TRF2ts MEFs were seeded onto eight-well chamber slides (Millipore). For IF detection of γH2AX, p-ATM, and 53BP1, cells were washed with PBS and fixed for 10 min with 2% paraformaldehyde (PFA). For p-DNA-PKcs, FK2 and BRCA1, cells were washed with PBS and pre-extracted with ice-cold 0.5% Triton/PBS on ice for 5 min prior to fixation in 2% PFA. Subsequent processing was done as before [32 (link)], further specified in Supplementary Materials and Methods .
For IF-FISH cells were pre-extracted with 0,5% triton/PBS, fixed for 10 min with 2% PFA and 10 min on ice with methanol. Staining with primary antibody for 53BP1 (NB100-305, Novus, 1:500) and secondary antibody (Alexa Fluor 568, Invitrogen), FISH detection of telomere repeats with a FITC-OO-(CCCTAA)3 PNA custom probe (Biosynthesis) and image acquisition were done as described [18 (link)].
For IF-FISH cells were pre-extracted with 0,5% triton/PBS, fixed for 10 min with 2% PFA and 10 min on ice with methanol. Staining with primary antibody for 53BP1 (NB100-305, Novus, 1:500) and secondary antibody (Alexa Fluor 568, Invitrogen), FISH detection of telomere repeats with a FITC-OO-(CCCTAA)3 PNA custom probe (Biosynthesis) and image acquisition were done as described [18 (link)].
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