Anti chd7

Embryos (E11.5-E17.5) were dissected and fixed in 4% PFA for one hour at room temperature, washed three times in PBS following a 10%−30% sucrose gradient, embedded in OCT (Optimal Cutting Temperature compound; Tissue Tek Sakura Finetek, Torrance, CA) and stored at −80°C until sectioning. Cryosections were cut on a Leica (Buffalo Grove, IL) CM1950 Cryostat at 12 μm or 14 μm (e17.5) in the transverse orientation and stored at −80°C. On the day of staining, sections were removed from the freezer and dried at room temperature for 30–60 minutes. Slides were briefly post-fixed with 4% PFA and washed in PBST (0.1% Tween20, Sigma-Aldrich, St. Louis MO). Sections were blocked in 1% BSA (Sigma-Aldrich, St. Louis, MO) for one hour, then incubated with anti-CHD7 (Cell Signaling Technology #6505, Danvers, MA) at 1:2500 overnight. Sections were incubated in secondary antibody (AlexaFluor 555) goat anti-rabbit IgG (1:200, Invitrogen, Molecular Probes, Thermo Fisher Waltham, MA) at room temperature for one hour, then counter-stained with DAPI (4′,6-diamidino-2-phenylindole; (Invitrogen, Molecular Probes, Thermo Fisher Waltham, MA) 1:20,000 in 1x PBS) and mounted. Sections were imaged using a Leica upright DM5000B microscope and Leica DFC310 FX Digital camera.