Ki67 Expression Quantification in Xenograft Tumors

Immunohistochemistry was done as described previously [19 (link)]. Xenograft tumor tissues were fixed with 4% PFA, dehydrated, and embedded in paraffin. The rehydrated tissue slices were treated by heat-mediated antigen retrieval in citrate buffer (Vector Laboratories, Burlingame, CA). The tissues were made permeable with PBS containing 0.2% TritonX-100, then treated with 3% hydrogen peroxide to inactivate endogenous peroxidase. The tissues were washed with PBS containing 0.05% Tween-20, and incubated for 2 h with blocking solution (MOM blocking buffer, Vector Laboratories, CA). Primary Ki67 antibody (550609, BD Biosciences, San Diego, CA, dilution, 1:2000) was incubated overnight at 4°C, and secondary antibodies were incubated for 1h at room temperature. Signals were amplified with ABC kit (Vector Laboratories) and visualized with 3,3′-diaminobenzidine substrate kit (SK-4105, Vector Laboratories). The tissues was further stained with hematoxylin, then dehydrated, and mounted (H5000, Vector Laboratories). Photograph was taken and Ki67 positive cell percentage was calculated.