Bf0711

Tissue sections were prepared as mentioned above and immunohistochemistry was performed according to the standard operating procedure. In brief, slices were deparaffinized in xylene, dehydrated in gradient ethanol, and treated with microwave for antigen retrieval. Then, the sections were pre-incubated in 3% hydrogen peroxide in methanol at room temperature for 10 minutes, blocked with 10% bovine serum albumin (BSA) and incubated with the primary antibodies (1:100 dilution for Ki67, cat.No. ab15580, Abcam; 1:100 dilution for CD31, cat.No. EP3095, Abcam; 1:100 dilution for Caspase 3, cat.No. BF0711, Affinity) overnight at 4 °C. Afterwards, the tissue slices were incubated with an HRP-conjugated secondary antibodies, stained with DAB and counterstained with hematoxylin. Images were obtained under a microscope in a 400×field and five representative fields were quantified using the software IPP 6.0 and assessed by two individual investigators. To analyze the cell proliferation, the number of Ki67 positive/proliferating cells (nuclei stained brown) were divided by the total number of cells in a field to calculate the proliferating index.^{60 (link)}