Af886

Survivin immunofluorescence analysis was performed on frozen tumor sections. Tumor sections were fixed with 4% of paraformaldehyde for 20 min at room temperature. After washing twice with PBS, slides were immersed in 0.5 % of H₂O₂/PBS solution at room temperature for 10 min to block the endogenous peroxidase activity. The slides were rinsed in PBS solution with two changes, 5 min each, and then, incubated in 1% of Triton X-100 solution for 10 min. Enzymatic activity and non-specific binding sites were blocked by incubating the slides in 10% of FBS for 1 hour at room temperature in a humidified chamber. Subsequently, replicate sections were incubated at 4°C overnight with a primary rabbit anti-survivin antibody (AF886; R&D System) at the final concentration of 10 µg/ml. Thorough rinsing was followed by the incubation with the red-fluorescent dye-labeled anti-rabbit IgG (10 µg/ml; T6391; Life Technologies) for 1 hour. Negative controls for each tissue section were performed leaving out the primary antibody. Finally, the nuclei were stained with Hoechst 33342 (5 µM) for 15 min at room temperature and the stained sections were observed and photographed using a Nikon Eclipse E400 microscope and a Spot Advanced software (Spot Imaging). To quantify the survivin protein levels from the images, the cell fluorescent intensity was measured with ImageJ.