Anti h4ac

Embryos were collected on grape agar plates, dechorionated for 2 min in 50% bleach, and fixed in methanol-heptane (1:1) for 5 min. The fixed embryos were stored in methanol at −20°C. Before immunostaining, the embryos were first rehydrated gradually (5 min each in 1:3, 1:1, and 3:1 PTA:methanol and then 10 min in PTA). PTA consisted of PBS supplemented with 0.1% Triton X-100, and 0.02% azide. The embryos were then blocked in PBTA (PTA plus 1% BSA) for 30 min and incubated with primary antibodies (1:100 in PBTA) for 1 h at room temperature or overnight at 4°C. The following primary antibodies were used: anti-Histone 3 (Abcam, ab1791), anti-H3K9me1 (Active Motif, 39249), anti-H3K9me2 (Active Motif, 39683 and 39375), anti-H3K9me3 (Active Motif, 39765; Millipore, clone CMA308), anti-H3K9ac (Abcam, ab10812), anti-H3K27ac (Abcam, ab4729), anti-H3K27me3 (Cell Signaling, 9733), and anti-H4ac (Millipore, 06-598). The embryos were washed three times for 5 min each in PBTA and incubated with the appropriate fluorescently labeled secondary antibodies (Molecular Probes) for 1 h in the dark at room temperature. They were then washed four times for 5 min each in PBTA and mounted in VectaShield mounting medium with DAPI (Vector Laboratories). For TALE-light stainings, GFP- or mCherry-tagged purified TALE-light protein (1:500) was included during the incubation with secondary antibodies.

Chromatin immunoprecipitation (ChIP) experiments were performed as previously described by Bruno et al. (29 (link)) using the following antibodies: anti-AATF/Che-1 (Bethyl), anti-UBF (H-300; Santa Cruz Biotechnology), anti-RPA194 (H-300; Santa Cruz Biotechnology), anti-HDAC1 (Sigma-Aldrich), anti-H3K9me3 (Abcam), anti-H3K27Ac (Millipore) and anti-H4Ac (Millipore). For sequential ChIP experiments (Re-ChIP), immunoprecipitated complexes were eluted in 25 μl 10 mM DTT for 30 min at 37°C. After centrifugation, the supernatant was diluted 10 times in Re-ChIP buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM TRIS pH 8.0) and subjected again to ChIP procedures. Immunoprecipitations with no specific immunoglobulins (Santa Cruz Biotechnology) were performed as negative controls. For quantitative ChIP analysis (ChIP-qRT), 1 μl of purified DNA was used for amplification on a 7500 Fast Real-Time PCR System (Applied Biosystems) using a SYBR Green 2× qPCR Master Mix (Primerdesign). Information on primers used in these experiments is provided in Supplementary Table S3.