Quantifying Telomere-PML Colocalization

Combined telomere FISH and PML immunofluorescence was performed on cytospins as described above. The TissueFAXS Plus (Tissue Gnostics) automated microscopy workstation was utilized for slide-based imaging. The platform contains an 8-slide ultra-precise motorized stage for high-throughput scanning and utilizes a Zeiss Z2 Axioimager microscope with high quality optics. A separate high-performance workstation with TissueQuest software (Tissue Gnostics) was used to analyze the fluorescent images. For each cell line, automated, precise nuclear segmentation was performed to generate total cell number. Images containing the segmentation masks, along with telomere and PML signals, were exported for analysis using using ImageJ software [60 (link)]. Background subtraction was performed for PML images using a rolling ball algorithm, and a threshold was applied to the remaining spots. Rolling ball and threshold values were empirically determined for each set of images. Colocalization between telomere foci and PML bodies was identified using the Colocalization plugin for ImageJ [46 (link)]. All analyses were limited to segmented nuclei.