Rabbit anti phh3

Embryos were first permeabilised overnight in PBS + 1% DMSO + 1% Triton. They were then blocked in PBS + 0.1% Triton + 0.1% BSA + 5% NGS, and incubated overnight in primary antibodies as follows: rabbit anti-laminin (Sigma, L9393) at 1:50, rabbit anti-PhH3(Abcam, ab5176) at 1:500, mouse anti-acetylated tubulin(T 6793, Sigma) at 1:250.
Primary wash was performed in PBS + 0.1% Triton + 0.1% BSA, before a secondary block in PBS + 0.1% Triton + 0.1% BSA + 5% NGS, and overnight incubation in goat anti-rabbit and/or goat anti-mouse secondary antibodies at 1:250. Staining with DAPI at 1:500 and rhodamine phalloidin at 1:250 was performed with the secondary incubation. Embryos were washed thoroughly with PBS + 0.1% Triton and mounted for confocal imaging on glassbottomed dishes in 80% glycerol. All imaging was performed on an Olympus V3000 inverted confocal microscope at 30X optical magnification.
For EdU labelling, EdU was applied to live embryos in seawater at a final concentration of 20µM for 2 hours prior to fixation. Fluorescent detection of incorporated EdU was performed following the manufacturer's instructions using a Click-it EdU Alexa Fluor 647 Imaging Kit (Invitrogen) prior to primary antibody incubation.