Cells were grown on Histogrip (Invitrogen) coated glass coverslips and fixed using ice-cold 100% methanol (β-tubulin) or with 3.7% formaldehyde diluted in PBS with 0.5% Triton X-100 for 10 min (Mad2, pSerCdk, Lamin A/C, Plk1, cyclin B1, and securin). All cells were washed and then blocked (3% BSA, 0,1% Tween 20 in PBS) for 30 min. Cells were incubated with primary antibodies were incubated for 2 h at room temperature in blocking solution. DNA was stained with DAPI. For Lamin A/C staining a Leica DM6000 SP8 confocal with a 63× lens was used. All other images were captured using Leica DM5500 microscope coupled with a Coolsnap HQ2 camera, using a Leica 100× or 40× APO 1.4 lens, powered by Leica LAS AF v3 software. To quantify pSer-CDK, cyclin B and secruin levels in cells, a single in-focus plane was acquired. Using ImageJ (v1.48, NIH), an outline was drawn around each cell and circularity, area, mean fluorescence measured, along with several adjacent background readings. The total corrected cellular fluorescence (TCCF) = integrated density – (area of selected cell × mean fluorescence of background readings), was calculated. This TCCF was then equalized against the mean TCCF of neighboring interphase cells in the same field of view, with results presented as fold increase over interphase levels. Box plots and statistical analysis (2-sided unpaired Student t tests) were performed using GraphPad Prism 5. For all other images, 0.3 µm z-sections were taken, de-convolved, and displayed as 2D maximum projections using ImageJ. False coloring and overlays were performed using Adobe Photoshop CS5 software.