872 domain were found in the CPs throughout development. While the highest expression was seen for ZO-2 with no selectivity among the preparations analyzed, ZO-1 tran- script displayed a clear enrichment in the MV preparations, and ZO-3 mRNA, found in both barriers, was selectively enriched in developing CPs, yet at very low levels. Claudin-1, -2 and -3 are detected at tight junctions of the blood–CSF barrier in human fetal brain The choroidal expression of the three major Clds highly expressed in the rodent BCSFB, Cld-1, -2 and -3, was investigated by IHC on sections obtained from 13 brains of human fetuses and neonates. Table 1 summarizes the clinical data, and indicates the duration of ﬁxation prior to parafﬁn embedding of the tissues. HPS (not shown) or hematoxylin (Fig. 8a) staining of the tissue, showed mor- phological differences between LVCP and 4VCP, espe- cially at the earlier stages investigated. LVCPs were characterized by a markedly enlarged core with an ‘‘edematous’’ appearance and were lined with columnar to cuboidal cells with a clear cytoplasm. Conversely, 4VCPs were composed of a thinner papillary core lined with columnar to cuboidal cells with an eosinophilic cytoplasm. Despite these histological differences, a clear apico-lateral staining typical of intercellular choroidal epithelial junc- tions was observed for Cld-1 throughout the surface of the epithelium in both LVCP and 4VCP already at 8 weeks of development (WD8) which was the earliest fetal stage analyzed (Fig. 8a). This staining was consistently seen at all developmental stages (Fig. 8; Table 1). A weak and discontinuous apico-lateral Cld-2 immu- noreactivity was associated with the CP epithelium in brain sections obtained from WD8 and -10 (Fig. 8a, inserts). This irregular staining became continuous at later stages (Fig. 8b, c; Table 1). A typical TJ-associated labeling was also observed in the choroidal epithelium of both LVCP and 4VCP for Cld-3. This staining was faint but continuous throughout the epithelium. Its intensity varied to some extent from case to case. This was independent of the gestational age and could be related to the duration of ﬁxation (Table 1). The choroidal speciﬁcity of Cld-1, -2 and -3 immunoreactivity was strengthened by the absence of staining of the contiguous neuroependyma. Choroidal vessels (e.g. Fig. 8c, arrowheads), and brain parenchyma were negative for these Clds in all 13 cases investigated. Discussion 123 Clds are transmembrane proteins that form the basis of TJ strands in both endothelial and epithelial cells. The identity of the main Clds located in TJs of the BCSFB remained so Histochem Cell Biol (2012) 138:861–879 Fig. 6 Claudin-5 and claudin-4 are expressed in endothelial cells in c the developing brain a–d. Cld-5 immunoreactivity in the developing and adult brain. Parenchymal vessels are labeled (green) in E19 (arrows a) or adult (arrows b, d) animals. In c, the anti-Cld-5 polyclonal antibody was used to show the colocalization of the TJ protein (red) with the endothelial marker RECA-1 (green) in a P2 animal. In all CPs immunodetection revealed a signal associated with the largest vessels penetrating the choroidal stroma (arrowheads b, d), but not with the choroidal epithelium, labeled in red by anti-Cld-1 Ab (d). Note that all cisternal vessels such as the vein of Galen are also labeled by anti-Cld-5 Ab. DAPI was used for nuclei staining. e Representative Western blot of Cld-4. Brain cortex (lane 1 20 lg) and MV (lanes 2 and 3 40 lg) were isolated from adult animals. Lane 4 was loaded with MDCK (10 lg) as positive control. A low, but speciﬁc Cld-4 signal was detected in MV (upper panel lane 3 band at 22 kDa), but not in cortex or in MV when the primary antibody was omitted (lane 2). Actin used as a loading indicator is shown in the lower panel (42 kDa band). Due to the high expression of Cld-4 in MDCK cells, lane 4 was treated separately from the other lanes for HRP detection. f–h Cld-4 immunoreactivity (red) in the developing and adult brain. Double immunolabeling with anti-RECA-1Ab (green) allowed visualizing the parenchymal vessels that were positive for Cld-4 in P2 animals (f left panel). Enlargements of Cld-4 staining associated with the two vessels are shown in the right panels. g, h Parenchymal vessels labeled for Cld-4 in P9 and adult rat brain. DAPI was used for nuclei staining. Scale bars 20 lm. 3 V choroid plexus of third ventricle, CP choroid plexus tissue, v vein of Galen. Other abbreviations as in Fig. 2 far uncertain as the anti-Cld-1 antibody used in previous immunohistochemical investigations was later shown to cross-react with other Clds (Wolburg et al. 2003). By combining qRT-PCR analyses with the use of speciﬁc antibodies that discriminate between Cld-1, -2 and -3, we have been able to demonstrate that besides Cld-2, already identiﬁed in CP (Lippoldt et al. 2000), both Cld-1 and Cld- 3 participate into the molecular structure of junctions between choroidal epithelial cells in the rat BCSFB. Cld-1 displayed the highest expression among all Clds in the CPs. Cld-1, -2, and -3 were highly enriched in both LVCP and 4VCP, compared to brain capillaries or brain tissue, and located at the choroidal epithelial junctions. In addition to these three proteins, and to Cld-11 pre- viously described in mouse (Wolburg et al. 2001), this work identiﬁes Cld-6, -9, -10, -12, -19, and -22 as addi- tional Clds expressed in the CP. The expression of Cld-6, - 9, -19 and -22 was highest in the choroidal tissue compared to MVs and cortex, especially during perinatal stages of development. In line with their involvement in TJ com- plexes at the BCSFB, immunoreactivity of Cld-9 and -19 was localized at intercellular junctions of the epithelium in both LVCP and 4VCP. The large number of Clds associ- ated with the choroidal tissue at the stage E19, and the concurrent expression of occludin and of ZO proteins necessary for the recruitment of Clds to the tight junction strands (Umeda et al. 2006), are in line with the apparent ability of the epithelial junctions to impede the diffusion of both large and small organic compounds, such as proteins