In this study, we used mice (BALB/c) at different ages. Validation of SV2A, SV2B and SV2C antibodies were performed on SV2A KO, SV2B KO and SV2C KO mice. The SV2C KO mice were generated in UCB (UCB Pharma SA, Braine l’Alleud). Double SV2A/SV2B KO mice were purchased from Jackson® Laboratory (Bar Harbor, Maine USA)
. Animal care was in accordance with the declaration of Helsinki and followed the guidelines of the Belgium ministry of agriculture in agreement with European Community laboratory animal care and use regulation (86/609/CEE, CE of J n°L358, 18 December 1986). Experimental researchs on animals were performed with the approval of ethics committee of the University of Liège. The number of the file from ethics committee is 1122 and accepted December 21, 2010.
Numbers of animals
Regarding the results presented in the section entitled “Immunohistofluorescence (non quantitative)”, 5 animals per age were observed in sagittal sections, 4 or 5 animals per age in coronal sections. For the section entitled “Quantitative confocal immunofluorescence” 3 animals per age were used (Figure
2A). For the section entitled “Laser micro dissection and quantitative Western blot” (Figure
2B), 3 animals per age were used. In the Figure
2C and Figure
3, 3 animals per age were used this quantification. This experiment was repeated once with three new animals per age.
Processing of tissue and sections
Mice were anaesthetized with a Nembutal® injection (Pentobarbital 60mg, Ceva Sante Animal®, Bruxelles, Belgium) before intracardiac perfusion with NaCl 0.9% (VWR International®, Prolabo), followed by paraformaldehyde (PAF) 4% (4.3g/l NaOH, 44g/l paraformaldehyde, 18.8 g/l NaH2PO4) at 4°C. Brains were removed and postfixed in PAF 4% at 4°C over night (o/n), then cryoprotected for 48h in azide phosphate buffer saline (PBS) solution containing 30% sucrose before freezing at −80°C in a 2-methylbutane solution (Aldrich®, Germany). Forty micrometer thick coronal and sagittal sections were cut on a cryostat and stored at −20°C.
Immunolabelling and histology
Permeabilization and blocking of unspecific binding sites were performed by incubation at room temperature (RT) during 30 min in the blocking solution (10% donkey serum and 0.3% Triton X-100 in phosphate buffer saline, PBS). Primary antibodies were diluted in a solution containing 10% donkey serum (Jackson Immunoresearch Laboratories®, West Grove, PA, USA) and 0.1% Triton X-100 in PBS (carrier solution). Commercially available antibodies directed against SV2A (1:200, Abcam®, Cambridge, UK), SV2B (1:500, SYSY®, Göttingen, Germany) and SV2C (1:500, SYSY®) were used. Brain sections or fixed cells were incubated with primary antibodies at RT for 2h or at 4°C o/n. Three 15 min washes were performed in PBS at RT. rhodamine-Red-X or RRX-conjugated secondary antibodies were used (Jackson Immunoresearch Laboratories®). All secondary antibodies were diluted 1:500 in the carrier solution. Finally, tissue sections were washed three times with PBS and coverslip added using DAPI-containing Vectashield® solution (Hard Set Mounting Medium®, Vector laboratory, Burlingame, CA, USA). The slides were stored in the dark at 4°C. Selectivity was confirmed by absence of staining on slices from SV2A KO, SV2B KO and SV2C KO mice. In addition, the specificity of the antibodies was confirmed by blocking peptides (Abcam® for SV2A peptide and SYSY® for SV2B and SV2C peptides). Incubation in absence of primary antibody resulted in a complete loss of detectable signal.
Image acquisition data analysis
Immunostained sections were examined using the Olympus Fluoview FV1000 confocal microscope (Olympus® Europa, GmbH, Hamburg, Germany). The level of immunohistofluorescence was semi-quantitatively assessed based on the signal intensity and scored in four classes (no labelling, low, medium and intense fluorescence and brightness) by two independent blinded observers. The quantification by confocal microscopy was performed using Olympus software F10 ASW.
