4.10. Immunocytochemistry

Fibroblasts in culture were seeded on coverslips and subsequently fixed with ice-cold 100% methanol at −20 °C for 10 min. Once fixated, the cells were blocked at 25 °C for 30 min in 10% FBS in PBS-T (0.2% Tween) and then incubated with the following primary antibodies: anti-p21 (MA5-14949, Invitrogen, Waltham, MA, USA, dilution 1:250), anti-lamin A/C (#E-1 sc-376248, Santa Cruz, dilution 1:2500), anti-cGAS (#15102, Cell Signaling, dilution 1:100), anti-progerin [56], anti-prelamin A (MABT858, Sigma-Aldrich, dilution 1:250), anti-P-H2A.X Ser139 (#05-636, Sigma-Aldrich, dilution 1:2000), and anti-lamin A (#L1293, Sigma-Aldrich, dilution 1:2000). After washing with PBS-T, the samples were incubated with the corresponding secondary antibodies at 25 °C for 1 h: affinity-purified Alexa Fluor 488 goat or donkey IgG antibodies (Life Technologies, Carlsbad, CA, USA) and Cy3-conjugated IgG antibodies (Jackson ImmunoResearch). All samples were counterstained with DAPI in Vectashield mounting medium (Vector Inc., Burlingame, CA, USA). Images were acquired using an Axio Imager D2 fluorescence microscope (AxioCam MRm, Objective 40× oil NA 1.4; Carl Zeiss, Oberkochen, Germany). For statistical evaluation, 800 to 1000 cells were screened in 3 independent experiments.