Hrp horseradish peroxidase conjugated secondary antibodies

The hippocampal tissues and neurons were homogenized in cooled RIPA buffer and centrifuged at 12, 000 g for 10 min at 4 °C. Protein concentration was determined using a BCA protein assay kit (Beyotime). Tissue homogenates (40 μg protein) from each mouse were boiled in loading buffer and electrophoresed on 10% sodium dodecyl sulfate-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5 % nonfat milk and incubated with primary antibodies overnight at 4 °C. After incubation at 37°C for 45 min with HRP (horseradish peroxidase)conjugated secondary antibodies (1:5000; Beyotime), the membranes were developed using the enhanced chemiluminescence reagent (Beyotime) and visualized with autoradiography film. The intensity of each band was quantified with Gel-Pro-Analyzer software (Media Cybernetics, Bethesda, MD, USA). The primary antibodies used in the present study were listed in table 1.