Map2b

Brain sections or glass coverslips were xed with 4% paraformaldehyde for 30 min, rupture of cell membrane was performed using 0.1% Triton X-100 for 10 min at room temperature, PBS containing 5% normal goat serum for 1 h at room temperature was used for blocking, and sections or coverslips were incubated overnight at 4 °C with the following primary antibodies: ErbB4, NRG, FGF2, Sirt1, syntaxin, Homer, and MAP2 (Abcam). Alexa Fluor 488 (green)/Alexa Fluor 594 (red) conjugated secondary antibody (Abcam, Cambridge, MA) were then used to detect primary antibodies for 1 h.
For dendritic spine analysis, the primary neurons coverslips were incubated with the primary antibodies: microtubule-associated protein 2B (MAP2B; 1:200; BD Transduction Laboratories, San Jose, CA, USA) and vesicular glutamate transporter 1 (vGlut1; 1:100; Neuromab, Davis, CA, USA) overnight at 4 °C. Alexa Fluor 488 (green)/Alexa Fluor 594 (red) conjugated secondary antibody (Abcam, Cambridge, MA) were then used to detect primary antibodies for 1 h. At least 10 cultured primary neurons per coverslip were used for quantitative analysis.

For dendritic spine analysis, we used immunocytochemistry with anti-microtubule-associated protein 2 (anti-MAP2) and anti-vesicular glutamate transporter 1 antibodies. After xation, the primary antibodies used were microtubule-associated protein 2B (MAP2B; 1:200; BD Transduction Laboratories, San Jose, CA, USA) and vesicular glutamate transporter 1 (vGlut1; 1:100; Neuromab, Davis, CA, USA). Primary antibodies were applied overnight at 4°C, followed by incubation with AlexaFluor-conjugated secondary antibody for 1 h (1:500; Life Technologies, Waltham, MA, USA) and glass cover slipping. A z stack of the optical sections was visualized on a confocal laser scanning microscope (FV10i-w; Olympus Corporation, Tokyo, Japan). At least 10 cultured neurons from two batches of cultures per group were used for quantitative analysis.
Functional labeling of presynaptic boutons with FM4-64 FM4-64 staining (Invitrogen) was performed according to the manufacturer's instructions. Brie y, primary neurons were incubated with 5 mg/mL FM4-64 (Invitrogen) and 50 mM KCl in Hank's balanced salt solution for 1 min at 4°C, and then washed with the solution to remove free FM4-64.