Immunofluorescence was performed as previously described (Ali et al., 2018 (link)). For most experiments, cells were suspended in 10 mM Tris, pH 7.4, and fixed with formaldehyde (4% final concentration) and Triton X-100 (0.5% final concentration) for 30 min. Cells were then resuspended in 4% paraformaldehyde and 3.4% sucrose solution and spread onto slides. Slides were stained with appropriate antibodies and mounted with Vectashield anti-fading agent (Vector Laboratories, Burlingame, CA) supplemented with 0.5 μg/ml DAPI. For visualization of HA-tagged proteins, either monoclonal mouse anti-HA (1:1000) or polyclonal rabbit anti-HA (1:200; Sigma, St. Louis, MO) was used. Other antibodies used for immunofluorescence were rabbit anti-Pdd1 (1:1000; Abcam, Cambridge, UK) and rabbit anti-H3K27me3 (1:1000; Millipore/Merck, Burlington, MA), monoclonal mouse anti-Dmc1/Rad51, clone 51RAD01 (1:50; Neomarkers, Fremont, CA), and monoclonal mouse anti-phosphorylated H2A.X (1:200; BioLegend, San Diego, CA).
To detect replication during development, cells were incubated in BrdU for 1 h and then fixed for immunostaining (as described above). Before incubation with anti-BrdU antibody (1:40; Abcam, Cambridge, UK), samples were denatured as previously described (Shodhan et al., 2017 (link)).
To detect replication during development, cells were incubated in BrdU for 1 h and then fixed for immunostaining (as described above). Before incubation with anti-BrdU antibody (1:40; Abcam, Cambridge, UK), samples were denatured as previously described (Shodhan et al., 2017 (link)).