Immunofluorescence and 3D Imaging of Nucleoli

Cells were washed with PBS, fixed in 4% PFA for 15 minutes and permeabilized with 0.5% PBS/Triton for 5 minutes. Incubation with primary antibody was performed for 1 h in PBS-5% BSA and cells were stained with AlexaFluor 488/568 conjugated anti-rabbit or -mouse secondary antibodies (Molecular Probes) and counterstained with DAPI. After mounting in 50% glycerol/50% 0.2 M Na-glycine, 0.3 M NaCl, 3D epifluorescent 3D image stacks were acquired using a Leica DMI6000B microscope equipped with an Orca C4742-80-12AG camera (Hamamatsu) and Volocity (Perkin-Elmer Improvision) and were subsequently deconvoluted (Iterative Restoration, Volocity). In a few cases 3D stacks were also obtained using a Leica SP5 II scanning confocal microscope. Nucleolar statistics were obtained from three independent immunofluorescence experiments in which ∼20 nuclei were analysed by a protocol established using the Volocity software. DAPI staining was used to define the nuclear volume and Fibrillarin staining to define the nucleoli and their individual volumes. 3D Immuno-FISH was performed as previously described [77] (link) using a Cy3 labeled fragment from the mouse rDNA, (positions 20138 to 23651 in Genbank Accession BK000964.3). Colocalization of FISH and protein signals were estimated using Volocity and given by the Pearson Global Correlation [78] .