Abe457

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.

Three-month-old male mice were anesthetized and transcardially perfused with 0.9% saline containing heparin (10 mg/L) followed by fixative (4% paraformaldehyde, 15% picric acid, 0.1% glutaraldehyde in 0.1 M PB). Brains were collected and post-fixed overnight before coronal sections were taken at every 50 μm. Sections were washed and then treated with 1% H₂O₂ for 15 min to remove endogenous peroxidase activity. After washing and blocking with 2% normal horse serum, sections were incubated with primary antibodies [anti-mouse GFAP, 1:1000 for 2 h at room temperature (RT), Sigma, G3893; anti-rabbit c-fos, 1:2000 for overnight at RT, Millipore, ABE457; anti-chicken GFP, 1/2000 for overnight at RT, Abcam, ab13970; anti-mouse NeuN, 1/1000 for overnight at RT, Millipore, MAB377; anti-rabbit Iba-1, 1/2000 for overnight at RT, Wako, 019-19741]. The following day, sections were extensively washed and incubated in biotinylated anti-rabbit secondary antibody, ABC reagent, and diaminobenzidine (DAB) substrate (Vector Laboratories). Crystal violet staining was performed to detect cell nuclei. Immunofluorescence was performed with a combination of Alexa Fluor 488–or Alexa Fluor 594–labeled anti-rabbit, anti-chicken, or anti-mouse secondary antibody (1:500 for 1 h at RT, Invitrogen). Representative images were selected from at least 3 times repeated experiments.