Embryoid bodies (EBs) were fixed with 4% paraformaldehyde (w/v) for 15 min at RT, then washed and permeabilized with 0.25% Triton X-100 solution for 5 min at RT. After that, they were blocked with 5% fetal bovine serum (FBS) in PBS. Primary antibodies were incubated with EBs overnight at 4°C. The next day, the EBs were washed and incubated with secondary antibodies and 3 μM DAPI for 1 hour at RT in the dark. After washing, the EBs were imaged with a Leica DMi8 inverted microscope.
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