Cellular characterisation of the apical layer of 2D enteroids. Immunolocalisation of (A–F) apical epithelial cell types in 2D enteroids grown on Matrigel coated cell culture plastic at day 6 of culture. All cells are counterstained with DAPI (blue). The cells are stained for A E-cadherin (epithelial cells with adherens junctions, green) and vimentin (red), but vimentin expression is not evident in the sub-epithelial layer after mild cell permeabilisation with saponin, B villin (enterocytes, green), C Lysozyme (Paneth cells, red), D Chromogranin A (enteroendocrine cells, red), E Muc5AC (Goblet cells, green), and F CD45+ (leukocytes, green). Representative images from 3–4 independent experiments. Scale bars: 50 µm. G mRNA expression of the stem cell marker Lgr5 in freshly isolated villi, at day 2 and 5 of culture, grown on uncoated cell culture plastic. Data represents the median and 95% CI of three independent experiments.