Histochem Cell Biol (2012) 138:861–879 871 Fig. 5 Detection of claudin-9 and claudin-19 in choroid plexus during rat brain development. a Representative Western blot of LVCP, 4VCP and cortex dissected from P2, and adult animals (10 lg per lane). Cld-9 protein was enriched in both 4V and LV compared to the cortex, and was higher in adult as compared to P2 animals (upper panel band at 25 kDa). Actin used as a loading indicator is shown in the lower panel (42 kDa band). b, c Cld-9 immunoreactivity (red) in the developing and adult brain. A distinct signal was associated with the CP epithelial cell membrane in selected areas of both LVCP and 4VCP in E19 animals (b). The staining was present at all intercellular junctions in P2 animals (insert c), and was observed throughout the CPs in adult animals (c). It was not associated with parenchymal vessels (arrowheads). d–f Cld-19 immunoreactivity (red) in the In addition, our qRT-PCR data identiﬁed Cld-16 and Cld-4 as highly enriched in the capillary fractions of both adult and P9 animals compared to cortical or choroidal tissue. To date, no information could be generated con- cerning Cld-16 protein level or localization in the brain. Cld-4 mRNA levels were very low compared to Cld-5 levels (Fig. 1c). The presence of Cld-4 protein in this fraction was conﬁrmed by Western blot analysis (Fig. 6e) and by IHC in developing and adult animals (Fig. 6f–h). developing and adult brain. Choroid plexus epithelial cell membranes were distinctly labeled in E19 animals (d), displaying a typical honeycomb pattern. The insert shows a negative control obtained by omitting the primary antibody. The arrow localizes the CP. A strong signal was also observed in the epithelium at later stages, as illustrated for 4VCP in P2 (e) and LVCP in P9 (f upper panel) animals. No choroidal staining was observed when the Ab was preincubated with excess of immunogen peptide (P) (f lower panel). The signal was absent from the choroidal vessels (arrowhead f). In b, c, f, double immunolabeling with anti-RECA-1 Ab allowed visual- izing the choroidal vessels. DAPI was used to stain nuclei. Scale bars 20 lm. Crtx cerebral cortex. Other abbreviations as in Fig. 2 Expression of occludin and zonula occludens proteins in barriers of the developing brain Occludin is a transmembrane TJ component at both the BBB and BCSFB. Accordingly, qRT-PCR showed high expression levels for this TJ protein in all LVCP, 4VCP and MV preparations compared to cortex. Levels did not change throughout development (Fig. 7). Transcripts for the three ZO proteins that bind Clds through their PDZ 123