Mouse anti afp

For the RPE characterization studies, iPSC-derived RPE was seeded and processed as previously described (19). For the differentiation assay, embryoid bodies were fixed with 4% PFA, permeabilized with 0.1% Triton X-100 and blocked in 10% donkey serum (Millipore) and 1% BSA (Sigma-Aldrich). Primary antibodies were diluted in 1% donkey serum and 1% BSA, and incubated overnight at 4 °C. Secondary antibodies were incubated 45 min at room temperature with 0.2 µg/ml bisBenzimide Hoechst (Sigma-Aldrich) prior to mounting in ProLong Diamond Antifade Mountant (Molecular probes, Life Technologies). Primary antibodies: 1:100 dilution rabbit anti-human ZO-1 (Invitrogen, Life technologies), 1:250 rabbit anti-human LRAT (Abcam), 1:500 mouse anti-human Bestrophin-1 (Abcam), 1:200 mouse anti-Nestin (Novus Biologicals, Lille, France), 1:200 mouse anti-SMA (Dako, Les Ulis, France) and 1:200 mouse anti-AFP (Sigma-Aldrich). Secondary antibodies: 1:500 dilution donkey anti-rabbit IgG-Alexa Fluor 594, and donkey anti-mouse IgG-Alexa Fluor 488 (Jackson ImmunoResearch). Images were taken using a Zeiss ApoTome 2 Upright wide-field microscope (Carl Zeiss SAS).