870 Histochem Cell Biol (2012) 138:861–879 Fig. 4 Differential expression of claudin-2 in choroid plexus during rat brain development. a Representative Western blot of LVCP and 4VCP dissected from E19, P2, and adult animals (10 lg per lane). Cld-2 expression increased in CPs during development, and was distinctively higher in LVCP than in 4VCP of E19 and P2 animals (upper panel band at 22 kDa). Actin used as a loading indicator is shown in the lower panel (42 kDa band). b–e Cld-2 immunoreactivity (red) in the developing and adult brain. LVCP and 4VCP sections have been labeled simultaneously and pictures generated with identical camera settings. Cld-2 staining was associated with the epithelium throughout the LVCP of E19 animals (arrow), while the staining in 4VCP was faint and patchy, with large portions of the epithelium remaining unlabeled (b). In P2 animals (c), the staining was more homogeneous in 4VCP and became continuous between epithelial cells (insert), as in the LVCP (right panel). The epithelial staining was strong in all CPs at P9 (d arrow) and in adult animals (e). In b, d, e double immunolabeling with anti-RECA-1 Ab allowed visualizing the choroidal vessels unlabeled by the anti-Cld-2 Ab (arrowheads). DAPI was used for nuclei staining. Scale bars 20 lm. Abbreviations as in Fig. 2 (Fig. 5d–f). Ab neutralization with the immunogen peptide completely abolished the signal (Fig. 5f). Cld-19 immu- noreactivity was absent from the choroidal vessels (e.g. arrowheads in Fig. 5f). It was neither detected in the parenchymal vessels nor in the neuropil at any stage of development (data not shown). Claudin-5, -4, and -16 are speciﬁc for the blood–brain barrier in the developing brain Cld-5 is a well-known landmark of the BBB. As expected, its expression level was high in cerebral MVs isolated from adult as well as from P9 animals. The more than tenfold difference in Cld-5 gene expression between cortex and MV conﬁrms the endothelial speciﬁcity of this protein. 123 In cortical tissue from E19 animals, mRNA expression levels were signiﬁcantly lower (p \ 0.001, Tukey’s multiple comparison test) than in other developmental stages (Fig. 1c). As IHC revealed a clear labeling of capillaries by anti-Cld-5 antibodies already at this fetal stage of develop- ment (Fig. 6a, b), this most likely results from the lower density of the capillary network in the brain of immature rats (Caley and Maxwell 1970). In contrast, qRT-PCR analysis showed low mRNA levels associated with the choroidal tissue. IHC of Cld-5 within this tissue was performed with the monoclonal antibody and showed that the protein is associated with the larger, penetrating vessels in the cho- roidal stroma. A fainter signal was also observed in some terminal vessel loops, but no signal was associated with the epithelium in our experimental conditions (Fig. 6c, d).