Histochem Cell Biol (2012) 138:861–879 Table 1 Description of human samples included in the study and claudin immunopositivity evaluation Case WD Clinical history 1 2 3 4 5 6 7 8 9 10 11 12 13 8 10 10 16 18 20 20–21 21 22 28–29 38 39 2 months and 17 days post-natal Spontaneous abortion Limb malformations Spontaneous abortion Spontaneous rupture of membranes Twin pregnancy Spontaneous rupture of membranes Acute chorioamniotitis Twin pregnancy Late spontaneous abortion Acute chorioamniotitis Acute chorioamniotitis Premature, pulmonary hypoplasia, refractory hypoxia Postpartum death, SIDS Postpartum death, SIDS SIDS Fixation (days) 3 3 7 13 43 40 35 67 8 58 54 17 38 CLD-1 LVCP ? CA ? CA ? CA ? CA ? CA ? CA ? CA ? CA ? CA ? CA ? CA ? CA ? CA 4 VCP ? CA ? CA ? CA ? CA ? CA ? CA ? CA ? CA ? CA ? CA ? CA ? CA ? CA CLD-2 LVCP ? DA ? DA ? DA ? CA ? CA ? CA ? CA ? CA ? CA ? CA ? CA ? CA ? CA 4 VCP ? DA ? DA ? DA ? CA ? CA ? CA ? CA ? CA ? CA ? CA ? CA ? CA ? CA CLD-3 LVCP ? CA ? CA ? CA ? CA ? CA ± CA ? CA ± CA ? CA 0 ? CA ? CA ? CA 865 4VCP ? CA ? CA ? CA ? CA ? CA ± CA ? CA ± CA ? CA 0 ± DA ? CA ? CA Immunolabeling was scored as follows: 0: no staining, ± : weak staining, ? and ?: deﬁnite staining. C continuous, D discontinuous, A apico- lateral LVCP choroid plexus from the lateral ventricle, 4VCP choroid plexus from the fourth ventricle, WD weeks of development, SIDS sudden infant death syndrome Protein content Choroidal tissues and brain cortices were homogenized in Cell lysis buffer (9803 from Cell Signaling, Boston, MA) containing 1 mM phenylmethylsulfonyl ﬂuoride (PMSF, igma, St. Louis, MO) with 10–15 strokes in a glass–glass homogenizer. Protein concentrations were determined by the method of Peterson (1977) using BSA for the standard curve. Western blotting Proteins were separated on 10 % Bis–Tris Criterion XT gels (Bio-Rad, Hercules, CA, USA) run in MOPS buffer (Bio-Rad) at 80 V for 10 min, followed by 120 V for 2 h. The gel was blotted to 0.45 lm Whatman PROTRAN nitrocellulose membrane (# NBA085C, Perkin Elmer, Waltham, MA) in Tris–glycine transfer buffer (Euromedex, Soufﬂeweyersheim, F), containing 20 % methanol for 50 min. After assessing transfer efﬁciency by Ponceau-red staining, the membrane was cut longitudinally to separate actin from the lower molecular weight Clds and incubated in a saturation buffer (5 % skimmed milk, 0.1 % Tween 20 in Tris-buffered saline; TBS) at RT for 1 h. When antibody quality was tested, the membranes were kept in one piece. All primary anti-Cld antibodies were diluted in saturation buffer at 1 lg/ml except Cld-9, which was used at 0.307 lg/ml. The rabbit anti-actin pAb was used at a ﬁnal dilution of either 0.533 or 0.267 lg/ml, depending on the amount of protein loaded. Membranes were incubated at 4 C overnight, then washed 3 9 10 min in TBS contain- ing 0.1 % Tween-20 (TBST) at RT. Horseradish peroxi- dase-conjugated secondary antibodies from Jackson ImmunoResearch (Baltimore, MA, USA) were either goat- anti-rabbit (111-035-045) used at a ﬁnal concentration of 0.04 lg/ml or goat-anti mouse (115-036-003) used at 0.08 lg/ml. After two washes in TBST and a ﬁnal one in TBS, chemiluminescent HRP substrates (Immobilon Wes- tern from Millipore, Molsheim, France, for Clds, and Pierce ECL substrate from Perbio Science, Brebie`re, France, for actin) were used and membranes were exposed to X-ray ﬁlms (Biomax, Kodak Rochester, NY). Results The expression of TJ-associated proteins was assessed in rats at E19, P2, P9 and adult stages in LVCPs and 4VCPs by qRT-PCR. For comparison, the expression levels of these genes were also measured in cortical tissues at all stages, and in cortical MVs isolated from P9 and adult brain (Figs. 1, 7). Differences in y scale values between 123