Samples were loaded into an automated cytospin machine (Shandon Cytospin 4, Thermo Electron Corporation, Thermofisher Scientific) following the manufacturer′s instructions, and centrifuged at 500–700 revolutions per min (rpm) for 5 min. The slides prepared by the cytospin technique were fixed by immersion in 95% ethyl alcohol for 20–30 min.
The primary antibodies for immunofluorescence staining were mouse anti-Puf-A [18 (link)], rabbit anti-p53 (sc-6243, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and rabbit anti-NPM1 (sc-5564, Santa Cruz). The secondary antibodies for immunofluorescence staining were donkey anti-mouse IgG (Invitrogen) and Alexa555-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA, USA). Briefly, the cells were fixed in 4% paraformaldehyde for 20 min at room temperature, permeabilized with 0.5% PBST for 5 min, and then blocked with 5% bovine serum albumin (BSA) for 30 min. Slides were incubated at 4 °C with primary antibodies. After overnight incubation, the cells were washed and incubated for 1 h at room temperature with secondary antibodies. The cells were then counterstained with DAPI (Sigma, Burlington, MA, USA).
The primary antibodies for immunofluorescence staining were mouse anti-Puf-A [18 (link)], rabbit anti-p53 (sc-6243, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and rabbit anti-NPM1 (sc-5564, Santa Cruz). The secondary antibodies for immunofluorescence staining were donkey anti-mouse IgG (Invitrogen) and Alexa555-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA, USA). Briefly, the cells were fixed in 4% paraformaldehyde for 20 min at room temperature, permeabilized with 0.5% PBST for 5 min, and then blocked with 5% bovine serum albumin (BSA) for 30 min. Slides were incubated at 4 °C with primary antibodies. After overnight incubation, the cells were washed and incubated for 1 h at room temperature with secondary antibodies. The cells were then counterstained with DAPI (Sigma, Burlington, MA, USA).