Anti gad 65

Cells and tissue samples were fixed (4% PFA), quenched (0.1 M glycine, 120 mM phosphate buffer, pH 7.4, 30 min, 4 °C), and permeabilized in blocking buffer (0.4% Saponin for cell cultures, 3% Triton X-100 for brain slices; 120 mM phosphate buffer, 1% BSA, pH 7.4, 1 h, 4 °C). Samples were then incubated with primary antibody (1:100–1:300 v/v in blocking buffer; 1–2 h at 23 °C or O/N at 4 °C) followed by secondary antibody (1:200 v/v in blocking buffer, 1–2 h at 23 °C). Samples were washed with blocking buffer (4 °C), rinsed in PBS, and mounted (Fluorsave, EMD Millipore). Primary antibodies used: anti-PSD95 (mouse IgG2a, monoclonal; clone 7E3-1B8, Merck Millipore), anti-c-Myc (mouse monoclonal clone 9E10; Abcam, Cambridge, UK), anti-GAD 65 (mouse IgG2a, monoclonal; clone GAD-6; Sigma), anti-p38 synaptophysin^{48 (link)} (mouse IgG1, monoclonal; clone 7.2; Synaptic Systems, Goettingen, Germany), anti-GluR-1 (rabbit polyclonal; generated in house), anti-SNAP25 (rabbit polyclonal; cat. 111002, Synaptic Systems), anti-p65 synaptotagmin (rabbit and goat polyclonal^{46 (link)}), anti-GFP (rabbit polyclonal; cat. A6455, Thermo Fisher Scientific). Secondary antibodies used: Donkey anti-Mouse, anti-Rabbit, and anti-Goat AlexaFluor488/568/647 (Jackson ImmunoResearch).

The cultures were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS, pH 7.4) for 10 min. After washing with PBS, the cultures were blocked and permeabilized with Blocking Buffer (0.3% Tween 20, 5% skimmed milk, and 2% goat serum in PBS). Cultures were incubated overnight with primary antibodies [anti-SYN1 (1:20,000; homemade), anti-VGLUT1 (1:2000; Synaptic Systems, 135 302), anti-GAD65 (1:5000; Sigma, G5638), anti-PSD95 (1:2000; NeuroMab, 75–028), anti-Homer-1 (1:1000; Synaptic Systems, 160,011), or anti-Gephyrin (1:1000; Synaptic Systems, 147 011)] diluted in Blocking Buffer. After washing three times for 5 min in PBS, cultures were incubated for 30 min at room temperature with secondary antibodies [Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 647 (1:200; Invitrogen, A32733) or Dylight 649, Goat Anti-Mouse IgG (1:200; Abbkine, A23610)]. Samples were placed in mounting medium (Southernbiotech, 0100–01) after washing them three times for 5 min in PBS and once in double distilled H₂O (ddH₂O).