Chemidoc mp chemiluminescent imaging system

Hippocampus and cortex tissues were homogenized and lysed in sample buffer (0.5 M Tris-HCl pH 6.8, 50% glycerol, 10% sodium dodecyl sulfate (SDS), and 1: 100 inhibitor proteases and inhibitor phosphatases cocktail). The protein concentration was determined using the Bio-Rad DC protein assay (BioRad Laboratories). The lysate was centrifuged at 12,000 rpm for 10 min at 4°C, and the supernatant was denatured by boiling at 100°C with 4:1 loading buffer. For each sample, an equal amount of protein (30 μg) was separated using SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore). The membranes were blocked in 5% bovine serum albumin (BSA) dissolved in Tris-buffered saline with Tween-20 (TBST) for 1.5 h at room temperature. Then, the membranes were incubated with primary antibodies against Aβ_1-42, APP, ADAM10, BACE, NEP, IDE, PreP, PS1, SAPPα, SAPPβ, DRP1, OPA1, MFN1, MFN2, SYN 1, SYN, PSD-95, β-Actin, and COX-IV overnight at 4°C. The membrane was then incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse for 2 h at room temperature. Bands were visualized using an ECL chemiluminescence kit (Millipore) on a ChemiDoc MP Chemiluminescent imaging system (Bio-Rad, USA).

The hippocampus tissues and HT22 cells were placed in ice-cold RIPA buffer for 15 min and 30 min, respectively. The hippocampus tissues were homogenized at 4 °C, and the supernatant was taken to prepare a sample. Protein samples were separated by SDS-PAGE analysis gel and transferred onto polyvinylidene diflouride (PVDF) membranes (Millipore). The membrane were blocked with 5 % skimmed milk for 70 min. Then the membrane was washed for three times, each time for 10 min, followed by overnight incubation at 4 °C with primary antibodies. The next day, the membrane were washed and hatched with secondary antibodies for 1 h at room temperature. Bands were scanned using an ECL chemiluminescence kit (Millipore) on a ChemiDoc MP Chemiluminescent imaging system (Bio-Rad, USA), and quantified using NIH Image J software.