868 Fig. 2 Western blot analysis and cellular distribution of claudin-1 in choroid plexus during rat brain development. a Representative Western blot of LVCP and 4VCP dissected from E19, P2, and adult animals (2 lg per lane). Cld-1 protein expression was steady throughout development in both types of plexuses (upper panel band at 22 kDa). Actin used as a loading indicator is shown in the lower panel (42 kDa band). b–d Cld-1 immunoreactivity (red) in CPs of the developing and adult brain. A strong signal was associated with epithelial cell membranes of both LVCP and 4VCP in E19 animals (b left and right panel, respectively). A similar signal was observed at later stages, as illustrated for 4VCP in a P2 animal (c), or LVCP in an adult rat (d). The inserts in b and d highlight the typical honeycomb pattern of choroidal epithelial cell junctions. Double immunolabeling with the anti- RECA-1 Ab (green) allowed visualizing choroidal vessels, and DAPI was used for nuclei staining. Scale bars 20 lm. LV and 4V choroid plexus from the lateral and fourth ventricle, respectively, E19 19-day-old embryo, P2 2-day-old, ad adult Histochem Cell Biol (2012) 138:861–879 and 4VCP was less prominent at P2, as Cld-2 labeling became continuous throughout the TJ network in the latter tissue (Fig. 4c). Cld-2 immunoreactivity was intense and regular in all CPs of P9 and adult rats (Fig. 4d, e). No staining of the MV was observed in the choroidal stroma or the brain parenchyma at any developmental stage. A mirror image was observed when Cld-11 expression levels were compared between LVCP and 4VCP (Fig. 1b). Cld-11 gene was expressed at a much higher levels in 4VCP than in LVCP at all E19, P2 and P9 stages. In adult animals, Cld-11 expression level decreased in CPs, while it increased largely in the cortex. This likely reﬂects axonal myelination, Cld-11 being a crucial component of the interlamellar strands in oligodendrocyte myelin sheaths (Morita et al. 1999a). These differences of choroidal expression could not be assessed at the protein level, as a Western blot signal could only be obtained on medulla 123 samples from myelinated brains. No other Cld showed such a difference in expression between LVCP and 4VCP. Claudin-9 and -19 are selectively enriched in choroid plexus epithelial cells Among the four additional Clds whose mRNAs were selectively enriched in CPs in comparison to the cortex or MV, Cld-6 displayed higher choroidal expression levels at perinatal than at P9 and adult stages, while Cld-22 was expressed more prominently in both CPs from P2 onwards (Fig. 1b). The protein level and cellular localization of these two Clds could not be investigated due to the lack of adequate antibodies. Cld-9 expression levels in CPs increased from E19 to adult stage (Fig. 1b). This proﬁle was also reﬂected at the protein level in both 4VCP and LVCP as illustrated by the large increase in Cld-9 signal by