Hippocampal Protein Extraction and Western Blotting

Mice were euthanized and had their hippocampus dissected and flash frozen. Flash-frozen tissue was homogenized in a T-PER tissue protein extraction reagent, and supplemented with protease (Roche Applied Science, IN, USA) and phosphatase inhibitors (Millipore, MA, USA). The homogenized tissues were centrifuged at 4 °C for 30 min. The supernatant (soluble fraction) was stored at − 80 °C. Western blotting was performed as previously described [49 (link)]. Protein extract was denatured and run in precast Novex gels (Life Technologies). Proteins were then transferred to nitrocellulose membranes using an iBlot (Life Technologies). Membranes were then blocked in 5% powdered milk in Tris-buffered saline with Tween (TBST). Primary antibodies were incubated in 5% milk in TBST overnight. Membranes were washed in TBST the following day and then incubated in fluorescent secondary antibody. The membranes were then washed again, and imaged and quantified using a LI-COR Odyssey CLx (LI-COR Biosciences) and Image Studio (version 1.0.11, LI-COR Biosciences).