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 ].
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 ].