Live organoids were cut in small pieces using surgical blade and cultured in a solution of transferrin conjugated to Alexa Fluor-488 (Alexa Fluor-488, Life Technologies Corporation, Grand Island, NY). Following a 10 minute incubation in 200 μg/ml transferrin solution in media, organoids were washed three time in PBS and fixed using 4% paraformaldehyde. The fixed organoids were then washed with PBS, stained with Hoechst, and whole-mounted onto glass coverslip. The outer (flat) surface of tissue sitting on the glass coverslip were imaged using confocal microscope. The images were processed in ImageJ, by removing the background (thresholding) and counting the size of Alexa Fluor-488 positive particles.
