Embryo Fixation and Immunostaining Protocol

Embryos were fixed immediately after collection following the removal of the zona pellucida with acid Tyrode's solution (Sigma) and processed as described.⁴³ (link) After permeabilization embryos were washed 3 times in PBS-T (0.1% Tween in PBS), blocked and incubated with the primary antibodies for ˜12 h at 4°C, followed by 2 washes in PBS-T, blocking for 30 min and incubation for 2 h at 25°C with the corresponding secondary antibodies: A488-conjugated goat anti rabbit IgG, (Life Technologies A11070) or Cy3-conjugated goat anti mouse IgG, Jackson ImmunoResearch (115–165–146) used at a 1:500 dilution. After washing, embryos were mounted in Vectashield (Vector Laboratories) containing 4′-6-Diamidino-2-phenylindole (DAPI). For visualizing cell-cell boundaries, blastocysts were stained with Alexa Fluor 635 phalloidin (Molecular Probes). The antibodies used were: H3K64ac (⁹ (link); 1:200 dilution); H3K122ac (ref. ¹¹ (link); 1:150 dilution), H3K56ac (Abcam ab76307; 1:250 dilution) and anti-BrU (SIGMA-Aldrich B8434-clone BU-33; 1:250 dilution)