Histochem Cell Biol (2012) 138:861–879 869 Fig. 3 Western blot analysis and cellular distribution of claudin-3 in choroid plexus and cerebral parenchyma during rat brain development. a Representative Western blot of LVCP and 4VCP dissected from E19, P2, and adult animals (10 lg per lane). Cld-3 protein levels decreased in both types of CP during development (upper panel band at 22 kDa). Actin used as a loading indicator is shown in the lower panel (42 kDa band). b–g Cld-3 immunoreactivity (red) in the developing and adult brain. Double immunolabeling with anti-RECA-1 Ab (green) allowed visualizing choroidal and parenchymal vessels (e.g. arrowheads in b, g), and DAPI was used for nuclei staining. During perinatal stages of development, immunodetection revealed a distinct signal associated with the CP epithelial cell membranes as illustrated for LVCP in E19 (b), or in P2 animals (c). The inserts in b and c highlight the typical honeycomb pattern of choroidal epithelial cell junctions. The area delimited by the rectangle in c, is enlarged in d, which shows Cld-3 in a parenchymal vessel of the developing brain (i Cld-3 (red), ii RECA-1 (green) and iii merge staining). Cld-3 immunoreactivity was observed at the epithelial junctions in CPs of P9 animals (e), and could still be detected in adult CP (g). f A negative control (NC), run in the absence of primary antibody, for a P9 animal (arrow CP). Scale bars 20 lm. Abbreviations as in Fig. 2 Western blot between P2 and adult animals (Fig. 5a). The immunohistochemical analysis revealed a patchy signal for Cld-9 already at E19, which was associated with the epi- thelium in both CPs (Fig. 5b). The labeling became con- tinuous at all intercellular epithelial junctions in P2 animals (Fig. 5c, insert), and was also observed throughout the CPs in adult animals (Fig. 5c). It was not found associated with parenchymal vessels at any stage (e.g. arrowheads in Fig. 5b). Cld-19 gene level of expression was close to the adult level throughout development as assessed by qRT-PCR (Fig. 1b). The choroidal localization of the protein deter- mined by IHC showed its association with the epithelial junctions in both LVCP and 4VCP at all stages investigated 123