Immunofluorescence analysis of alpha-fetoproprotein (AFP) (green) in the endoderm derived from human ES cells. Embryoid bodies (EBs) were generated from the H9 embryonic stem cell line (WiCell Research Institute.) After four days in suspension culture, EBs were plated on Geltrex-coated tissue culture-treated polystyrene plates and continuously cultured for 21 days. EB cultures were then fixed and permeabilized according to the 3-Germ Layer Immunocytochemistry Kit and stained with anti-alpha-fetoprotein at 4°C overnight. Secondary staining was completed using Alexa Fluor 488-conjugated anti-mouse IgG and DAPI for nuclear DNA (blue) for 1 h at room temperature. Stained wells were imaged at 40X.