Ab21061

EBs (6, 8, and 12 days old) were fixed with 1% paraformaldehyde during 15 min at room temperature (RT). Fixed EBs were cryoprotected in 15% sucrose in PBS, embedded in a solution containing 7.5% gelatine (Sigma-Aldrich) and 15% sucrose in PBS, frozen and cryosectioned (8–10 μm). EB sections were immersed in PBS at 37°C until gelatine was completely dissolved, and then processed for immunocytochemistry. Sections were blocked with 10% FBS and 0.05% Tween in PBS for 1 h, followed by incubation overnight with the following primary antibodies: Anti-MyoVIIa (1:400, HPA028918, Sigma), Anti-MyoVI (1:50, 25-6791, Proteus Biosciences) Anti-Pou4f3 (1:50, HPA038215, Sigma), Anti-Espin (1:1,000, gift of A. J. Hudspeth), Anti-Gfi1 (1:2,000, gift of Hugo Bellen), Anti-Gfi1 (ab21061 or ab290, Abcam), Anti-Lhx3 (1:100, ab14555, Abcam), Anti-Tuj1 (1:500, MMS-435P, Covance), Anti-GFP (1:500, ab13970, Abcam), Anti-Doublecortin (1:1,000, AB2253, Merck Millipore). Sections were washed 3 times in PBS followed by incubation for 1 h at RT with AlexaFluor-conjugated secondary antibodies (1:400, Molecular Probes) and 0.15% DAPI (Sigma-Aldrich). Slides where then mounted with prolong gold (Life technologies). Fluorescent images of fixed sections were captured with Widefield Zeiss observer or using a Leica TCS SP8 Confocal 4 Detectors. All digital images were formatted with Adobe Photoshop CS and ImageJ.