Goat anti rabbit fluorescein isothiocyanate fitc conjugated secondary antibodies

Immunofluorescence analysis was performed on synaptosomes as described previously [29 (link),30 (link)]. The synaptosomes were attached to the polylysine-coated coverslips for 40 min, fixed with 4% paraformaldehyde for 5 min, and permeabilized with 0.2% Triton X-100 in phosphate-buffered saline (PBS) for 1 h. Subsequently, the synaptosomes were incubated with primary antibody solutions containing anti-GABA_B receptor antibody (1:100; Abcam, Cambridge, UK) and anti-vesicular glutamate transporter 1 (VGLUT1) antibody (1:100; Abcam, Cambridge, UK) overnight. Synaptosomes were then washed with PBS and incubated in a mixture of goat anti-mouse DyLight 549- and goat anti-rabbit fluorescein isothiocyanate (FITC)-conjugated secondary antibodies (1:200; Invitrogen, Carlsbad, CA, USA) for 2 h at room temperature. Immunoreactivity was visualized on a Image Xpress Micro confocal microscope (Molecular Devices, San Jose, CA, USA). The estimation of the percentage of glutamatergic terminals positive for GABA_B receptor was counted three randomly selected areas (255 × 255 µm²) from each coverslip and averaged using ImageJ (Bio-Rad, Hercules, CA, USA).

The synaptosomes were attached to the polylysine-coated coverslips for 40 min at 4 °C, fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 30 min, permeabilized with 0.2% Triton X-100 for 60 min and incubated for 24 h with a mixture primary antibodies against vesicular transporter of glutamate type 1 (VGLUT1, 1:200; Abcam, Cambridge, UK) and GABA_A receptorα1 subunit(1:100; Abcam, Cambridge, UK) for 90 min at room temperature. After rinsing with blocking buffer, the synaptosomes were incubated with a mixture of goat anti-mouse DyLight 549-and goat anti-rabbit fluoresceinisothiocyanate (FITC)-conjugated secondary antibodies (1:200; Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature. The synaptosomes were then washed three times with phosphate buffer and 0.1 M carbonate buffer (pH 9.2), and the coverslips were mounted with fluoromount (DAKO North America, Inc., Carpinteria, CA, USA). Images were acquired in an Image Xpress micro confocal (Molecular Devices, San Jose, CA, USA).