Anti sox17

The encapsulated spheroids were fixed with 4% paraformaldehyde and immersed in 30% sucrose in PBS for 24 h. The spheroids were then imbedded in OCT compound (Fisher Healthcare, Waltham, MA, USA) and sectioned into 10 μm thick slices using a cryostat instrument (Leica CM1950; Leica Biosystems Inc., Buffalo Grove, IL, USA). Then the sections were permeabilized by immersion for 20 min in 1x PBS supplemented with 0.1% Triton X-100 in 2% BSA and blocked with 2% BSA in 1x PBS for 2 h at 25 °C. After thorough washing with 1x PBS, the slides were incubated in 5 μg/mL solution of anti-Sox2 antibodies (R&D Systems) or 5 μg/mL solution of anti-Sox17 (SantaCruz, Dallas, TX, USA) for 1 h at 25 °C. Both antibody solutions were prepared in a blocking buffer of 2% BSA in 1x PBS. The sections were washed again three times for 5 min each, then incubated with the corresponding secondary antibodies conjugated to Alexa Fluor 488 for Sox2 and 694 for Sox17 (2 μg/mL; Invitrogen) for 1 h at 25 °C in the dark. A sectioned slice was immersed in 50 μL of mounting medium containing DAPI (Vectorlab, San Francisco, CA, USA), placed under a coverslip (170 μm from Fisher Healthcare), and imaged using an Olympus fluorescence microscope (see above for microscope information).