Immunohistochemistry in RIF-1 tumours after everolimus or vehicle treatment. C3H mice bearing RIF-1 tumours were treated with vehicle (A, left column) or everolimus at 10 mg/kg/day (B, right column) for 5 days prior to sacrifice (n = 7 per group). Tumours were ablated and prepared for immunohistochemistry as described in Methods. The entire sections were scanned. Ki67-stained slices of one representative tumour from each treatment (scalebar = 1 mm) are shown in row 1. Viable tumor tissue is outlined in red and necrosis regions are marked with asterisks. Percentage of Ki67-positive cells was 46% and 26% and the total tumour area was 43.9 mm2 (8% necrosis) and 22.4 mm2 (19% necrosis) for the vehicle-treated and everolimus-treated mice, respectively. Magnified sections stained for Ki67 (scalebar = 50 μm), cleaved caspase-3 (CASP3, scalebar = 25 μm), and CD31 (scalebar = 25 μm) are shown below in row 2, 3, and 4, respectively. There was no difference between the groups in apoptosis (CASP-3 staining) and blood-vessel density (CD31 staining).