Rabbit polyclonal anti ha

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)).