Immunodetection of Brain Proteins

PRP was prepared and washed as above. Platelet pellets were resuspended in lysis buffer containing 50 mM Tris-HCl (pH 7.5), 2 mM K₂EDTA, 0.05% Triton X-100, and protease inhibitors (Sigma (St. Louis, MI, USA)). The suspension was incubated on ice for 30 min and vortexed every 5 min. Rat brain lysates (from the excised hippocampus) and human brain lysates (from the visual cortex) were prepared by tissue homogenisation in the lysis buffer (above). Twenty g proteins were separated on 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels, transferred to a nitrocellulose membrane (Hybond-ECL, Amersham, Piscataway, NJ, USA), and processed as previously reported [6 (link)]. Membranes were first incubated with diluted human sera (1:500) followed by secondary horseradish-peroxidase–linked anti-human antibodies (1:20,000; Jackson Laboratories). Signals were developed using ECL Plus substrate (Thermo Fisher Scientific, Waltham, MA, USA) in a FujiFilm LAS-3000 phosphoimager (Life Science, Stamford, CT, USA).