Brdu labelling and detection kit 1

Cells were allowed to incorporate 5-bromo-2-deoxyuridine (BrdU) (10 μM; Roche) for 24 h. Cells were fixed in PFA for 20 min and additionally fixed in 5% acetic acid in ethanol for 20 min. BrdU was detected using reagents from the BrdU Labelling and Detection Kit I (Roche) according to the manufacturer’s instructions. The numbers of BrdU-positive nuclei were blindly counted relative to the Iba1- or IB4-positive cells (at least 150 cells per condition).

Embryo cryosectioning, staining and immunofluorescence was performed as described previously (Huang et al., 2012 (link)). Primary antibodies (supplier, catalogue number and working concentration) were: anti-Timp3-loop1 (Abcam; ab39184; 5 µg/ml), anti-Sox2 (Abcam; ab92494; 1:200), anti-T (R&D; AF2085; 1 mg/ml) and anti-GFP (Abcam; ab13970; 10 mg/ml). For S-phase analysis, E8.5 (2-5 s) embryos were cultured ex vivo in rat serum-containing medium at 37°C for 5 h, containing 31 µg/ml BrdU (BD Biosciences) (Bellomo et al., 1996 (link)). Similarly, tail buds (including the PSM and the last two formed somite pairs) from E10.5-E13.5 embryos were cultured for 4 h, but in N2B27 culture medium (Invitrogen). Samples were fixed overnight in 4% paraformaldehyde in PBS at 4°C and cryosectioned. Antigen retrieval was performed with 10 mM sodium citrate (pH 6.0) for 10 min (Tang et al., 2007 (link)) and sections were stained with a BrdU Labelling and Detection Kit I (Roche). Cells were counted using Photoshop (Adobe) and ImageJ software (NIH).