Immunohistochemical Analysis of Tumor Xenografts

Tumors were paraffin-embedded and 4 μm sections were used for immunohistochemistry. The sections were deparaffinized and treated with BLOXALL endogenous blocking solution (Vector Laboratories, CA, USA) to inhibit endogenous peroxidase activity. The epitope retrieval was conducted with Retrieve-All Antigen 1 (pH 8), followed by blocking with PBS supplemented with 2% normal horse serum and 3% BSA. Sections were incubated with individual primary antibodies at 4 °C overnight and with the appropriate secondary antibody at room temperature for 1 h. Peroxidase activity was revealed using 3,3-diaminobenzidine (Vector Laboratories), and sections were counterstained with hematoxylin (Vector Laboratories). After dehydrogenation with alcohol and xylene, sections were viewed and photographed with an Olympus BX51 microscope (Olympus, Tokyo, Japan) and Mosaic2.1 software. The following antibodies were used: Ki-67 (1:100, Abcam, Cambridge, UK), cleaved caspase-3 (1:100, Cell Signaling Technology, MA, USA), and KIT (1:200, Abcam). Ki-67, cleaved caspase-3, and KIT. Positive staining in xenografted tumors were quantified using ImageJ software (NIH). Three HPF (high-power fields, at ×400 magnification) were used for each tumor [42 (link)].