H2ax phospho s139 antibody

The acromegaly model and WT zebrafish larvae (9 dpf) were washed with PBS, and then fixed in 4% paraformaldehyde (PFA) overnight at 4 °C. The larvae were treated with methanol for 2 h at −20 °C to enhance permeabilization, and then washed three times in PDT (0.1% Tween-20, 0.3% Triton-X, 1% dimethyl sulfoxide (DMSO) in PBS) with gentle shaking, with a total time of 30 min per wash. The larvae were incubated in blocking solution with 5% BSA for 1 h at room temperature with gentle shaking prior to overnight incubation with rabbit H2A.X (phospho S139) antibody (Abcam; cat No.# ab81299) at a 1/350 dilution in blocking solution. To remove residual primary antibody, larvae were sequentially washed three times with PDT (30 min each wash) and then incubated again in blocking solution with 5% BSA in PBST for 1 h at room temperature with gentle shaking prior to incubation with anti-rabbit, Alexa Fluor 488 (ThermoFisher Scientific; cat. No. A-11034) antibody at a 1/150 dilution in the dark for 2 h at room temperature, and then washed three times in PDT for 30 min each. For counterstaining, larvae were incubated with DAPI at 5/250 dilution in the dark, and then stored in blocking buffer at −4 °C until examination.