Fig. 2.

Histological, immunohistological and ultrastructural analyses of tonsil epithelial organoids. (A) Histology of human tonsil epithelium tissues and organoids on day 15. Each sample was stained with H&E for morphological analysis, Alcian blue to visualize the mucosal layer and mucous-producing goblet cells, PAS to visualize mucosubstances, and Masson's trichrome to visualize connective matrix. (B) Immunohistological analyses of surface and crypt areas of human tonsil epithelium tissues along with tonsil organoids on days 5, 10 and 15. Each sample was immuno-stained with antibodies specific for epithelium markers, including NGFR, ITGA6, CD44, MUC1, and E-cadherin, as well as a cell proliferation marker, Ki67. (C) Immunofluorescence to compare distribution of suprabasal/superficial layer markers, MUC1 and CK4 (green), between the tonsil tissues and the organoids on day 15; the epithelial marker E-cadherin (red), was used as a control. Merged images with Hoechst 33342 staining (blue) are shown in the right-hand panels. (D) Immunofluorescence images showing a time course of organoid development on days 5, 10, and 15. Representative markers (green) for basal layer cells (NGFR and CD44; upper panels) and suprabasal/superficial layer cells (MUC1 and CK4; lower panels) were labeled with the corresponding antibodies; Ki67 (red) was used as a control for active proliferation. Merged images with Hoechst 33342 staining (blue) are shown in the right-hand panels. (E) TEM images illustrating ultrastructural morphology of human tonsil epithelial organoids. Keratohyaline granules are indicated by white arrows (right upper panel). The image of desmosome–tonofilament complexes in the red rectangle in the left lower panel is enlarged on right. In all panels except for (E), scale bar is 100 μm. In (E), scale bars are 10 μm (upper left) and 1 μm (upper right and lower left). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)