Anti pml

FFPE tissue samples were fixed in 10% buffered formalin, dehydrated, embedded in paraffin wax and sectioned at 4 μm. Tissue sections were deparaffinized in xylene and re-hydrated through a series of decreasing ethanol concentrations up to water. Immunofluorescence (IF) was performed on deparaffined tissue sections processed with 10 mM sodium citrate (pH 6.5) cooked under pressure for 2 min. Tissue sections were permeabilized with 0.5% Triton in PBS and blocked with 5% BSA in PBS. Samples were incubated overnight at 4 ºC with rabbit polyclonal anti-PML (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA, H-238). Q-FISH was performed on IF-stained slides fixed with 4% formaldehyde for 20 min. The DAPI images were used to detect telomeric signals inside each nucleus. Immunofluorescence images were obtained with a TCS-SP8 STED 3X confocal microscope equipped with a 63×/NA 1.4 oil immersion objective, a white light laser and LAS X v3.5 software (Leica-Microsystems). Z-stacks of the samples were acquired and then analyzed with Definiens Developer XD 64 v2.5 software (Definiens Inc., Munich, Bayern, Germany).

Immunofluorescence was performed as described previously [10 (link)]. Anti-PML (mouse, 1:100 dilution) was purchased from Santa Cruz Biotechnology. Anti-γH2AX (Ser-139, mouse, 1:100) was purchased from BioLegend, and anti-Ki67 (mouse, 1:100) was from BD Biosciences. Cells were incubated with a goat anti-mouse (clone Poly 4043) or a donkey anti-rabbit (clone Poly 4064) secondary antibody conjugated with Cy3 (BioLegend, San Diego, CA) for 1 h in the dark, washed with PBS, and mounted on microscope slides using Vectashield mounting medium containing DAPI for fluorescence (Vector Laboratories, Burlingame, CA). Images were captured on a Leica SP2 confocal microscope using the appropriate filter sets.

Cells were grown on coverslips, fixed with 4% paraformaldehyde and permeabilized with ice-cold methanol. Cells were rinsed with phosphate-buffered saline, blocked with 10% goat serum and then incubated with primary antibody overnight, followed by incubation with Alexa Fluor-conjugated secondary antibodies (Life Technologies). Coverslips were mounted with ProLong Gold Antifade reagent with DAPI (Life Technologies). The following primary antibodies were used for immunofluorescence: anti-Flag (Sigma-Aldrich, F1804, 1:400), anti-HA (Santa Cruz Biotechnology, sc-805, 1:400), anti-PML (Santa Cruz Biotechnology, sc-966, 1:400) and anti-PP1α (Bethyl Laboratories, A300-904A, 1:400). The stained slides were visualized by a bright-field or confocal microscope. Senescence-associated β-galactosidase activity in prostate tissue was measured with the senescence detection kit (Calbiochem) on 5 μm thickness frozen section.

Approximately 35,000 cells were seeded on 18 mm round coverslips the day before treatment. 24 h after treatment, cells were fixed and stained as previously described [66 (link)]. The following antibodies were used: anti-PML (1:200, Santa Cruz, Dallas, TX, USA), Cy3 anti-mouse (1:400, Thermo Fisher, Waltham, MA, USA). Cells were eventually stained with DAPI (1:10,000, Sigma Aldrich, Steinheim, Germany). Final stainings were stored at 4 °C and analyzed using an Olympus IX83 inverted microscope in combination with Olympus CellSens Dimension software (Olympus, Tokio, Japan).