Gephyrin

Sections were blocked with 5 % normal goat serum (NGS) or normal horse serum (NHS) in PBS-T for 1 h at RT. The primary antibodies (PV; 1:1000, Swant CS56; 1:100, Sigma-Aldrich; Biotin-WFA; 1:100, Sigma-Aldrich, Gephyrin; 1:200, Synaptic system) were incubated overnight at 4 °C. Following three washes in PBS they were incubated for 2 h at RT with the appropriate secondary antibody conjugated with Alexa fluor 647, Alexa fluor 488 or Alexa fluor 568 or Streptavidin-Alexa fluor 647 (Molecular Probes, Invitrogen) diluted 1:500 in PBS-T. incubated with secondary antibodies for 2 h. Sections were rinsed and mounted on 1% gelatin-coated slides with FluorSave™ Reagent (Merck Millipore, Germany).
For synaptic puncta, quantification images were captured using a Leica SPE confocal microscope using ×63 objectives with a 1024 × 1024 image resolution (n = 6 per group). At least 3 z-stack images (total 5 µm) were taken per section with at least three sections analyzed per animal (~360 µm apart). Images contained at least 5 PV positive neurons. At least 50 PV⁺ neurons per animal were analysed for Gephyrin (+) puncta quantification. Synaptic puncta analysis was performed with an automated custom script using an imageJ 1.29 plugin (available from c.eroglu@cellbio.duke.edu) [64 ].

Proteins of mice hippocampus and frontal cortex was prepared after 3 injections of SCE, AA, SCE and AA mixture (10 mg/kg) using an RIPA buffer consisting of 50 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.1% SDS, 0.5% deoxycholate and 1% Nonidet P-40. A 10% phosphatase inhibitor and 10% protease inhibitor cocktail (Roche, Basel, Switzerland) was added before using the RIPA buffer. Next, 12 μg of protein was mixed with a 5X sample buffer and loaded on SDS-PAGE gel. The Protein sample was run on gel by electrophoresis and transferred by 200 mA to polyvinylidene fluoride (PVDF) membrane (Millipore, MA, USA). The membrane was blocked by 5% skim milk and incubated with primary antibodies including anti-PSD95 (Thermo Scientific, MA, USA), GluR1 (Abcam, Cambridge, UK), GAD65 (Abcam, Cambridge, UK), Gephyrin (Synaptic Systems, Goettingen, Germany), and α-Tubulin (Sigma-Aldrich, MO, USA) antibodies at 4 °C overnight. The membrane was incubated with a secondary anti-mouse or rabbit IgG horseradish peroxidase antibody (HRP, Pierce Biotechnology, MA, USA), which corresponds to the primary antibody host, for 1 h at room temperature and each protein band was visualized by the ECL system (Thermo Scientific, MA, USA). For ECL detection, we used medical X-ray film blue (AGFA CP-BU NEW, Belgium), developer solution, and fixer solution for the ECL detection.