864 rabbit anti-Cld-3 pAb (34-1700), rabbit anti-Cld-4 pAb (36-4800), rabbit anti-Cld-5 pAb (34-1600) and the mouse anti-Cld-5 monoclonal antibody (mAb) (35-2500) were purchased from Zymed Laboratories (Invitrogen, Carlsbad, CA). Rabbit anti-Cld-9 pAb (16196-1-AP) was bought from Protein Tech Group (Chicago, IL). Rabbit anti-Cld-11 pAb (AP06062PU-N) was ordered from Acris Antibodies (SanDiego, CA). Antibody quality was assessed by Wes- tern blot of CPs or MVs. Only one speciﬁc band for Cld-1, -2, -3 (all 22 kDa) or Cld-9 (25 kDa) was observed in adult LVCP. The anti-Cld-4 pAb and anti-Cld-5 monoclonal Ab detected only one band (22 kDa) in adult MVs (Online Resource 2). The rabbit anti-Cld-5 antibody also selec- tively labeled parenchyma MVs and was used for double immunostaining with the endothelial cell antibody RECA- 1. The rabbit anti-Cld-19 pAb, custom-produced by Zymed Laboratories as an IgG preparation, was a kind gift of Dr. Yu (Angelow et al. 2007). In our hands, the antibody stained the tight junctions of the thick ascending limb of Henle on rat kidney sections, as previously described in mouse kidney cortex (Angelow et al. 2007). It was not applicable to Western blot analysis of cerebral prepara- tions. The mouse anti-RECA-1 mAb (MCA970GA), directed against a rat endothelial cell-surface antigen, and the rabbit anti-actin pAb (A-2066) were purchased from AbD Serotec (Oxford, UK), and Sigma (St-Louis, MO, USA), respectively. Immunohistochemical analysis of claudins in rat brain Rat brain sections (10 lm thick) were cut in a cryostat, mounted on glass-slides, air-dried at room temperature (RT), and immediately used for IHC or kept at -20 C. Slices were ﬁxed in acetone/methanol (V/V) at -20 C for 90 s except for Cld-4. For this protein, sections were ﬁxed in 4 % paraformaldehyde at RT for 10 min and microwave-treated (3 9 5 min at 600 W) in 0.01 M cit- rate buffer, pH 6 for epitope retrieval. After 1 h of satu- ration at RT in a blocking solution containing 0.2 % BSA, 0.2 % Triton X-100 and 10 % normal goat serum (NGS) in phosphate buffered saline (PBS, pH 7.4), sections were incubated at 4 C overnight with primary antibodies diluted in a PBS solution containing 1 % BSA, 0.3 % Triton, 1 % NGS. Primary antibodies were diluted and used at a ﬁnal concentration of 0.625 lg/ml for Cld-1, -2, -3,- 4. The anti-Cld-5 mAb and pAb were used at 1.25 lg/ ml and 0.625 lg/ml, respectively. Cld-9 pAb was diluted to 0.383 lg/ml and Cld-19 pAb to 4 lg/ml. In some cases, double labeling was performed with one of the anti- Cld pAb and the anti-RECA-1 mAb used at a ﬁnal con- centration of 2.5 lg/ml. After 5 washes for 10 min at RT in the Ab-solution without NGS, sections were incubated 123 Histochem Cell Biol (2012) 138:861–879 with either Alexa Fluor 488-conjugated secondary goat- anti-mouse Ab (A-10667), Alexa 555 -conjugated goat- anti-mouse secondary Ab (A-21424) or Alexa 555 -con- jugated secondary goat-anti-rabbit Ab (A-21428) all from Invitrogen at a ﬁnal concentration of 2 lg/ml at RT for 1 h. Diamidine-2-phenylindole-dihydrochloride (DAPI, 236276 from Roche Diagnostics) was used as ﬂuorescent nuclear stain at 0.1 lg/ml in PBS. Sections were washed again four times in Ab-solution without NGS and rinsed once in PBS. Negative controls were performed by omitting the primary Ab. After mounting the slides with Fluoroprep (bioMerieux, Marcy l’Etoile, F), immunoﬂu- orescence was viewed and analyzed using a Zeiss ﬂuo- rescence microscope equipped with a Digital Camera and the software AxioVs40 V 4.7.2.0. For direct comparison of expression and localization between either develop- mental stages or between 4VCP and LVCP at a given stage, appropriate sections were mounted on the same slide and treated simultaneously under identical condi- tions. Pictures were taken at the same exposure time to assess potential expression differences. The staining speciﬁcity of the custom-produced Cld-19 pAb was evaluated using a blocking peptide on brain slices. The IgG preparation was incubated with a tenfold molar excess of peptide that corresponds to the epitope recog- nized by the Ab (H-ERANSIPQPYRSGPSTAAREYV- OH) at 4 C for 8 h under agitation. The staining protocol was then performed as described above. Immunohistochemical analysis of claudins in human fetal brain Fetal brains were ﬁxed in 4 % formaldehyde. The dura- tion of ﬁxation is provided in Table 1. Coronal slices of the supratentorial brain and horizontal slices of the brainstem and cerebellum were performed and embedded in parafﬁn. Sections (4 lm thick) were cut from the blocks with a Leica microtome (Leica Instruments GmbH, Hubloch, Germany) and transferred to SuperFrost Plus slides (VWR International bvba, Leuven, Belgium) for morphological and immunohistochemical studies. Hema- lum phloxin saffron (HPS) staining was performed for morphological analysis. An automated IHC analysis of the sections was performed on a BenchMark XT (Ventana Medical Systems Inc, Tucson, AZ, USA) using anti-Cld antibodies at a ﬁnal concentration of 2.5 lg/ml according to the manufacturer’s protocol, and revealed by avidin– biotin–peroxidase complex, and a Ventana kit including DAB reagent and an ampliﬁcation system. Sections were counterstained with hematoxylin. The localization, conti- nuity and intensity of Cld expression were assessed in all 13 cases.