Anti gaba

After retrograde labeling, the fixed brains were processed using a standard immunocytochemistry protocol: Fixed brains were blocked for 20 minutes in a solution of 5% normal goat serum (NGS) and 0.4% PBST. The brains were incubated with primary antibodies in 5% NGS blocking solution at 4 degrees for 1–3 days. Brains were next rinsed 3 × 10 minutes in PBST and placed in secondary antibodies in 5% NGS blocking solution at 4 degrees for 1–3 days. Finally, brains were rinsed 3 × 10 minutes in PBST and mounted in Vectashield (Vector Labs) for confocal imaging. Primary antibodies used were mouse anti-nc82 (1:40, DSHB), rat anti-mCD8 (1:40, Invitrogen MCD07800), rabbit anti-GFP (1:500, Invitrogen A11122), and rabbit anti-GABA (1:100, #A2052; Sigma, St. Louis, MO). Secondary antibodies used were goat anti-mouse 633 (1:400, Invitrogen A21050), goat anti-rat 488 (1:400, Invitrogen A11006), goat anti-rabbit 488 (1:500 when amplifying anti-GFP, 1:250 when amplifying anti-GABA; Invitrogen A11034) and streptavidin 568 (1:500, Invitrogen S11226).

After the behavioral testing and EEG recording, the brains were removed, post-fixed in 4% PFA overnight, and incubated in 30% sucrose in PBS at 4°C until they sank. Coronally sectioned slices (30 µm) were cut on a cryostat (Leica CM 1950). Sections were incubated with rabbit anti-GABA (Invitrogen, United States) primary antibodies overnight at 4°C. Slides were rinsed with distilled water and labeled with Alexa Fluor 594 goat IgG (Invitrogen, United States) for 2 h. Images were captured using a BX51WI microscope (Olympus, Japan).