Af2400

Wholemount immunohistochemistry was performed as described previously (Osorno et al., 2012 (link)). Embryos were fixed in 4% PFA in PBS at 4°C for 2 hrs (HF) or overnight (>E8.5 embryos). Samples were costained against Sox2 (Abcam, United Kingdom; ab92494; 1:200) and T (R&D, Minneapolis, MN; AF2085; 1 µg/ml), followed by overnight incubation in PBS containing 4’,6-diamidino-2-phenylindole (DAPI, Life Technologies). Confocal microscopy was performed after dehydration through a PBS/methanol series (10 min each), three 5 min washes in 100% methanol, clearing in 1:1 v/v methanol/BA:BB (2:1 benzyl alcohol:benzyl benzoate), and two washes in BA:BB. Selected embryos were embedded, sectioned and stained as described in (Huang et al., 2012 (link)). Primary antibodies (supplier, catalogue number and working concentration) were as follows: anti-Sox2 (Abcam; ab92494; 1:200, Santa Cruz, Dallas, TX; sc-17320; 1 mg/ml or Merck Millipore, Germany; AB5603; 5 mg/ml); anti-T (R&D; AF2085; 1 µg/ml or Santa Cruz; sc-17743; 1 mg/ml); anti-Foxa2 (Santa Cruz; sc-6554; 1 mg/ml or R&D; AF2400; 1 µg/ml); anti-GFP (Abcam; ab13970; 10 µg/ml); anti-Pax3 (DSHB, Iowa City, IA; 1:20); anti-Pax6 (DSHB; 1:20); anti-PDGFRβ (Abcam; ab32570; 1:100); anti-β-catenin (Sigma; C2206; 1:1000); anti-Active β-catenin (Millipore; 05–665, clone 8E7; 0.1 µg/ml); anti-N-cadherin (Sigma; C3865; 1:400).