866 graphs provide an approximation of the respective abun- dance of the various gene products (see ‘‘Materials and methods’’). Overall, Cld-1, -2 and -3 were the most highly expressed Clds in the choroidal tissue and displayed a distinct speciﬁcity for this tissue in comparison to age- matched MVs and cortices (Fig. 1a). The latter tissue dis- played 10- to 100-fold lower expression levels than CPs (p \ 0.001). Cld-6, -9, -10, -11, -12, -19 and -22 were expressed at lower, but still signiﬁcant levels in the cho- roidal tissue (Fig. 1b). While Cld-19 and Cld-22 displayed a high degree of choroidal speciﬁcity at all stages investi- gated (statistically different from age-matched cortex mRNA levels, p \ 0.001 for P2, P9, adult), and Cld-9 followed a similar pattern of choroidal enrichment (p \ 0.05 for LVCP and 0.01 for 4VCP at E19, p \ 0.001 at P2 and in adult), Cld-6 was found enriched in CP as compared to cortex preparations only in the perinatal developmental stages (p \ 0.001 at E19 and 0.001 for 4VCP at P2, Fig. 1b). Cld-4, -5, and -16, whose expression in the choroidal tissue was very low, were rather selec- tively enriched in the MVs forming the BBB (Fig. 1c). Cld-14 was not detected in any sample of CP, cortex and MV. Its mRNA was found in liver, used as positive control (data not shown). The developmental qRT-PCR analysis of Clds in CPs revealed overall high expression levels in the tissues sampled at prenatal and postnatal stages. We observed however variations in the choroidal expression and localization of Clds during development and investi- gated them in more detail using protein analysis when antibodies of sufﬁcient quality were available. Claudin-1 and -3 expression is high in both types of choroid plexus in the developing brain Quantitative RT-PCR analysis showed that Cld-1 and -3 are already highly expressed in both LVCPs and 4VCPs of E19 and P2 rats compared to P9 and adult animals. Cld-1 and Cld-3 mRNA levels were not statistically different between 4V and LVCP at all stages investigated (Fig. 1a). Western blot analysis (see method section and Online Resource 2 for details on the quality of the antibodies used) conﬁrmed the expression of Cld-1 in CPs of E19 and P2, and showed no obvious difference in protein levels between 4VCP and LVCP, or between stages (Fig. 2a). This result and all subsequent Western blot analyses presented have been conﬁrmed on at least three batches of tissues. The cellular protein localization was then determined by IHC. Cld-1 was found strictly associated with the interepithelial junctions of both 4VCP and LVCP at all stages investigated (Fig. 2b–d). Cld-1 was not detected in the choroidal or parenchymal vessels at any stage of development. Western blot analysis of Cld-3 in CPs also conﬁrmed the expression of the protein in E19 and P2 animals, and 123 Histochem Cell Biol (2012) 138:861–879 Fig. 1 Developmental analysis of claudin transcript levels in choroid c plexuses, cerebral cortex and cerebral microvessels of the rat. RT-PCR was performed on tissues sampled at four developmental stages. The results, expressed relative to the bacterial AraB gene added as an external standard, are shown as mean ± SD, n = 4, except for MV, for which values represent the mean of two preparations. Gene expression levels did not differ by more than 13.8 % between the two age-matched MV preparations. The results are expressed as arbitrary units, and are all normalized to the expression level found for Cld-1 in one sample of LVCP obtained from P2 animals, and set arbitrarily at 100 (see ‘‘Materials and methods’’ for details of calculations). a Clds displaying the highest speciﬁc expression in CP throughout development. b Clds expressed at lower, but signiﬁcant levels in the CP by comparison to other brain preparations. Some displayed a strong speciﬁcity for the choroidal tissue. c Clds speciﬁcally enriched in the MV preparations, see ‘‘Results’’ for a detailed description of the data. *, **, ***Statistical differences in expression levels between developmental stages, analyzed by one-way ANOVA followed by a Tukey’s multiple comparison test, p \ 0.05, 0.01, 0.001, respectively. For clarity, only differences observed for LVCPs and 4VCPs are shown on the graphs. §, §§Statistical differences in expression levels between age-matched 4VCPs and LVCPs, two-tailed paired Student’s t test, p \ 0.05 and 0.01, respectively. Other relevant statistical analyses are stated in ‘‘Results’’. LVCP and 4VCP choroid plexus of the lateral and fourth ventricle, respectively, Crtx cerebral cortex, MV cerebral microves- sels, E19 19-day-old embryo, P2 and P9 2-day-old and 9-day-old, respectively; ad adult additionally showed a clear decrease of Cld-3 protein level in the CPs of adult animals (Fig. 3a). This reﬂects the strong and statistically signiﬁcant down-regulation of Cld-3 mRNA observed between P2 and adult stages (Fig. 1a). IHC analysis showed that the protein is associated with the inter-epithelial junctions of both 4VCP and LVCP in developing animals (Fig. 3b, c, e, f). Cld-3 immunoreac- tivity was also observed in both CPs in the adult (Fig. 3g), with a lower intensity than in developing animals. The protein was not detected in the choroidal vessels (e.g. arrowheads in b). In contrast, parenchymal vessels were labeled (Fig. 3d), in line with the previously reported Cld-3 immunoreactivity in parenchymal vessels of the adult mouse brain (Wolburg et al. 2003). Cld-3 labeling was however weaker in parenchymal vessels than in the CP epithelium at all stages investigated. The developmental proﬁle of claudin-2 and -11 expression differs between the choroid plexuses of the lateral and fourth ventricles Cld-2 expression largely increased throughout develop- ment in the 4VCP. A developmental Cld-2 upregulation also occurred in the LVCP to a lesser extent, as mRNA levels in perinatal stages were already higher in this tissue than in 4VCP (Fig. 1a). These differences between either CPs or stages were reﬂected at the protein level. Western blot analysis showed a signal in LVCP of E19 rats, which became progressively more intense in older animals. Cld-2