Immunofluorescence (IF) and Image Analysis

Cells were fixed on a coverslip with 4% formaldehyde (Agar Scientific) for 10 min. Cells were permeabilized with 0.1% Triton-100 (Fisher), 0.1% Tween-20 (NBS Biologicals) in 1× PBS (PBS-Triton-Tween) for 10 min and blocked with 1% BSA (Fisher Scientific) in PBS-Triton-Tween for 30 min. CREST antisera (Europa FZ90C-CS1058) was diluted 1:1000 in the blocking solution, and cells were incubated in humidified chambers for 1 h at room temperature. The cells were then washed with blocking solution, and the cells were incubated with Alexa Fluor-conjugated secondary antibodies (Life Technologies, 1:500) for 30 min. Cells were washed thrice in the blocking solution and mounted in the 4,6-diamidino-2-phenylindole (DAPI) containing medium (Vectashield). The samples were stored in dark at 4 °C before microscopy. The fixed cell images were captured using a Leica SP5 confocal microscope using a 63× or 100× 1.4 NA/oil objective with Z-stacks of confocal slices taken at 1 μm intervals. Pixel intensities were never saturated; laser exposure and detector settings were identical between samples across an experiment. The ImageJ software was used for image analysis. CREST staining was used as a mask to determine the average GFP-PLK1 staining intensity in CREST-stained kinetochores.