Cultured hippocampal neurons were fixed with 4% paraformaldehyde and 0.4% sucrose in PBS for 10 min, permeabilized with 0.2% Triton X-100 in PBS for 10 min, washed with PBS, blocked with 10% normal goat serum in PBS for 1 h, and then incubated at 4 °C overnight with primary antibodies: pan-GIRK2 (1:500, Alomone Labs), PSD-95 (2 μg/mL, Thermo Fisher Scientific), MAP2 (1:1000, PhosphoSolutions; Aurora, CO) and CaMKIIα (1:1000, EMD Millipore; Billerica, MA). After washing 3–5 times in PBS, cells were incubated with secondary antibodies tagged with FITC (1:2000, Jackson ImmunoResearch Laboratories, Inc.; West Grove, PA) or Alexa-595/647 (2 μg/mL, Thermo Fisher Scientific) for 1 h at room temperature and protected from light. After 3 washes in PBS, coverslips were mounted onto glass slides for image acquisition. Fluorescent images were captured under a 40X or 60X (oil-immersion) objectives from a BX51W1 upright microscope with Disk Spinning Unit confocal system (Olympus; Center Valley, PA) and digital CCD camera (C10600-10B, Hamamatsu Photonic System Corp; Bridgewater, NJ). Images were processed and analyzed with Metamorph Advanced 7.7.7.0 (Molecular Devices) and NIH ImageJ software.
