At pre-defined time points (60 days (60d) and 90–100 days (90d)) the organoids were fixed overnight in 4% paraformaldehyde (diluted from 32%, Electron Microscopy Sciences, Hatfield, PA) and cryoprotected in a 30% sucrose solution prior to embedding in optimal cutting temperature compound (OCT), VWR International, Bridgeport, NJ). Frozen tissue was sectioned at 30 μm using a cryostat and collected on ultra-frosted glass microscope slides. Sections were stored at -20C°. For immunolabeling, sections were permeabilized for 30 minutes in phosphate-buffered saline (PBS) containing 0.3% Triton-X100 (Sigma-Aldrich, Natick, MA) and then blocked in 10% v/v horse serum solution in PBS contain 0.1% Triton-X100 (PBST) for 1 hour followed by incubation with primary antibodies overnight at 4°C in PBST containing 5% horse serum (source). Sections were received three 15 min washes in PBST containing 5% horse serum. Secondary antibodies was prepared in PBST containing 5% horse serum and Hoechst (for nuclear immunoreactivity, source) and sections were incubated in secondary for 1 hour at room temperature before washing as before. Sections were then coverslipped in Fluoromount-G (Electron Microscopy Sciences, Hatfield, PA) mounting medium and
