Anti phospho smad

Embryos were fixed in 4% paraformaldehyde for 3 h. Primary Abs against Sox9 (1:500, Millipore #AB5535) and β-catenin (1:500, BD Bioscience #610153) in TBST were applied to 5 μm paraffin sections or 10 μm OCT frozen sections and binding visualized with Alexa Fluor 488- or 594-secondary Abs (1:500, Invitrogen). For pSmad immunofluorescence, fixed limb buds were embedded in 7% low-melting agarose and 100 μm vibrotome sections were treated with anti-phosphoSmad1,5 (1:200, Cell Signaling #9516), visualized with Alexa Fluor 594 secondary Ab and imaged using confocal microscopy. Anti-Cyclin D1 (1:50, Thermo Scientific #RM9104) and Anti-Caspase 3 (1:250, Cell Signaling #9661) were detected with horseradish peroxidase-secondary Abs (Vector Labs) on paraffin sections. For immunoblots, 1% SDS lysates of distal digital plates (digit rays and interdigits) dissected from limb buds were used, and blots were probed with affinity-purified polyclonal anti-Hoxd13 (1 μg ml⁻¹ final, gift from Scott Stadler), anti-Gli3 (ref. 25 (link); 1 μg ml⁻¹ final) and anti-Vinculin (1:1,000, Sigma #V4139). Band intensities were quantitated with the Image J software or with the Odyssey Li-Cor system to quantify fluorescence signals and were normalized to Vinculin; at least three independent samples were analysed for each genotype. Significance of differences was determined using the two-tailed, Student's t-test.