Rabbit anti sox6

Mouse or zebrafish embryos were fixed in 4% paraformaldehyde, extensively washed in PBS, and incubated overnight in PBS containing 15% sucrose. Samples were then frozen in nitrogen-chilled isopentane and kept at −80°C until use. Cryostat sections (8 μm thick) were permeabilized in 1% BSA, 0.2% Triton X-100 in PBS for 30 min at room temperature and then incubated for 1 hr in blocking solution (10% goat serum in PBS) and overnight with the primary antibody or with mock PBS. After incubation for 45 min with the fluorescent-conjugated secondary antibody (Jackson ImmunoResearch Laboratories), sections were washed in PBS 0.2% Triton X-100 and mounted, and fluorescent immunolabeling was recorded with a DM6000 Leica microscope. The following primary antibodies were used: rabbit anti-Sox6 (Abcam, 1:300), rabbit anti-Nfix (Novus Biological, 1:200), mouse anti-MyHC-I (Sigma, 1:200), BAD5 (monoclonal, 1:2), MF20 (monoclonal, 1:2), and F59 (monoclonal, 1:10). Nuclei were stained with Hoechst (1:1,000).