Figure 6
Morphological, histological and immunocytochemical appearance of mature P 0 and P 1 EBEC ALI cultures. [a] Phase contrast microscopy of mature confluent epithelial cell layers showing typical mosaic-like appearance (objective magnification: ×4), at higher magnification in the inset (×40). [b] Representative image of immunofluorescence staining of mature cultures for cytokeratins 5/6/18 (objective magnification: ×40). [c-d] Representative H&E staining of sections of epithelial layers grown on membrane inserts at P0 (c) and P1 (d) (objective magnification: ×40), indicating pseudo-stratified organization with columnar (c) or cuboidal (d) cells and flat basal cells. Asterisk indicates the porous membrane support. [e-f] PAS-positive cells in P0 (e) and P1 (f) mature EBEC insert cultures. Asterisk indicates the porous membrane support. [g-l] Representative scanning electron micrographs of mature P0 (g, i, k) and P1 (h, j, l) EBEC ALI-cultures, showing the presence of mucus-like amorphous material (g, h) (scale bars = 3 μm), cilia (i, j) (scale bars = 10 μm) and microvilli (k, l) (scale bars = 5 μm) at the apical cell surface. Inset of picture (l) shows the presence of particularly elongated microvilli-like projections in some surface areas of P1 cultures (scale bars = 2 μm). [m, n] Representative images of immunofluorescence staining of P0 and P1 cultures with antibodies directed against the cell type-specific cytokeratins 18 (m) and 10/13 (n) (objective magnification: ×40). Nuclei are blue stained with DAPI.