Fig. 7. The numerous pRb granules are not preferentially associated with perinucleolar regions and do not colocalize with CldU-labeled replication sites during any stage of S-phase. An asynchronous culture of WI38 cells was pulse-labeled for 5 minutes with 10 µM CldU, fixed with formaldehyde and stained with anti-pRb rabbit polyclonal antibodies and anti-nucleolin mouse monoclonal antibodies. The cells were briefly fixed again with formaldehyde before mild HCl hydrolysis and labeling of CldU-substituted DNA with rat monoclonal anti-BrdU antibodies. DNA was stained with DAPI. Images corresponding to double- and triple-labeling protocols were pseudocolored as indicated within each panel. Sites that co-localize appear yellow for red/green, light blue or blue-green for blue/green, and magenta for blue/red merged images, respectively. Based on the appearance of the CldU replication patterns, the cells were classified into early-S (A), mid-S (B) and late-S (C) categories. Besides the strong nucleolar signal, nucleolin exhibits a weaker diffuse nucleoplasmic staining within formaldehyde-fixed cells. This soluble nucleoplasmic form of nucleolin can be removed completely by mild extraction with Triton X100 prior to fixation (not shown). Note the nearly complete lack of colocalization (yellow color) between the pRb (red) and CldU (green) signals in the double-labeled images.