Axiovison software v4

OCI-AML3, IMS-M2, and K562 or patient cells were fixed for 20 min with methanol at −20 °C, then cytospined on polylysine-L slides (#045797, Dominique Dutscher, Brumath, France), permeabilized using Triton X-100 0.5% for 15 min and blocked for 1 h with 3% BSA in DPBS (pH 7.4). Cells were incubated with a mouse anti NPM1 antibody recognizing its wild type and mutated form (#sc-47725, CliniSciences, Nanterre, France) 1 h at room temperature, slides were then washed and incubated for 1 h at room temperature with a secondary antibody goat anti-mouse IgG-coupled Alexa 488 fluorochrome (#A-11017, Life Technologies, Courtaboeuf, France). Nuclei were counterstained with DAPI (#H-1200, Eurobio, Courtaboeuf, France). Images were acquired by immunofluorescence microscopy on a Zeiss Axiovert microscope with a Plan-Apochromat X40 N.A.1.4 oil immersion objective using the Axiovison software v4.2 (Carl Zeiss, Marly le Roi, France).

Cells were allowed to adhere to polylysine-L slides (Thermo Scientific) and analyzed using an Image-iT^® Fixation/Permeabilization Kit (Life Technologies). Briefly, cells were fixed for 15 minutes with 4% paraformaldehyde in PBS (pH7.3) at room temperature, then permeabilized with Triton X-100 0.5% for 15 minutes and blocked for 1 hour with 3% BSA in DPBS (pH7.4). Cells were incubated with cleaved caspase-3 antibody (rabbit monoclonal Ab 9664; Cell Signaling Technology) or cytochrome c antibody (mouse monoclonal Ab 556432; Becton Dickinson) overnight at 4°C. Secondary antibodies were goat anti-rabbit and goat anti-mouse IgG coupled with Alexa 568 (red) or Alexa 488 (green) fluorochromes (Life Technologies) incubated at 37°C for 2h. Nuclei were counterstained with DAPI (Vector). Images were acquired by immunofluorescence microscopy on a Zeiss Axiovert microscope with a Plan-Apochromat X63 N.A.1.4 oil immersion objective using the Axiovison software v4.2 (Carl Zeiss).