Immunocytochemistry
Cells were fixed with 10% formalin for 20 minutes at room temperature,
washed with 0.01M phosphate buffer (pH 7.4) three times, permeabilized
in phosphate buffer with 0.5% Triton X-100 for 10 minutes at room
temperature, washed three times with PBS, blocked with blocking buffer
(BSA + Triton X-100) for 1 hour at room temperature, and labeled with
1:1000 dilution of primary antibodies (Sox9, Sox17, FoxA2, Brachyury,
PDX-1, NGN3, NeuroD1, MafA, NKX2-2, NKX6-1, insulin) in blocking buffer
overnight at 4° C. Samples were then incubated for an hour with 1:1000
donkey anti-mouse IgG labeled with Alexa Fluor 488 and/or donkey
anti-goat, donkey anti-rabbit or donkey-anti rat IgG labeled with Alexa
Fluor 594 (Thermofisher) as appropriate for the primary antibody.
Sampled were mounted with Prolong diamond antifade mountant
(Thermofisher) and imaged on a Zeiss Axio observer Z1 fluorescence
microscope.