Quantitative Analysis of Nucleolar Proteins

Cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked using 1% bovine serum albumin and 1% heat-inactivated goat serum in phosphate-buffered saline (PBS). Primary antibodies were directed against fibrillarin (#ab5821; 1:500), nucleolin (#ab22758, 1:250), UBF (#ab61205, 1:250; all from Abcam) or p53 (1:200, R135, gift of N. Krupenko), followed by secondary antibodies conjugated to Alexa-488 (1:1000, Invitrogen); nuclei were visualized using DAPI. Digital images were made under uniform exposure, capturing three images per sample, and the number of nucleoli per nucleus and percentage of fibrillarin+, nucleolin+, or UBF+ nucleolar area per DAPI+ nucleus were calculated using ImageJ [36 ].