Goat anti human sox17

Cells at different time points of differentiation were fixed using 4% paraformaldehyde (PFA). Samples were then subjected to treatment with NH₄Cl and blocking with 0.3% TritonX-containing BSA before incubation with the primary antibodies. Mouse anti SSEA-1 (Santa Cruz Biotechnology, USA), 1:200, over-night at 4 °C; rabbit anti-PKD1 (Santa Cruz, USA), 1:200, 2 h at room temperature (RT); rabbit anti-PKD2 (Orbigen, USA), 1:1000, 1 h at RT; mouse anti-Oct3/4 (Santa Cruz, USA), 1:200, overnight at 4 °C; rat anti-CD31, (Becton Dickinson, USA), 1:100, 1 h RT; mouse anti α-Actin (Sigma Aldrich, Germany) 1:100, 1 h 37 °C. Samples were further incubated with fluorescence labelled secondary antibodies Alexa Fluor® 488 (green), Alexa Fluor® 568 (red), Alexa Fluor® 647 (magenta) (Life-technologies, all diluted 1:500). For germ layer-specific staining we used the chicken anti β–tubulin-III antibody ((TUBB3), Millipore, Billerica, MA), 1:1000, goat anti-human Brachyury (R&D Systems, Minneapolis, MN, USA, www.rndsystems.com), 1:100, o.N. 4°, AF2085 and goat anti-human SOX17 (R&D Systems), 1:500, o.N. 4°, AF1924. Nuclei were stained with DAPI (blue) (1:20,000). Images were captured using an upright fluorescence Zeiss Axioimager Z1 microscope and analysed using Axiovision software (Zeiss, Germany).

The cells were harvested on days 5, 13, and 20, and were fixed in phosphate-buffered saline (PBS) containing 4% paraformaldehyde for 15 min at room temperature, followed by three washes with PBS containing 0.1% bovine serum albumin (BSA). The cells were permeabilized using 0.1% Triton X-100 in PBS containing 0.1% BSA and 4% normal goat serum (Gibco-BRL), or 10% donkey serum for Sox17. The cells were incubated with the primary antibodies overnight at 4ºC, followed by a 1 h incubation with the secondary antibodies at room temperature. The following antibodies and dilutions were used: Goat anti-human sox17, 1:40 (R&D Systems); guinea pig anti-human pdx1, 1:200 (Abcam, Cambridge, MA, USA); mouse anti-human insulin l:200 (Sigma-Aldrich). Donkey anti-goat antibody was used at 1:300 (Sigma-Aldrich); goat anti-guinea pig antibody was used at 1:500 (abcam) and fluorescein isothiocyanate anti-human insulin antibody was used at 1:400 (Chemicon, Temecula, CA, USA). The cells were mounted in ten random fields of vision using DAPI (BD Biosciences, Franklin Lakes, NJ, USA) dye, and examined using a fluorescence microscope (Nikon Inc., Melville, NY, USA). Each vision contained >200 cells and totaled >2000 cells per sample.