Tissues were fixed in zinc-formalin, paraffin-embedded and sectioned at 5 microns, and were stained with Hematoxylin and Eosin (H&E), or prepared for immunostaining as previously described [61 (link), 72 (link)]. Images were captured with 10, 20 or 40x objectives, using a Nikon Eclipse NI-U with a DS-QI1 or DS-Ri1 camera and NIS Elements software, and adjusted in Adobe Photoshop. Antibodies were as follows: Rabbit and chicken anti-Krt5, mouse anti-Krt8 (Covance), rabbit anti-Krt8 (Abcam), rabbit anti-Ki-67 (Abcam), rabbit anti-AR (AR-441; Abcam), mouse anti-p63 (Biocare medical), rabbit anti-Syp (Thermo Fisher Scientific), rabbit anti-Krt10 (Covance), rabbit anti-Tgfbr2 (Novus), mouse anti-pAkt (Cell Signaling), rabbit anti-pSmad2 (Millipore), rabbit anti-Smad4 (Millipore) and chicken anti-GFP (Abcam). Alexafluor 488, 546 and 647 secondary antibodies were from Invitrogen. DNA was stained with Hoechst 33342. Confocal images were captured using a Zeiss LSM710 Multiphoton Confocal microscope and adjusted in Adobe Photoshop. Two tissue microarrays (TMA) contained four 0.6mm tissue cores from each of 93 cancers from patients who had radical prostatectomy. A third TMA contained four 0.6mm cores from each of 45 cancers from TURP samples. TMAs were examined by IHC, as in [20 (link)], or by IF for Krt5 and Krt8. Following IF and imaging, the same slide was stained with H&E.
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