2.5. Immunofluorescence

For promyelocytic leukemia protein (PML) and TRF2 co-localization, interphase nuclei were used. The cells were fixed with 3.7% formaldehyde in PBS for 20 min in 1.5 mL tube. After 3 times washing with PBS, the cells were transferred on Polysine Slides (Thermo Scientific™ Shandon™ Polysine Slides, Thermo Scientific, Waltham, MA, USA) and permeabilized with 0.3% Triton X-100 in PBS for 5 min at RT and next blocked with 1% BSA in PBS-T (PBS-Tween20) at RT for 30 min. After washing with PBS-T, the cells were incubated with antibodies: anti-PML (1:200, ab96051) and anti-TRF2 (1:100, ab108997) (Abcam, Cambridge, UK) overnight at 4 °C. After incubation, cells were washed twice with PBS and incubated with secondary antibodies: FITC-conjugated and TexasRed-conjugated, respectively (all at 1:000, F2761, T6390) (Life Technologies, Carlsbad, CA, USA) for 1 h at RT in the dark. Cells were then washed three times with PBS and nuclei were stained with DAPI. Images were taken using an Olympus BX61 fluorescent microscope (Shinjuku, Japan) with objective 20×. To analyze co-localization PML/TRF2, ImageJ software http://rsbweb.nih.gov/ij/ with JACoP plugin was used [16]. The Pearson’s coefficient was used to calculating a set of co-localization indicators.