Fig. 2: Fluorescent Wheat Germ Agglutinin (WGA) conjugate and Nile Red enable validation of the apical-out topology in live small intestinal organoids.

a Representative 3D confocal images of WGA-Alexa Fluor 488 and phalloidin–Texas Red (F-actin labeling) co-stained organoids with apical-out (AO), apical-basal (AB) and basal-out (BO) topology. The scale bar is 100 μm. b Large field mosaic scan of PFA-fixed pig intestinal organoids after 18 h of polarity reversion, co-stained with fluorescent WGA and phalloidin. Left: representative images of AO, AB and BO organoids, indicated on mosaic image (right). The scale bar is 100 μm. c Analysis of the AO, ABO and BO topology confirms a major population of polarity reverted organoids, estimated with WGA staining and F-actin labeling, respectively. Histogram plot shows average percentage of WGA/F-actin topology types counted from 4 independent mosaic scan experiments (see table S2). d Topology analysis based on WGA and Nile Red labeling for polarity reverted organoids, quantified from the mosaic scan experiment: AO with lipid droplets (AO LDs) and AO without lipid droplets (AO no-LDs), AB and BO. e Lipid droplets display a characteristic distribution in AO organoids, contrasting with BO. f Co-staining with WGA and Nile Red reveals organoid structure in apical-basal organoid (AB). The scale bar is 50 μm