Histochem Cell Biol (2012) 138:861–879 as described above. CPs collected from several animals (between 2 and 5, depending on the developmental stage) were pooled. Following CP isolation, brain cortices were sampled at all stages and cleaned of apparent meninges under a stereomicroscope. These tissues were either used as homogenates or for RNA extraction at all stages of development, or for MVs preparation at P9 and adult stages. The cortical MVs were isolated and assessed for purity as previously described (Gazzin et al. 2008). Brieﬂy, cortices were homogenized in a Dounce-type glass–glass homogenizer after the addition of 5 vol/g tissue of 1 % bovine serum albumin (BSA)-supplemented Krebs–Ringer. The MVs were separated from larger vessels and brain parenchyma material by a sequence of a 17.5 % 70 kDa- Dextran gradient followed by ﬁltering steps through decreasing pore diameter mesh sieves. The microvessel fraction retained on the 40-lm sieve was recovered in 0.1 % albumin in Krebs–Ringer buffer. All steps were performed at 4 C. For gene expression analysis, the tissue collection and MV isolation were performed under RNase- free conditions. Samples collected for gene expression analyses and Western blot (WB) were snap-frozen in liquid nitrogen and kept at -80 C. For immunohistochemistry, brains sampled from E19 to adult animals were snap-frozen in isopentane at -50 C, embedded in Tissue-Tek (Sakura Finetek Europe, The Netherlands) and stored at -80 C. Human fetal brain tissues were obtained from autopsies performed at the Centre de Pathologie Est, Groupement Hospitalier Est, Hospices Civils de Lyon, France, after obtaining an informed consent from the parents. Quantitative RT-PCR Total RNA was isolated from four batches of 4VCP, LVCP as well as cortices sampled from E19, P2, P9 and adult rats using the RNeasy Micro Kit (Qiagen, Valencia, CA, USA), and DNAse-treated according to the manufacturer’s protocol. For comparative purpose, RNA was also isolated from two batches of MVs prepared from P9 and adult rat brain. Total RNA was quantiﬁed using OD260nm on a NanoDrop 2000c spectrophotometer (ThermoScientiﬁc, Baltimore, MA, USA) and quality was assessed with the Agilent 2100 Bioanalyser (Agilent Technologies, Palo Alto, CA, USA). RNA (1 lg) was spiked with 25 pg of bacterial AraB RNA from E. coli used as an external standard (GE Healthcare Bio-Sciences Freiburg, Germany) and reverse transcribed using the iScript Reverse Transcription Super- mix for RT-qPCR (Bio-Rad, Hercules, CA, USA). This external bacterial standard was used for normalization as the expression of conventionally used house-keeping genes including glyceraldehyde-3-phosphate deshydrogenase or hypoxanthine-phosphoribosyl transferase proved to be variable between tissues or developmental stages. 863 Quantitative real-time PCR (qRT-PCR) was performed with the LightCycler FastStart-DNA Master SYBR Green I kit and the LightCycler 1.5 Instrument (Roche Diagnostics GmbH, Mannheim, Germany). All primers were designed using NCBI Primer-BLAST and selected to generate amplicons with a length of 100–200 bp (Online Resource 1). The LightCycler experimental run protocol consisted of an initial Taq activation at 95 C, for 8 min followed by a ‘‘touch down’’ ampliﬁcation program. The ﬁrst cycle of the program consisted of 15 s at 95 C, 5 s at 68 C and 8 s at 72 C. The annealing temperature was reduced by 0.5 C every cycle until 62 C was reached. This was followed by generic PCR ampliﬁcation for 27 additional cycles keeping the annealing temperature at 62 C. Melting-curve analysis was then performed to verify the ampliﬁcation of a single product with a speciﬁc melting temperature. MgCl2 con- centration was optimized for each gene and negative PCR controls without cDNA template were included in every run. A standard curve was generated using the LightCycler Software 4.1 by non-linear regression analysis of crossing points (Cp) measured from serial dilutions of a cDNA pool for each gene analyzed and for the external standard AraB. Cp values of unknown samples were used with the appro- priate standard curve to determine in each sample the rel- ative cDNA concentration of the target gene. Potential variability in sample-to-sample reverse transcription efﬁ- ciency and RT-PCR processing was corrected by normal- izing the data to AraB expression. To provide an approximate ranking of the different Cld gene product abundance, expression levels of all genes were estimated ﬁrst in a reference sample, arbitrarily chosen as P2 LVCP #3 as follows: AraB efﬁciencyAraB CP/ Target efﬁciencyTarget CP, where efﬁciencies of ampliﬁca- tion were calculated from the linear part of the standard curves using the LightCycler Software 4.1 (http://www. roche-applied-science.com/sis/rtpcr/lightcycler/index.jsp?id =lct_0106010302). The obtained values were expressed as a percentage of Cld-1 value after correction for variation in the size of ampliﬁcation product. For each target gene, the expression levels of all individual samples were ﬁnally expressed relative to the value of the reference sample. The signiﬁcance of differences in expression levels between tissues or between developmental stages was assessed for each gene by one-way ANOVA followed by a Tukey’s multiple comparison test. The signiﬁcance of difference in expression levels between age-matched 4VCP and LVCP was assessed by two-tailed paired Student’s t test. Primary antibodies Polyclonal rabbit anti-Cld-1 Ab (pAb) (51-9000, not cross- reacting with Cld-3), rabbit anti-Cld-2 pAb (51-6100), 123