Immunofluorescence Staining Protocol

Cells were permeabilized with 0.2% Triton X-100 (t-octylphenoxypolyethoxyethanol, Sigma-Aldrich) in DPBS for 10 min and rinsed with DPBS. Cells were blocked with 5% normal donkey serum (EMD Millipore)/0.02% (v/v) Triton X-100 in DPBS for 4 h at room temperature or overnight at 4 °C. Primary antibodies [rabbit anti-p53 (7F5) (Cell Signaling 2527S, Lot 8), mouse anti-Mdm2 (2A10) (Abcam ab16895, Lot GR324625–5)] were applied at 1:1000 and 1:200 dilutions, respectively, in blocking buffer and incubated overnight at 4 °C. Cells were washed with DPBS six times for 5 min each. Secondary antibodies [donkey anti-rabbit IgG (Jackson ImmunoResearch) and donkey anti-mouse IgG (Jackson ImmunoResearch)] were labeled as previously described with ATTO 488 (ThermoFisher Scientific) or Alexa Fluor 647 (ThermoFisher Scientific) for a dye ratio of ∼1:1 (51 (link)). Secondary antibodies were added at 3 μg/ml each in blocking buffer and incubated for 2 h at room temperature in the dark. All subsequent steps were performed in the dark. Cells were washed with DPBS six times for 5 min each. Antibody stacks were crosslinked by 4% (v/v) paraformaldehyde in DPBS for 15 min. Remaining fixative was quenched and washed with DPBS-G twice for 5 min each, followed by DPBS twice for 5 min each. Stained cells were stored at 4 °C for up to 2 weeks before imaging.