Anti phospho h3antibody

16a and 16e were microinjected into one-cell stage zebrafish embryos using a PV820 Pneumatic PicoPump (WPI, Sarasota, FL, USA). For immunostaining, embryos were fixed in 4% paraformaldehyde. Embryos were rinsed three times in phosphate-buffered saline (PBS), in cold methanol for 5 min, and then rinsed in MeOH/PBS-Tween 20 (PBST) and PBST for each 5 min. Embryos were blocked for 3 h using Blocking Reagent (5% BSA/ 10% goat serum in PBS/1% DMSO/0.5% Triton-X100/0.1% Tween 20) with mild rotating. Embryos were incubated overnight at 4 °C with a polyclonal anti-phospho-H3 antibody (1:1000, Cell Signaling Technology, Danvers, MA, USA). Embryos were washed 4× in PBDTT (1 h each) before addition of biotin-labeled goat anti-rabbit antibody (1:2000) in 1% BSA/PBDTT after blocking for 4 h. Antibody staining was detected using the Vectastain Elite ABC reagent and Vector DAB substrate kit to the manufacturer’s specifications. Embryos used in this study were obtained from the Zebrafish Center for Disease Modeling (ZCDM; Daejeon, Republic of Korea). Animal experiments were conducted according to guidelines approved by the Animal Care and Use Committee of Chungnam National University.

Guts were dissected at indicated ages in ice-cold PBS and immediately
fixed in 4% formaldehyde for 30 minutes. The staining was performed essentially
as described38 with anti-phospho-H3
antibody (Cell Signaling, 9701), anti-Prospero (Developmental Studies Hybridoma
Bank) or anti-HRP48 . Guts were mounted in
mounting medium with DAPI (Vectastain). pH3 positive cells per midgut were
counted on a fluorescence microscope. Representative images were acquired with
the Zeiss LSM700 confocal microscope.