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. 2013 Apr 24;9:86. doi: 10.1186/1746-6148-9-86

Figure 3.

Figure 3

A-H: 3D cell culture of canine endometrial glands with surrounding stromal cells cultured for 48 hr in standard medium (M199 + 10% FCS). (A) H&E-stained histological section of 3D-cultured canine endometrial glands (asterisk) and surrounding stromal cells (arrows) featuring physiological morphology with luminal formation and secretory activity(s). (B) Immunohistochemical characterization of stromal cells by anti-vimentin staining (green); glandular epithelial cells lack any staining reaction,counterstained with DAPI (blue). (C) Glandular epithelial cells were identified by immunohistochemical anti-cytokeratin staining (green), counterstained with DAPI (blue). (D) Demonstration of the intact basement membrane (green) surrounding the glandular structures with anti-laminin immunohistochemical staining, counterstained with DAPI (blue). (E) Transmission electron micrograph of 3D-cultured canine endometrial epithelial cells to demonstrate apical polarity (mv, microvilli)and intact junctional complex (arrows) featuring tight junctions, zonulaadherens and desmosomes, characteristic for differentiated glandular epithelial cells. (F) Basolateral polarization of the 3D-cultured glandular epithelial cells, ensuring correct luminal formation and function,demonstrated with anti-b-catenin immunohistochemical staining (green),counterstained with DAPI (blue nuclei). (G) Combined demonstration of apical polarization, with histochemical WGA lectin binding to the glycocalyx (red), and basolateral polarisation demonstrated with immunohistochemical anti-b-catenin staining (green) on 3D-cultured canine endometrial glands(nuclei demonstrated by DAPI counterstaining, blue). (H)The same combined double-staining method (compare with panel G)of the canine endometrium in vivo, featuring apical and basolateral polarization of the endometrial glands demonstrated histochemically byapical WGA lectin binding to the glycocalyx (red), and immunohistochemically bybasolateralanti-b-catenin staining (green),comparable to the 3D in vitro cultured glandular structures (panel G); DAPI counterstaining (blue). Scale bars A-D, F-H25 µm; E 1 µm.