Immunocytochemistry of Fibroblast Cells

For immunocytochemistry, fibroblasts were grown directly on coverslips. Cells were fixed in 100 % methanol at -20 °C for 10 min. Subsequently, cells were further processed for immunohistochemistry as previously described [45 (link)]. The following primary antibodies were used: anti-progerin S9 (1 µg mL^-1) [47 (link)], anti-lamin A (Abcam, 1/500), anti-lamin B1 (Santa Cruz Biotechnology, 1/75), anti-γH2AX (Millipore, 1/200), anti-53BP1 (A300-272A, Bethyl, 1/1000), and anti-Rad51 (Novusbio, 1/300). The secondary antibodies were affinity-purified Alexa Fluor 488 goat or donkey IgG antibodies (Molecular Probes) and Cy3-conjugated IgG antibodies (Jackson ImmunoResearch). DAPI in Vectashield mounting medium (Vector Inc.) was used to counterstain the samples. Images were acquired by using an Axioplan fluorescence microscope (Carl Zeiss).