S extracted and any fluid remaining within the organ was removed.

S extracted and any fluid remaining within the organ was removed. The heart was weighed and the ratio of body: heart weights was calculated (Table 1). Heart and body weights were not significantly different between saline or dantrolene-treated NonTg and 3xTg-AD mice (one-way ANOVA, body weights: p.0.05; heart weights: p.0.05; n = 4 for all groups). Body weights were obtained for the TASTPM mice, and were also not different between treatment groups, or from the NonTg and 3xTg-AD groups (p.0.05).RNA Extraction and Quantitative RT-PCRTotal mouse hippocampal RNA was extracted and purified from 20?0 mg samples (wet weight) dissected from AD-Tg and NonTg animals using TRI Reagent and the corresponding protocol (Invitrogen). RNA was constituted in sterile, nucleasefree water, and sample concentrations were determined spectrophotometrically at OD260 nm. After DNase treatment of 1 mg of total RNA (DNAfree kit; Ambion), dNTP and random primers were used for cDNA synthesis using the High Capacity Reverse Transcription kit (buy AKT inhibitor 2 Applied Biosystems) and associated protocol. 1 ml of the resulting cDNA products was evaluated using realtime PCR. Target genes were amplified and evaluated using the 7500 RT PCR System and SYBR green detection (Applied Biosystems). Cycling parameters were as follows: 2 min at 50uC, 10 min at 95uC, and then 40 cycles at 95uC for 15 s, Gracillin followed by 60uC for 60 s. A dissociation phase was added to the end of each cycle to determine product purity. Oligonucleotide primers were 1379592 synthesized by Invitrogen for target gene amplification. Cyclophilin A was used as an endogenous control gene. Primer sequences are as follows: RyR1: forward (F), 59-TCTTCCCTGCTGGAGACTGT- 39; reverse (R), 59-GTGGAGAAGGCACTTGAGG39; RyR2: (F), 59-TCAAACCACGAACACATTGAGG-39; (R), 59AGGCGGTAAAACATGATGTCAG-39; RyR3: (F), 59TGGCCATCATTCAAGGTCT-39; (R), 59GTCTCCATGTCTTCCCGTA-39; CycloA: (F), 59-GGCCGATGACGAGCCC-39; (R), 59-TGTCTTTGGAACTTTGTCTGCAAAT-39. Primer specificities were validated by the presence of a single amplicon for each primer set at the predicted sizes (95, Table 1. Ratio of heart and body weights from control and dantrolene-treated mice.Immunohistochemistry and Ab DepositionMice were transcardially perfused with ice-cold PBS (3 ml) followed by 4 paraformaldehyde (5 ml). Brains were extracted and fixed overnight in 30 sucrose-cryoprotectant solution. Coronal hippocampal sections 40 mm thick were cut on a cryostat and collected in TBS (0.1 M Tris, 0.9 saline, pH 7.4). Thioflavin S Staining: Free-floating hippocampal sections were washed with TBS (463 minutes). The sections were soaked in 0.5 thioflavin S (in 50/50 ethyl alcohol/distilled water, SigmaAldrich) for 10 minutes. This was followed by 263 minute washes with 50 ethyl alcohol. Sections were washed again with TBS (263 minutes), mounted with minimal drying, and coverslipped with anti-fade mounting medium PVA-DABCO for microscopy. 