Hippocampal Synaptic Protein Quantification

The hippocampus was homogenized as previously described [43 (link)] with some modifications. Briefly, we used 120 μl of TBS buffer (20 mM Tris-HCl, 137 mM NaCl, pH 7.6) containing protease and phosphatase inhibitors and 1% Triton-X100 in a dounce homogenizer. After 30-min incubation on ice, it was centrifuged at 14000g at 4 °C for 30 min. The supernatant was collected. Protein concentrations were determined (Pierce microplate BCA Protein Assay kit, thermofisher.com). Western blot was used as previously described [44 (link)]. The levels of the synaptic proteins PSD-95 (1:3000, MAB1596, Millipore) and synaptophysin (1:1000, Ab14692, Abcam,) were measured and normalized to beta-actin.