PMC:4829102 / 26506-28036 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/4829102","sourcedb":"PMC","sourceid":"4829102","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4829102","text":"Immunofluorescence microscopy\nAntibodies employed for immunofluorescence are listed in Supplementary Table 1. For visualisation of RPA foci, cells were pre-extracted on ice for 2 min (10 mM PIPES, pH 6.8, 300 mM Sucrose, 100 mM NaCl, 1.5 mM MgCl2 and 0.5% TritonX-100) and fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. Coverslips were washed 3 × in PBS and blocked in 10% FBS in PBS for 30 min before incubation with primary antibody in 0.1% FBS in PBS for 1h at room temperature. Unbound primary antibody was removed by washing 4 × 5 min in PBS at room temperature followed by incubation with the indicated secondary antibody for 45 min at room temperature. Slides were then washed 4 × 5 min in PBS before mounting with Vectashield mounting medium (Vector Laboratories) with DAPI.\nIn order to visualise ssDNA foci, the same protocol for RPA focus staining was used, preceded by treatment of cells growing on coverslips with 10 μM BrdU for 24 hours before fixation.\nIn order to visualise RAD51 foci, cells grown on coverslips were fixed with 4% paraformaldehyde in PBS for 10 min at room temperature followed by permeabilisation with 0.5% TritonX-100 in PBS for 10 min at room temperature. Cells were then blocked, incubated with primary and secondary antibodies and mounted for analysis as described above. Confocal microscopy was carried out using a Zeiss LSM 510 laser scanning confocal microscope with Zen 2009 software using a 63x objective. Image analysis was carried out with FIJI (ImageJ) software.","divisions":[{"label":"Title","span":{"begin":0,"end":29}}],"tracks":[]}