Sc 53108

Western blot analysis was performed, as described elsewhere [8] . Briefly, at least 16 pooled organoids per each condition were washed three times with PBS (pH 7.4) and lysed in buffer containing 50 mM Tris-HCl (pH 6.8), 10% glycerol and 1% sodium dodecyl sulfate (SDS). The lysates were homogenized by sonication and protein concentrations were determined using DC Protein Assay (Bio-Rad). The lysates were then supplemented with 0.01% bromophenol blue and 1% β-mercaptoethanol and denatured at 100 °C for 5 minutes. The prepared samples were separated by SDSpolyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride (PVDF) membrane (Merck Millipore). The PVDF membrane was blocked in 5% skimmed milk in Tris-buffered saline containing Tween for 1 hour and incubated with primary antibodies overnight at 4 °C. The following antibodies were used: PAX6 (sc-53108, Santa Cruz, 1:1000), SOX2 (sc-365823, Santa Cruz, 1:1000), SOX1 (bs-12276R, Bioss, 1:1000), β-ACTIN (#4970S, Cell Signaling Technology, 1:1000).

The organoids were fixed in 4% paraformaldehyde for 30 minutes at room temperature, washed with PBS, cryopreserved in 30% sucrose (16104, Sigma Aldrich) in PBS for 1 hour at room temperature, embedded in Tissue-Tek ® O.C.T. Compound medium (Sakura) and sectioned on a cryostat (CM1850, Leica), (~10 μm). The sections were washed with PBS and blocked in Blocking buffer (PBS with 0.3% Triton X-100 (T8787, Sigma Aldrich) and 5% normal goat serum (G9023, Sigma Aldrich)) for 1 hour at room temperature in a humidified chamber. The primary antibodies were diluted in Antibody diluent (PBS with 0.3% Triton X-100 and 1% bovine serum albumin (A9647, Sigma Aldrich)) and applied to the sections overnight at 4 °C in a humidified chamber. The following primary antibodies were used: PAX6 (sc-53108, Santa Cruz, 1:200) and RAX (sc-271889, Santa Cruz, 1:200). The sections were washed with Antibody diluent and a secondary antibody (anti-Mouse AlexaFluor 488 (Thermo Fisher Scientific, 1:1000)) was applied for 1 hour at room temperature in a humidified chamber. The nuclei were stained with 1 μg/ml DAPI in PBS for 4 minutes at room temperature and the sections were mounted using Fluoromount TM Aqueous Mounting Medium (F4680, Sigma Aldrich).