Western Blot Analysis of Hippocampal Proteins

Synaptoneurosomes were prepared from mouse hippocampus as described17 (link) and western blot analysis was performed as previously27 (link). Hippocampal proteins were separated by SDS-PAGE and immunoblotted using the following primary antibodies: P2X7R (1:200, Cat n: APR-004, Alomone labs), IL-1β (1:2000, Cat n: ab9772. Abcam), synaptophysin (1:1000, Cat n: S5768. Sigma-Aldrich), α-tubulin (1:2000, Cat n: T6199. Sigma-Aldrich) and β-actin (1:2000, Cat n: A5441. Sigma-Aldrich). Membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch, Plymouth, PA, USA) and bands visualized using Supersignal West Pico Chemiluminescence Substrate (Pierce, Rockford, IL, USA). Images were captured using a Fuji-Film LAS-300 (Fuji, Sheffield, UK), and densitometry was performed using AlphaEaseFC4.0 gel-scanning integrated optical density software (Alpha Innotech, San Leandaro, CA, USA).