Sc 648

Animals were sacrificed with ketamine and xylazine, and perfused with 10 ml of PBS followed by 10 ml of 4% PFA in PBS (pH 7.4). Brains were dissected and post-fixed overnight in 4% PFA in PBS. 100 μm coronal brain sections were prepared using a vibratome (VT-1000S, Leica). After blocking with 10% FBS/0.2% Triton X-100 in PBS in the presence of 0.2% Triton X-100 for 1 hr, sections were incubated with primary antibodies overnight at 4C. The primary antibodies (1:500 dilution) used in these studies were: rabbit anti-CamKII (Abcam, ab5683), goat anti-c-Fos (Santa Cruz, SC-52G), goat anti-GFAP (Abcam, ab53554), rabbit anti-nNOS (Santa Cruz, sc-648), goat anti-nNOS (abcam ab72428), rabbit anti-ETV-1 (Abcam, ab81086), and chicken anti-GFP (Abcam, ab13970). Sections were washed twice with PBS, followed by a >3 hr incubation with fluorophore-conjugated secondary antibodies (1:500 dilution, Jackson Immuno Research). Fluorescent images were acquired and processed using a confocal microscope (FV1000, Olympus). In some experiments, brain sections were counterstained with DAPI (Sigma Aldrich).

Mice were anaesthetized with CO₂ and then transcardially perfused with PBS followed by 4% PFA in PBS (pH 7.4). The brain was dissected and fixed in 4% PFA at 4^○C for overnight. Fixed brains were sectioned into 100 mm coronal sections using a vibratome (Leica, VT-1000 s). For immunohistochemistry (IHC), brain sections were incubated in a blocking buffer (10% Donkey serum, 0.2% Triton-X) for 1–2 h. Sections were then incubated overnight with the following primary antibodies: rabbit anti-GAD65+GAD67 (1:500, Abcam, ab183999), rabbit anti-NOS1 (1:500, Santa Cruz, sc-648), chicken anti-GFP (1:1000, Abcam, ab13970). Sections were washed three times with PBS, then were incubated with secondary antibodies (1:500 dilutions, Jackson laboratory) in blocking buffer for 4 h. GAD65+GAD67 staining was performed without Triton-X.