Anti c fos

After deep anesthesia with pentobarbital sodium (20 mg/kg, i.p), mice underwent transcardial perfusion with saline followed by 4% paraformaldehyde (PFA). Brains were extracted, post-fixed in 4% PFA overnight at 4°C, and cryoprotected in 30% sucrose solution. Coronal brain sections (40 μm) were sliced on a cryostat (Leica CM1860) and stored in a cryopreservant solution of PBS, ethylene glycol, and glycerol at −20°C. For immunofluorescence staining, brain sections were washed with PBS before incubation in a blocking solution of 10% donkey serum mixed with 0.5% Triton X-100. Overnight incubation at 4°C with primary antibodies followed this—anti-GABA (1:500, Sigma-Aldrich), anti-cFos (1:500, Synaptic Systems) in 3% donkey serum with Triton X-100. The sections were then incubated by the corresponding Alexa Fluor-conjugated secondary antibodies at room temperature for 1.5 h. For nuclear staining, slides were counterstained with DAPI (1:1,000, Sigma-Aldrich). After final washings, tissue sections were mounted and imaged on LSM880 and LSM980 confocal microscopes (ZEISS), with fluorescence signal quantification conducted using ImageJ (NIH). Fluorescence-positive cells were quantified by applying a threshold to grayscale images within 10% of the average intensity. Cells meeting or exceeding this threshold were considered positive.

Adult male mice (8–12‐week old, 3–5 mice for each genotype) were anesthetized and transcardially perfused with ice‐cold PBS containing 4% paraformaldehyde. Brains were dissected, fixed overnight at 4°C, and processed as described previously by Cho et al. (2012 (link)). Sections (20 μm) were harvested on microscope slides and incubated overnight at 4°C with the following primary antibodies: anti‐Gαo, 1:500 (MBL); anti‐olfactory marker protein (OMP), 1:500 (WAKO Chemicals); anti‐Npn2, 1:20 (R&D Systems); anti‐c‐Fos, 1:500 (Synaptic Systems); and anti‐VGlut2, 1:500 (Synaptic Systems). After rinsing in tris‐buffered saline, primary antibodies were detected with the appropriate Alexa‐488‐conjugated secondary antibody at 1:500 (Molecular Probes). Bandeiraea simplicifolia (BS) lectin, 1:1500 (Vector Laboratories) was applied with the secondary antibody.

Brain sections were rinsed in 0.1M PBS with 3% H₂O₂. Then, brain sections were incubated for 1 h in a blocking solution of 0.1M PBS containing 3% normal goat serum and 0.1% triton X-100, followed by incubation with the primary antibody polyclonal guinea pig anti-c-Fos (Synaptic Systems, ref 226 004, 1:1000) at 4 °C for 72 h. All steps following incubation with the primary antibody were performed at room temperature (RT).
Next, brain sections were rinsed in 0.1M PBS-T and incubated with biotinylated goat anti-guinea pig secondary antibody (Vector, ref BA-7000-1.5, 1:500) for 1 h at RT. Samples were washed in PBS and incubated with VECTASTAIN® Elite® ABC HRP (PK-6100, Vector, A reagent 1:250 and B reagent 1:250) in PBS for 1 h at RT. Tissues were rinsed with PBS and then 0.05 M Tris buffer. Revelation was performed in 0.003% H₂O₂ with 0.05% 3,3′-diaminobenzidine (DAB) in 0.05 M Tris buffer for 11 min. Frozen sections were dehydrated in an ethanol series at 50%, 70%, 90%, and 100% and SaveSolv (VWR Q-Path) for 10 min each. Finally, the sections were mounted onto glass slides with Eukitt mounting medium (Sigma Aldrich) and coverslipped.