Figure 4.

Specific imaging of pancreatic cancer cells expressing mutant K-ras and survivin mRNAs on frozen tissue sections of pancreatic cancer tissues. Frozen tissue sections were incubated with K-ras MB1 or K-ras MB2 and counterstained with Hoechst 33342. All fluorescent images were taken by Nikon Eclipse E800 fluorescence microscope under a 40 × lens using an Optronics Magnafire digital imaging system. (A) Detection of expression of specific mutant K-ras genes in pancreatic cancer cells on frozen sections using K-ras MBs. K-ras MB1 detected the cancer cells expressing a GGT to GAT mutant K-ras gene on frozen sections of pancreatic cancer tissues from patients #1 and #2. However, bright red fluorescent cells were found on frozen sections of pancreatic cancer tissues from patient #5, which had a K-ras GGT to GTT mutation, only after incubation with K-ras MB2. The frozen sections from normal pancreatic tissues (patient #1 and #5) did not show bright red fluorescence signals following incubation with either K-ras MB1 (patient #1) or K-ras MB2 (patient #5). (B) Frozen sections from patient #2 were incubated with either K-ras MB1 or K-ras MB2 and the fluorescent images were taken under a fluorescence microscope. The sections were then stained with H&E and observed under a bright field of the microscope. The same areas were identified and compared with previous fluorescent images. Yellow arrows indicate pancreatic cancer cells expressing a GGT to GAT mutant K-ras gene. White arrows show the absence of fluorescence in normal stromal cells in K-ras MB 1 labeled section or in pancreatic cancer cells in K-ras MB2 labeled section. (C) Bright fluorescence was detected in normal pancreatic tissues after incubation with GAPDH MB-Cy3, suggesting that the absence of fluorescence in K-ras MB labeled normal pancreatic tissues was not a result of degradation of mRNAs in the frozen sections.