Immunofluorescence

Immunostaining of rat hippocampal neurons (DIV14) was performed as described previously [13]. Briefly, neurons were fixed with 4% paraformaldehyde and 4% sucrose in PBS for 20 min. These cells were permeabilized by incubation in PBS containing 1% bovine serum albumin (BSA) and 0.1% Triton X-100 for 30 minutes at room temperature. After washing, neurons were incubated in buffer containing antibodies against mouse ErbB4 (1:100) overnight at 4℃. These neurons were washed and incubated with an appropriate fluorescein isothiocyanate-conjugated secondary antibody for anti-ErbB4. Nuclei were labeled with 10 µM Hoechst dye. Images were visualized using a LSM 510 Meta system (Zeiss LSM 510 laser scanning microscope, Carl Zeiss).