Anti rabbit igg conjugated with horseradish peroxidase

Cells were treated for 24hrs, harvested with lysis buffer and protein concentration was determined using DC Protein Assay Kit (Bio-Rad, 5000111). Samples were heated at 95 °C in Laemmli buffer, loaded for fractionation to 10% SDS-PAGE gels (Bio-Rad, 4561033), and then transferred into Immuno-Blot PVDF membranes (Bio-Rad, 1620177). 5% skim milk in TBST was used as blocking buffer. Membranes were incubated overnight at 4◦C with the following antibodies; LC3B (Cell Signalling Technology, #2775), and p62 (Cell Signalling Technology, 5114) in TBST containing 1% milk. Anti-rabbit IgG conjugated with horseradish peroxidase was used as the secondary antibody (Santa Cruz Biotechnology). We used ECL detection reagents (Sigma-Aldrich, RPN3004) and captured high resolution images with Amersham Gel Imager (GE Healthcare life Sciences).

Hippocampal tissue (50 mg) was homogenized in 0.5 ml tissue lysate and centrifugated at 12,000 g at 4°C for 5 min. The supernatant was (20 μg protein) was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked in 5% nonfat milk for 1 hour at room temperature. After washing in phosphate buffer solution (PBS) for 3 times, the membrane was incubated with a rabbit-anti-rat PPAR-α antibody (1:1000; Abcam, England) [30 ], PPAR-β antibody (1:1000; Santa Cruz, CA) [24 ], PPAR-γ antibody (1:1000; Abcam, England ) [30 ] at room temperature overnight, and then incubated with an anti-rabbit IgG conjugated with horseradish peroxidase (1:1000; Santa Cruz, CA) at 37°C for 1 hour. The color reaction was carried out using ECL reagents (Pierce, USA). A Bio-Rad imaging system was used to quantify the PPARs band.

After the PCP or olanzapine treatments, cells were harvested with lysis buffer (containing NP40, Protease Inhibitor Cocktail, 1 mM PMSF and 0.5 mM β-glycerophosphate). Total protein concentrations were determined by DC-Assay (Bio-Rad, Hercules, CA), and detected using a SpectraMax Plus384 absorbance microplate reader (Molecular Devices, Sunnyvale, CA). Samples were heat-treated in Laemmli buffer at 95 °C, loaded to 8% SDS-PAGE gels for fractionation, and then transferred onto Immun-BlotTM PVDF membranes (Bio-Rad, Hercules, CA). The block consists of 5% BSA in TBST. The membranes were then incubated with synaptophysin, PSD95 (Life Technologies, NSW; dilution factor 1:1000), BDNF, NRG1 (Santa Cruz Biotechnology, Santa Cruz, CA; dilution factor 1:500), Akt, p-Akt, GSK3β, and p-GSK3β (Cell Signalling Technology, Beverly, MA; dilution factor 1:1000) antibodies in TBST containing 1% BSA overnight at 4 °C. Secondary antibodies were anti-rabbit IgG conjugated with horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA; dilution factor 1:5000). For visualization, ECL detection reagents were used and films were exposed on the AGFA CP1000 Tabletop Processor (AGFA Healthcarem Scoresby, VIC). Films were analysed using Quantity One software connected to a GS-690 Imaging Densitometer (Bio-Rad, Hercules, CA).