Gtx127342

Pools of 50 embryos were collected. Cells were lysed in Pierce IP Lysis Buffer (Thermo Scientific) with protease inhibitor cocktail (Merck), and protein was extracted. Proteins were separated by SDS gel electrophoresis as previously described (Sun et al., 2011 (link)) and incubated overnight at 4°C with anti-H2A.X (phospho Ser139) (1:1,000; GTX127342; Genetex) followed by goat anti-rabbit IgG (H1L)-horseradish peroxidase conjugated secondary antibody (1:5,000; 1,706,516; Bio-Rad). Staining was revealed by using Western Bright Sirius (1:1; Adventa) with an exposure of 1 min and 15 s.

Larvae were fixed for 2 h in 4% paraformaldehyde (PFA) in PBS at 4 °C, and washed in PBS + 0.5% Triton. The larvae were then blocked with 10% fetal bovine serum diluted in PBS for 1 h at room temperature. After blocking, the larvae were incubated in primary antibody: rabbit anti-EGFP (SC-8334, Santa Cruz Biotechnology, Santa Cruz, CA), 1:250 dilution; mouse anti-γH2AX (GTX127342, GeneTex), 1:100 dilution, in blocking buffer overnight at 4 °C. On the following day, larvae were washed in PBS + 0.5% Triton and blocked for 1 h. Anti-GFP was detected with a donkey polyclonal secondary anti-rabbit IgG H&L (Alexa Fluor® 488, 2 mg/ml, ab150061, Abcam), and anti-γH2AX was detected with donkey anti-mouse IgG H&L (Alexa Fluor® 594) (2 mg/ml, ab150064, Abcam), in blocking buffer overnight at 4 °C. Next, larvae were washed in PBS + 0.5% Triton, fixed for 30 min in 4% PFA in PBS, and washed in PBS + 0.5% Triton.

Transgenic fluorescent embryos were embedded in 1% agarose dissolved in 1xE3 in a glass-bottom dish. For immunofluorescence, double staining was performed using chicken-anti-GFP (1:500; Life Technologies), rabbit-anti-H2A.X (phospho Ser139) (1:500; GTX127342; Genetex) rabbit, and pH3 (1:250; Abcam) antibodies. We used AlexaFluor488-conjugated anti-chicken (1:1,000; Life Technologies) and AlexaFluor594-conjugated anti-rabbit (1:1,000; Life Technologies) secondary antibodies to reveal primary antibodies. Confocal imaging was performed using a Nikon inverted A1r spectral.