IHC analysis of MB tumors in Ptch1-CKO mice. MB tumors were analyzed using IHC for Ki67 and p27, as markers of proliferation and differentiation, respectively (N = 12). DAB staining for Ki67 at low (× 3; A and B) and high (× 20; C and D) magnification showed the presence of multiple Ki67-negative areas in some tumors (B and D) but in not others (A and C). (Note: the red boxes show the approximate location of C and D on A and B, respectively.) Within the same two tumors, Ki67 (red; E and F)/p27 (green; G and H) double fluorescent staining revealed that the Ki67-negative areas (F) were regions of differentiation with high p27-expression (H and J). Conversely, tumors with uniform Ki67 expression (E) had scattered p27-expressing cells (G) mixed among the Ki67-expressing cells (I). Scale bars, 1 mm in A and B; 50 μm in C to J.