Rabbit anti gad65 gad67

Mice were anaesthetized and were 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 samples were sectioned into 100 μm 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 hrs. Then sections were incubated with primary antibodies diluted in blocking buffer: goat anti-c-Fos (1:500, Santa Cruz, SC-52G), rabbit anti-c-Fos (1:1000, Millipore ABE457), rabbit anti-GAD65+GAD67 (1:500, Abcam, ab183999), chicken anti-GFP (1:1000, Abcam, ab13970), rat anti-mCherry (1:500, Thermo Fisher, M11217), sheep anti-Foxp2 (1:2000, R&D systems, AF5647), and rabbit anti-HSD211β2 (1:300, proteintech, 14192–1-AP). Samples were incubated with primary antibodies overnight. After washing three times with PBS, the sections were incubated with secondary antibodies (1:500 dilutions, Jackson laboratory) in blocking buffer for 4 h. For an exception, GAD65+GAD67 staining was performed without Triton-X. Fluorescence in situ hybridization (FISH) was carried out in frozen brain sections using the RNAscope fluorescent multiplex kit (Advanced Cell Diagnostics) following the manufacturer’s instructions. IHC staining was applied for eYFP after FISH.

Mice were deeply anaesthetized with carbon dioxide and then transcardially perfused with PBS followed by 4% paraformaldehyde in PBS (pH 7.4) at 4 °C. The brains were extracted and fixed in 4% paraformaldehyde at 4 °C overnight. 100 μm coronal sections were prepared using a vibratome (Leica, VT-1000 s) for antibody staining. The primary antibodies (1:500 dilution) used were: goat anti-c-Fos (Santa Cruz, SC-52G), rabbit anti-NOS1 (Santa Cruz, sc-648), rabbit anti-GAD65⁺GAD67 (Abcam, ab183999), chicken anti-GFP (Abcam, ab13970) and rat anti-mCherry (Thermo Fisher, M11217). After washing three times with PBS, the sections were incubated with secondary antibodies (1:500 dilution) in blocking buffer for 4 h. The GAD65/67 primary/secondary antibody incubation solution was prepared without detergent. Fluorescence in situ hybridization was carried out using the RNAscope fluorescent multiplex kit (Advanced Cell Diagnostics) in accordance with the manufacturer’s instructions. Glp1r-cre/Ai9 mice were used with probes targeted to tdTomato and GLP1R.