Western Blotting of Hippocampal Proteins

Western blotting was performed using the Simple Western Jess system (ProteinSimple, USA), a combination of capillary electrophoresis and immunodetection techniques, following the manufacturer’s protocols (Beekman et al. 2018 (link)). The left hippocampal samples were collected and were frozen at − 80℃ at 6 h and 7 d after microwave exposure. The hippocampus was homogenized and lysed in a mixed liquid containing radio-immunoprecipitation assay (RIPA) lysis buffer and 1% protease inhibitor after all the samples were collected. The whole process was carried out at a low temperature (0–4℃). A fully automated western blotting was carried out on an array of protein samples after the protein concentration was determined with a bicinchoninic acid (BCA) protein assay kit. Proteins were detected with the following primary antibodies: PSD-95 (ab18258, Abcam), CaMKII (ab134041, Abcam), CREB (ab32515, Abcam), (ab181602, Abcam), GluN1 (MAB363, Millipore Sigma), GluN2A (MAB5216, Millipore Sigma) and GluN2B (ab93610, Abcam). Chemiluminescent signals were captured by a charge-coupled device (CCD) camera, and the resulting images were analysed by Compass software (ProteinSimple, USA) and expressed as peak intensities. For quantification, the areas under the protein peaks were normalized to GAPDH (ab181602, Abcam) and target protein loading controls.