Transfected neurons were fixed at 3 days post-transfection. Immunocytochemistry was performed as described previously11 (link),51 (link). First, hippocampal neurons were fixed in 2% formaldehyde/1 × PBS/4% sucrose for 10 min followed by incubation in cold methanol (−20 °C) for 10 min. Fixed neurons were incubated with primary antibodies diluted in 1 × GDB (0.1% gelatin, 0.3% Triton X-100, 0.45 M NaCl, 17.7 mM sodium phosphate buffer, pH 7.4) in a humidified container overnight at 4 °C. Antibodies used and their dilution factors are: anti-GABAAR γ2 (1:250; Synaptic Systems), anti-GABAAR α2 (1:250; Synaptic Systems), anti-vGAT (1:800; Synaptic Systems), anti-NL2 (1:500; Synaptic Systems), anti-gephyrin (1:1000; Synaptic Systems), anti-β-Gal (Promega), and anti-myc (1:100; Santa Cruz Biotechnology).
Alexa 488, Cy3- or Cy5 conjugated secondary antibodies were used to visualize bound primary antibodies.
Alexa 488, Cy3- or Cy5 conjugated secondary antibodies were used to visualize bound primary antibodies.