SV2A, B and C RNA quantification was performed with the branched DNA-based QuantiGene 2.0 assay Kit (Panomics, Inc.) [24, 25] following the manufacturer’s procedure. The specific probe sets for SV2A, B and C were designed and supplied from Panomics. Gene expression was normalized to the housekeeping gene GAPDH. For the selection of the best housekeeping gene, five references (HPRT1, GUSB, GAPDH, PPIB and SDHA) were tested on four controls and 10 samples from epileptic patients. The coefficients of variability across samples were calculated. Based on this, the best one was SDHA with GAPDH close behind. For some samples, the signals obtained for SDHA were Selleckchem PLX4032 too close to the background and
given that the quantity of the samples was limited, rather than use more Selleck C59 wnt sample volume, GAPDH was chosen as reference. In all cases, consecutive sections (5 μm) from formalin-fixed paraffin embedded tissue were stained with commercial antibodies against NeuN, synaptophysin, SV2A, SV2B, SV2C, ZnT3 and
dynorphin. Briefly, sections were deparaffinated in xylene and rehydrated through graded alcohols (100%, 80%, 60%). Endogenous peroxidase was blocked by 0.3% hydrogen peroxide in de-ionized water (10 min). Next, slices were washed twice in running tap water and immersed in citrate buffer (pH 6) during 12 min at 126°C for antigen retrieval. After washing with TBS, slices were incubated with the primary antibodies (listed in Table 2) during 1 h at room temperature except for dynorphin for which the incubation was overnight at 4°C. After three washings with TBS, sections were incubated in secondary antibody during 30 min at room temperature and immunoreactivity (IR) signal was developed with DAB (3,3′-diaminobenzidine). Haematoxylin was used to counterstain nuclei and sections
were analysed using a Zeiss Axioplan bright-field microscope. For all antibodies, negative controls were obtained by omitting the primary antibody and positive controls by staining known immunopositive tissues [2, 22, 28]. For SV2A, SV2B and SV2C, brain tissue from knockout mice was also used as negative control [2, 5, 13].. Additional negative and positive controls out were carried out for SV2C. The consistent positive staining of the striatum and pallidum in the mouse and the human was used as a positive control (supplementary data Figure S1a). Western blot analysis (see supplementary material and methods) on pallidum extracts showed that the protein identified by the polyclonal antibody had the expected molecular weight of 82 kDa according to the antibody manufacturer, and presented as a heterogeneous set of bands due to its N-glycosylation as previously reported  (supplementary data Figure S1b). The positive immunostaining in the pallidum was not seen anymore after specific blocking with SV2C recombinant peptide at 100 ng/ml (SYSY®, Goettingen, Germany). Moreover, NCBI blast of protein sequence (http://blast.ncbi.nlm.nih.gov/Blast.