Ab32570

The following primary antibodies were used for IF at the indicated dilutions: anti-P0 (1/500, Abcam, ab39375), anti-p75 (1/500, Millipore, ab1554), anti-S100 (1/1000, Dako, Z0311), anti-Iba1 (1/500, Wako, 019-19741), anti-CD31 (1/100, BD Biosciences, 553370), anti-neurofilament 200 kD (1/1000, Abcam, ab4680), anti-laminin (1/500, Abcam, 11575), anti-collagen III (1/1000, Abcam, ab7778), anti-fibronectin (1/500, Sigma-Aldrich, clone FN-3E2), anti-NG2 (1/500, Millipore, ab5320), anti-PDGFRβ (1/500, Abcam, ab32570), anti-αSMA (1/1000, Sigma-Aldrich, C6198), anti-GFP (1/1000, Abcam, ab13970), anti-Glut1 (1/500, Abcam, ab652), anti-F4/80 (1/100, Bio-Rad, MCA497G) and anti-NG2 (1/100, Thermo Fisher Scientific, MA5-24247). For further details of primary antibodies used in this study, see Table S1.

MVNs were fixed with 4% PFA (Thermo Scientific, 11490570) for 1 h at RT, permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, T8787) for 10 min at RT and blocked with 10% Donkey Serum (PAN-Biotech, PANP30-0101). MVNs were then stained with primary antibodies diluted 1:100-1:200 in blocking buffer, overnight at 4 °C. We used anti-CD31 (Abcam, ab24590), anti-PDGFRβ (Abcam, ab32570), anti-S100b (Sigma, S2532), anti-desmin (ab15200), anti-GFAP (Invitrogen, MA5-12023), anti-CLDN5 (Invitrogen, 341600), anti-ZO-1 (Thermo Fisher, 339100), anti-VE-cadherin (Abcam, ab33168), anti-laminin (Abcam, ab7463) and anti-collagen type IV (Sigma, MAB1910) antibodies. Devices were then washed 5 times with 1× PBS for >5 min, and stained with corresponding secondary antibodies Alexa Fluor 488 donkey anti-rabbit (Invitrogen, A-21206) or Alexa Fluor 647 donkey anti-mouse (Invitrogen, A-31571) diluted 1:250 in blocking buffer, overnight at 4 °C. Devices were washed again 5 times with 1X PBS for >5 min, stained with lectin Ulex europaeus agglutinin I (UEA I) Rhodamine (Vector Laboratories, RL-1062-2) or phalloidin (Invitrogen, R37112) and DAPI (Invitrogen, R37606), and washed overnight at 4 °C.

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).