4G8 Staining: Free-floating hippocampal sections were washed with TBS (3610 minutes) followed by a 10 minute wash with 70 formic acid. This was followed by a 10 minute wash with TBS+ (TBS +0.1 Triton-X) followed by TBS (3610 minutes). Tissue sections were blocked with 10 goat serum in TBS for 1? hours, then incubated with primary antibody (4G8, 1:1500, Covance) diluted in TBS++ (TBS +3 goat serum +0.1 18325633 Triton-X) for 72 hours at 4uC. The sections were washed again with TBS (3610 minutes) and incubated in secondary antibody (Alexa Fluor 488 goat anti-mouse, 1:250, In.S extracted and any fluid remaining within the organ was removed. The heart was weighed and the ratio of body: heart weights was calculated (Table 1). Heart and body weights were not significantly different between saline or dantrolene-treated NonTg and 3xTg-AD mice (one-way ANOVA, body weights: p.0.05; heart weights: p.0.05; n = 4 for all groups). Body weights were obtained for the TASTPM mice, and were also not different between treatment groups, or from the NonTg and 3xTg-AD groups (p.0.05).RNA Extraction and Quantitative RT-PCRTotal mouse hippocampal RNA was extracted and purified from 20?0 mg samples (wet weight) dissected from AD-Tg and NonTg animals using TRI Reagent and the corresponding protocol (Invitrogen). RNA was constituted in sterile, nucleasefree water, and sample concentrations were determined spectrophotometrically at OD260 nm. After DNase treatment of 1 mg of total RNA (DNAfree kit; Ambion), dNTP and random primers were used for cDNA synthesis using the High Capacity Reverse Transcription kit (Applied Biosystems) and associated protocol. 1 ml of the resulting cDNA products was evaluated using realtime PCR. Target genes were amplified and evaluated using the 7500 RT PCR System and SYBR green detection (Applied Biosystems). Cycling parameters were as follows: 2 min at 50uC, 10 min at 95uC, and then 40 cycles at 95uC for 15 s, followed by 60uC for 60 s. A dissociation phase was added to the end of each cycle to determine product purity. Oligonucleotide primers were 1379592 synthesized by Invitrogen for target gene amplification. Cyclophilin A was used as an endogenous control gene. Primer sequences are as follows: RyR1: forward (F), 59-TCTTCCCTGCTGGAGACTGT- 39; reverse (R), 59-GTGGAGAAGGCACTTGAGG39; RyR2: (F), 59-TCAAACCACGAACACATTGAGG-39; (R), 59AGGCGGTAAAACATGATGTCAG-39; RyR3: (F), 59TGGCCATCATTCAAGGTCT-39; (R), 59GTCTCCATGTCTTCCCGTA-39; CycloA: (F), 59-GGCCGATGACGAGCCC-39; (R), 59-TGTCTTTGGAACTTTGTCTGCAAAT-39. Primer specificities were validated by the presence of a single amplicon for each primer set at the predicted sizes (95, Table 1. Ratio of heart and body weights from control and dantrolene-treated mice.Immunohistochemistry and Ab DepositionMice were transcardially perfused with ice-cold PBS (3 ml) followed by 4 paraformaldehyde (5 ml). Brains were extracted and fixed overnight in 30 sucrose-cryoprotectant solution. Coronal hippocampal sections 40 mm thick were cut on a cryostat and collected in TBS (0.1 M Tris, 0.9 saline, pH 7.4). Thioflavin S Staining: Free-floating hippocampal sections were washed with TBS (463 minutes). The sections were soaked in 0.5 thioflavin S (in 50/50 ethyl alcohol/distilled water, SigmaAldrich) for 10 minutes. This was followed by 263 minute washes with 50 ethyl alcohol. Sections were washed again with TBS (263 minutes), mounted with minimal drying, and coverslipped with anti-fade mounting medium PVA-DABCO for microscopy. 4G8 Staining: Free-floating hippocampal sections were washed with TBS (3610 minutes) followed by a 10 minute wash with 70 formic acid. This was followed by a 10 minute wash with TBS+ (TBS +0.1 Triton-X) followed by TBS (3610 minutes). Tissue sections were blocked with 10 goat serum in TBS for 1? hours, then incubated with primary antibody (4G8, 1:1500, Covance) diluted in TBS++ (TBS +3 goat serum +0.1 18325633 Triton-X) for 72 hours at 4uC. The sections were washed again with TBS (3610 minutes) and incubated in secondary antibody (Alexa Fluor 488 goat anti-mouse, 1:250, In.