IonAt the designated time point post-op., the animals were decapitated without

IonAt the designated time point post-op., the animals were decapitated without anaesthesia, and the hippocampal subregions (CA1, CA2/3 and the dentate gyrus (DG)), were dissected out using the methods described previously [17,30], and stored in a 280uC freezer until use. The 6 month post-op. rats were sacrificedGlutamate Receptors after Vestibular DamageFigure 2. Mean normalized density of expression of NR1, NR2B, GluR1, GluR2, GluR3, CaMKIIa and pCaMKIIa in the CA1, CA2/3 and DG regions of the hippocampus at 6 months following BVD or sham surgery for animals trained in a T maze or not trained in a T maze. Error bars represent 95 confidence intervals for the mean. doi:10.1371/journal.pone.0054527.gat 24 h after the last behavioural test and the different groups were counter-balanced for the order of sacrifice in order to control for potential post-training time effects. At the time of processing, tissue buffer (containing Complete Proteinase Inhibitor, 50 mM Tris Cl pH 7.6) was added to the samples on ice, then the tissue was homogenised using ultrasonification (Sonifier cell disrupter B-30, Branson Sonic Power Co.) and centrifuged at 12,000 g for 10 min at 4uC. The protein concentration in the supernatant was measured using the Bradford method and equalized, then the supernatants were mixed with gel loading buffer (50 mM Tris-HCl, 10 SDS, 10 glycerol, 10 2mercaptoethanol, 2 mg/ml bromophenol blue) in a ratio of 1:1 and boiled for 5 min.Western BlottingTen mg of protein from each sample was loaded in each well on a 7.5 SDS-polyacrylamide mini-gel and pre-stained protein markers (10?50 kDa; Bio-Rad, Precision Plus: Dual colour) were used as molecular MedChemExpress 256373-96-3 weight markers on each gel. In order to control for between gel variations, an internal standard made of pooled cerebellar samples from sham rats was loaded on each gel. The samples were electrophoresed with a 90 V variable current (BioRad, PowerPack 3000) until protein flattened at the stacking/resolving interface, and 180 V thereafter. The proteins were transferred to polyvinylidene-difluoride (PVDF) membranes using a Licochalcone A transblotting apparatus (2.5 L; Bio-Rad). The transfer was performed overnight in transfer buffer (25 methanol, 1.5 glycine and 0.3 Tris-base) at a 10 V variable current (Bio-Rad PowerPack 3000). Non-specific IgG binding was blocked by incubation with 5 dried milk protein (Pams) and 0.1 bovine serum albumin (BSA) (Sigma) for 6? h at 4uC. The membranes were then incubated with affinity-purified polyclonal goat antibodies raised against GluR1, GluR2, GluR3 and GluR 4, and affinity-purified polyclonal rabbit antibodies raised against NMDA e1 (NR1), NMDA e2 (NR2A), NMDA f1 (NR2B), CaMKIIa and pCaMKIIa, overnight at 4uC (see antibody details in Table 1). The specificity of these antibodies has been demonstrated in previous studies (NR1 [31]; NR2A [32]; NR2B [33]; GluR1 [34]; GluR2 [35]; GluR3 [36]; GluR4 [37]; CaMKIIa [38]; pCaMKIIa [39]; b-actin [40]) and the dilutions were optimised for the current study. The secondary antibodies were anti-goat IgG linked to horseradish peroxidase and antirabbit IgG linked to horseradish peroxidase (see details in Table 1). Detection was performed using the enhanced chemiluminescence (ECL) system (Amersham Biosciences, NZ). Hyperfilms (Amersham Biosciences, NZ) were analyzed by densitometry to determine the quantity of protein expressed in each group usingGlutamate Receptors after Vestibular DamageFigure 3. Example of western blots for.IonAt the designated time point post-op., the animals were decapitated without anaesthesia, and the hippocampal subregions (CA1, CA2/3 and the dentate gyrus (DG)), were dissected out using the methods described previously [17,30], and stored in a 280uC freezer until use. The 6 month post-op. rats were sacrificedGlutamate Receptors after Vestibular DamageFigure 2. Mean normalized density of expression of NR1, NR2B, GluR1, GluR2, GluR3, CaMKIIa and pCaMKIIa in the CA1, CA2/3 and DG regions of the hippocampus at 6 months following BVD or sham surgery for animals trained in a T maze or not trained in a T maze. Error bars represent 95 confidence intervals for the mean. doi:10.1371/journal.pone.0054527.gat 24 h after the last behavioural test and the different groups were counter-balanced for the order of sacrifice in order to control for potential post-training time effects. At the time of processing, tissue buffer (containing Complete Proteinase Inhibitor, 50 mM Tris Cl pH 7.6) was added to the samples on ice, then the tissue was homogenised using ultrasonification (Sonifier cell disrupter B-30, Branson Sonic Power Co.) and centrifuged at 12,000 g for 10 min at 4uC. The protein concentration in the supernatant was measured using the Bradford method and equalized, then the supernatants were mixed with gel loading buffer (50 mM Tris-HCl, 10 SDS, 10 glycerol, 10 2mercaptoethanol, 2 mg/ml bromophenol blue) in a ratio of 1:1 and boiled for 5 min.Western BlottingTen mg of protein from each sample was loaded in each well on a 7.5 SDS-polyacrylamide mini-gel and pre-stained protein markers (10?50 kDa; Bio-Rad, Precision Plus: Dual colour) were used as molecular weight markers on each gel. In order to control for between gel variations, an internal standard made of pooled cerebellar samples from sham rats was loaded on each gel. The samples were electrophoresed with a 90 V variable current (BioRad, PowerPack 3000) until protein flattened at the stacking/resolving interface, and 180 V thereafter. The proteins were transferred to polyvinylidene-difluoride (PVDF) membranes using a transblotting apparatus (2.5 L; Bio-Rad). The transfer was performed overnight in transfer buffer (25 methanol, 1.5 glycine and 0.3 Tris-base) at a 10 V variable current (Bio-Rad PowerPack 3000). Non-specific IgG binding was blocked by incubation with 5 dried milk protein (Pams) and 0.1 bovine serum albumin (BSA) (Sigma) for 6? h at 4uC. The membranes were then incubated with affinity-purified polyclonal goat antibodies raised against GluR1, GluR2, GluR3 and GluR 4, and affinity-purified polyclonal rabbit antibodies raised against NMDA e1 (NR1), NMDA e2 (NR2A), NMDA f1 (NR2B), CaMKIIa and pCaMKIIa, overnight at 4uC (see antibody details in Table 1). The specificity of these antibodies has been demonstrated in previous studies (NR1 [31]; NR2A [32]; NR2B [33]; GluR1 [34]; GluR2 [35]; GluR3 [36]; GluR4 [37]; CaMKIIa [38]; pCaMKIIa [39]; b-actin [40]) and the dilutions were optimised for the current study. The secondary antibodies were anti-goat IgG linked to horseradish peroxidase and antirabbit IgG linked to horseradish peroxidase (see details in Table 1). Detection was performed using the enhanced chemiluminescence (ECL) system (Amersham Biosciences, NZ). Hyperfilms (Amersham Biosciences, NZ) were analyzed by densitometry to determine the quantity of protein expressed in each group usingGlutamate Receptors after Vestibular DamageFigure 3. Example of western blots for.