Donkey serum

Severely immune-compromised NOD/SCID IL2 receptor gamma chain knockout (NSG) mice (Jackson Laboratories) were injected with 1 million ESC in 100 μL of PBS subcutaneously in the dorsal flanks of each mouse using a 28 gauge needle. After 30 days, the mice were euthanized by cervical dislocation and the teratomas harvested and sent out for histological processing (IDEXX Laboratories). The prepared slides were then dehydrated in graded alcohol, de-paraffinized using three washes of xylene, then rehydrated in graded alcohol and placed in PBS until imaging with a confocal microscope. After deparaffinization, the GFP and RFP were either directly visualized, or counterstained in 1% BSA (Sigma), 5% donkey serum (Fitzgerald), 0.7% Triton X-100 (MP Biomedical) followed by a goat anti-DsRed antibody (Santa Cruz, 1:50 dilution) followed by donkey-anti goat PE (Abcam, 1:100 dilution). DAPI (4′,6-Diamidino-2-Phenylindole, Dilactate) was used to visualize the nuclei and images were taken on a Nikon TE 2000 fluorescent microscope. To verify endothelial and smooth muscle cell identity, goat anti-VE (vascular endothelial)-cadherin (Santa Cruz, 1:100) with a donkey anti-goat (Santa Cruz, 1:100) or rabbit anti-smooth muscle myosin heavy chain (Abcam, 1:50) with a donkey anti-rabbit FITC (Abcam, 1:50) was also used in conjunction with the direct visualization of the GFP or the counter stained RFP.

A3 and B2 ESC were plated at 10,000 cells/cm² on 50 ng/mL of collagen IV-coated or fibronectin-coated plates (BD Biosciences) and cultured in Stage 1 Differentiation Medium, called NSD12b, for A3 and B2, respectively.24 (link) Briefly, our serum-free Stage 1 Differentiation Medium, preoptimized for B2 and A3 cells included alpha-DMEM (Invitrogen), 15% KSR (Invitrogen), 1x penicillin-streptomycin (Invitrogen), 1x nonessential amino acids (Invitrogen), 2 mM L-glutamine (Invitrogen), 0.1 mM 2-mercaptoethanol (Calbiochem), 5 ng/mL of bone morphogenetic protein-4 (R&D Systems) and 0 or 20 ng/mL of VEGF (R&D Systems) for A3 and B2, respectively. At 0, 1, 2, 3, and 4 days, the cells were collected and stained for upregulation of mesoderm marker, Flk-1, and downregulation of pluripotent stem cell marker, Oct3/4. Briefly, cells were fixed 4% paraformaldehyde overnight before being placed in a solution of 1% bovine serum albumin (BSA) (Sigma), 0.7% Triton X-100 (MP Biomedicals LLC), and 5% donkey serum (Fitzgerald) for 1 hour prior to staining. Cells were stained with Alexa Fluor 647 anti-mouse Flk-1 (Biolegend), or anti-Oct3/4 PE conjugated antibody (Becton Dickinson [BD]) overnight. Cells were rinsed and analyzed on an LSR II flow cytometer (BD). All results were gated to 5% of an isotype control.