Af2085

H1 hESCs treated with FLAg and CHIR99021 for 3 days were washed with cold PBS, and fixed for 20 min with 4% PFA. Fixed cells were rinsed with PBS, permeabilized with 0.3% triton for 10 min, and blocked with 1% BSA for 1 hr. Cells were incubated for 2 hr with primary Abs for BRACHYURY (R and D systems AF2085; goat; dilution 1:100) or OCT3/4 (Santa Cruz sc5279; mouse; dilution 1:100) in 1% BSA and 1 hr with Alexa Fluor 488-labeled donkey anti-goat or Alexa Fluor 568-labeled donkey anti-mouse Ab. Coverslips were mounted using Fluoromount (Sigma-Aldrich) and analyzed on a Zeiss LSM700 confocal microscope using a 60 × oil objective. Images were assembled using ImageJ and Photoshop CS6 (Adobe Systems, Mountain View, CA).

Fixed cells were permeabilized with 0.05% TritonX-100/0.075% Sodium dodecyl sulfate (Fisher Scientific, BP151-100; Sigma Aldrich, 436143) for 20 min and blocked with 10% normal goat serum (Abcam, ab7481; for staining with Oct4 and Nanog) or 5% BSA (Sigma Aldrich, A8806; for staining with Sox1, Tbra, FoxA2, Otx2, YAP) for 1 h. Cells were incubated with 1:200 primary antibody against Sox1 (Cell Signaling Technology, 4194 S), Tbra (RD Systems, AF2085), FoxA2 (Santa Cruz, sc-374376), Oct4 (Santa Cruz, sc-5279) Nanog (Cell Signaling Technology, 8822 S), YAP (Cell Signaling Technology, 14074) or Otx2 (R&D Systems, AF1979) in blocking buffer for 2 h at room temperature, washed three times with 0.01% TritonX-100 (Fisher Scientific, BP151-100) and incubated with Alexa-488, −568 or −647 conjugated secondary antibody (1:1000, Thermo Fisher Scientific) and NucBlue (Thermo Fisher Scientific, R37605) in blocking buffer for 1 h at room temperature. Cells were washed 3 × 15 min with 0.01% TritonX-100 and incubated in PBS for imaging.

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).