti-MISP antibody (1:250, 26338-1-AP) or rabbit monoclonal anti-Ki-67
antibody (418071, Nichirei Biosciences, Tokyo, Japan) overnight at 4°C. Sections were
reacted with peroxidase-conjugated anti-rabbit IgG polyclonal antibody (418261, Nichirei
Bioscience) for 30 min, and stained with 3,3-diaminobenzidine (DAB; 040-27001, Wako,
Tokyo, Japan). The intensity of DAB staining was measured with ImageJ software, and the
percentage of positive cells was calculated.