Chromosome positioning in proliferating interphase nuclei. Proliferating human dermal fibroblasts (HDFs) cultures were subjected to 2D- or 3D-fluorescence in situ hybridization (FISH) to delineate and analyze the nuclear location of chromosomes 10, 13, 18, and X. In panels (a-d), the chromosome territories are revealed in green with a single chromosome territory for chromosome X, because the HDFs are male in origin. The red antibody staining is the nuclear distribution of the proliferative marker anti-pKi-67, the presence of which denotes a cell in the proliferative cell cycle. DAPI (4',6-diamidino-2-phenylindole) in blue is a DNA intercalator dye and reveals the nuclear DNA. Scale bar = 10 μm. The histograms in panels (e-h) display the distribution of the chromosome signal in 50 to 70 nuclei for each chromosome for 2D FISH, as analyzed with erosion analysis. This analysis divides each nucleus into five concentric shells of equal area, with shell 1 being the most peripheral shell, and shell 5 being the most interior shell [4–6, 9]. The percentage of chromosome signal measured in each shell was divided by the percentage of DAPI signal in that shell. Bars represent the mean normalized proportion (percentage) of chromosome signal for each human chromosome. Error bars represent the standard error of the mean (SEM). Panels i and j display 3D projections of 0.2-μm optical sections through 3D preserved nuclei subjected to 3D-FISH and imaged with confocal laser scanning microscopy. The chromosome territories are displayed in red, and proliferating cells also were selected with positive anti-pKi-67 staining (not shown in reconstruction). Scale bar = 10 μm. The line graph in panel (k) displays a frequency distribution of micrometers from the geometric center of the chromosome territories to the nearest nuclear periphery, as defined by DAPI staining. Images for 20 nuclei were analyzed.