Fig. 1
a Hematoxylin and Eosin (HE) staining of a longitudinal section of small intestine from a WT and CR−/− mouse showing a normal mesothelium. Primary mesothelial cells of both genotypes grown in vitro maintain their typical ‘cobblestone-like’ morphology, when grown to confluence as evidenced on the brightfield images (b). c HE staining of primary mesothelial cells grown in vitro revealed that CR−/− cells tended to be more flattened (larger diameter) and more “giant” cells (largest diameter > 100 μm) were observed, a typical example is shown in (h) Scale bar = 100 μm. The TEM pictures (d) demonstrate the presence of microvilli typical for mesothelial cells; no apparent differences with respect to microvilli number, size or length were noticed. e Genotyping of WT and CR−/− mice with primers recognizing the Calb2 WT allele (left panel) and the mutated allele (right panel). For each PCR analysis, a positive control (c+) and a negative control (c-) from genomic DNA of previously identified WT and CR−/− mice was amplified, as well as a PCR reaction without template DNA (H20 control). f Immunofluorescence images of primary mesothelial cells in vitro stained for cytokeratin and calretinin (left) and for vimentin and desmin (right), derived from WT (upper panel) and CR−/− (lower panel) mice. Nuclei are stained with DAPI (blue). Note the presence of all three intermediate-filament markers and the absence of a specific CR signal in cells from both genotypes. g Western blot analysis of CR in protein extracts from cerebellum (lanes 4 & 5) and prMC grown in vitro (lanes 2 & 3) from WT (2 & 4) and CR−/− (3 & 5) mice. Molecular weight markers (25 and 37 kDa) are shown in lane 1, purified recombinant CR (control) is shown in lane 6. Upper bands (>40 kDa) in lane 2 and 3 are non-specific bands not corresponding to the size of CR. i Mixed population of WT and CR−/− EGFP-labeled mesothelial cells: note the increase in cell surface area covered by a single CR−/− mesothelial cell (green boundaries) compared to WT cells (red boundaries). j Quantification of the average surface area covered by a WT or a CR−/− prMC is shown (n = 25 cells for CR−/− and n = 24 cells for WT, ** p < 0.01). For the analysis giant cells were omitted