Non-fixed mouse brain was cut using a vibratome (Leica-microsystems®, Grand-Bigard, Belgium) to obtain slices. The microdissection of the slices were used to dissect region of interest in the hippocampus. Protein extraction was performed on whole tissue using lysis buffer (Triton X-100, PBS 0.1 M, NaCl 1.5 M, EDTA 0.5 M). After incubation for 15 min on ice, the lysate was centrifuged for 10 min at 4°C at 10 000 G. Supernatant was collected and stored at −80°C for western blots analysis. Samples were diluted in loading buffer (Tris 106 mM, Tris base 141 mM, LDS 2%, Glycerol 10%, EDTA 0,51 mM, SERVA Blue G250 0.22 mM and red Phenol 0.175 mM, pH: 8.5) and boiled for 5 min. Microdissected hippocampus were directly added to the loading buffer and incubated for 30 min in an ultrasonic bath. Samples were then boiled for 10 min. Proteins were separated using a 10% polyacrylamide commercial gel (NuPage®, Invitrogen®, Merelbeke, Belgium) for 55 min at 200 volts (buffer: MOPS 50 mM, Tris Base 50 mM, SDS 0.1% and EDTA 1 mM, pH: 7.7) and transferred on a PVDF membrane (Roche®, Basel, Switzerland) for 60 min at 30 volts (buffer: Bicine 50 mM, Tris Base 50 mM and SDS 0.1%, pH 8.24). Membrane blocking was performed by incubation for 1 h in a blocking solution (0.2% I-Block in PBS-Tween, Tropix®). Then, the membranes were incubated for 2 h at RT in the presence of primary antibody directed against SV2A (1:2000, Abcam®), SV2B (1:10000, SYSY®) or SV2C (1:5000, SYSY®). After three washing steps in PBS-Tween solution (Tris 50mM, NaCl 120mM and Tween 0.2%, pH 7.6), the membranes were incubated for 1h with a secondary fluorescent antibody (Cy-5 conjugated anti-rabbit IgG, Jackson Immunoresearch Laboratories®) at RT. After three additional washes, the membranes were incubated at 37°C for 1 h in the dark in SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific®, Erembodegem, Belgium). Western blots were revealed using typhoon 9400 (Amersham Pharmacia Biotech®, GE Healthcare Lifesciences, Diegem, Belgium). The quantification of western blots was performed by Image Master 1D Elite software (Amersham Pharmacia Biotech®, GE Healthcare Lifesciences).
RNA purification and quantification
RNA extraction was performed with the RNeasy (Qiagen®, Venlo, Netherlands). One μg of total RNA was used to synthesize cDNA with the Applied Biosystems high capacity cDNA reverse transcription kit in a total volume of 100 μl following the manufacturer’s protocol (Life Technologies Corporation®, Carlsbad, California). Taqman Real-Time Quantitative PCR reactions (qPCR) were performed with the ABI 7900HT Sequence Detection System. Undiluted, 10× and 100× diluted cDNA were analyzed in duplicate for SV2A, SV2B and SV2C expression, using inventoried (SV2A and SV2B) and made to order (SV2C) Applied Biosystems TaqMan gene expression assays. Cq values were obtained (Applied Biosystems® SDS 2.3 software) using automatic threshold and baseline. To normalize the Cq values to the amount of cDNA per well (ΔCq), mouse β-actin was used as endogenous control. Relative gene expression was calculated with the formula 2–ΔΔCq, with the mean of the ΔCq of the P5 samples as calibrator to obtain the ΔCq.
All numerical analysis were performed using GraphPad Prism software. Statistical analysis was performed using two-way analysis of variance (ANOVA) followed by a Student’s t post-test. Data are presented as mean with standard deviation of mean (Mean +/− SD). p value < 0,05 was considered significant. For qPCR: One-way ANOVA followed by a Bonferroni’s Multiple Comparison Test was used.