Organoids were fixed in 4% paraformaldehyde (PFA) at 4 °C for 15 min, washed 3 times with PBS (10 min incubation at RT for each wash), and transferred to 30% sucrose solution for incubation overnight at 4°C. Sucrose solution was then removed and organoids were equilibrated with O.C.T compound at RT for 15 min. Organoids were then transferred to tissue base molds and embedded within O.C.T compound on dry ice. Organoids blocks were then stored at -80°C or used for cryosectioning to obtain 40 μm slices. Cryosection was washed with PBS, incubated with 0.1% Triton-100 for 15 min at RT, blocked with 3% bovine serum albumin for 2 hours at RT, and then incubated with primary antibody diluted in 3% BSA overnight at 4°C. After washed with PBS, cyrosection was incubated with secondary antibody diluted in 3% BSA for 1 hour at RT. Alexa Fluor Dyes were used at 1:1000 dilution and nuclei were stained by DAPI. After staining, cryosection was mounted with ProLong Gold Antifade Reagent and imaged with Leica TCS SP5 confocal microscope.
