The process of immunofluorescence analysis was performed as described by our previous studies26 (link). Briefly, brain slices were permeabilized with 0.3% Triton-X 100 and 5% BSA in PBS and incubated with primary antibodies at 4 °C overnight. After washing with PBS three times, samples were incubated with Alexa Fluor-555 secondary antibodies (goat-anti-mouse, A32727; goat-anti-rabbit, A32732; 1:1000, Thermo Fisher) for 1 h at room temperature. Samples were mounted with Vectashield mounting medium (Vector Labs) and images were taken by Leica TCS SP8 confocal microscope. The following primary antibodies were used: rabbit anti-NeuN (1:500, Abcam, ab177487), mouse anti-GABA (1:1000, Invitrogen, PA5-32241), and rabbit anti-neurogranin (1:1000, R&D, MAB7947). Unbiased stereology TissueFAX Plus ST (Tissue Gnostics, Vienna, Austria)73 (link) was applied to count EGFP-positive and NeuN-positive cell number in brain slices.
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