Localization of survivin in mouse MKs in anaphase. Mouse bone marrow cells were cultured in IMDM supplemented with 10% FBS, 1% penicillin/streptomycin, and 25 ng/mL thrombopoietin for 3 days. For immunostaining, cytospun cells were fixed in 4% fresh paraformaldehyde at 4°C overnight, washed with buffer and permeabilized with 0.25% Triton X-100 in PBS for 20 minutes at room temperature. Slides were blocked with 3% bovine serum albumin in PBS for 60 minutes at room temperature followed by overnight incubation with primary antibodies (full-length polyclonal antibody to survivin, sc-10811, 1:250 dilution; α-tubulin [Sigma-Aldrich], 1:600 dilution) in staining buffer (0.5% BSA, PBS, 0.25% Triton X-100) at 4°C. After 3 washes with 0.25% Triton X-100 in PBS, slides were incubated with appropriate secondary antibodies (red signal for survivin and green for α-tubulin), washed, and then stained with DAPI (blue) to visualize chromosomes. (A) Shown is a representative 16N MK analyzed of 50 MKs in anaphase examined. The intensity of staining was variable (from dim to strong), but all showed staining. (B) An 8N MK in anaphase. In 4% of MKs (2 of 50 MKs in anaphase), a portion of survivin translocated from the chromosomes to the microtubules as indicated by the orange color. White arrows point to distinct overlap of survivin (red) and midzone microtubules (green) in upper left expanded area. However, the majority of survivin remains associated with DNA (yellow arrows) as indicated by the pink color (red and blue overlap, expanded area in lower right corner). (C) A low-ploidy (4N) MK in anaphase; 6 4N MKs showed a similar profile. (D) A diploid NIH 3T3 cell in anaphase demonstrates typical midzone localization of survivin. Magnification, 900×.