2.5. c‐Fos immunohistochemistry

Mice were sacrificed 2 hours after the withdrawal time point of interest (0, 8, 24, and 72 hours or 7 days), because maximal levels of c‐Fos protein occur 1 to 3 hours after cellular activation. 22 Mice were deeply anesthetized with urethane (1.5 mg/kg, i.p.) and then transcardially perfused with 4‐mL phosphate buffer and then 20‐mL 4% formaldehyde (formalin diluted in phosphate buffer) at a 10 mL/min flow rate. Brains were removed, post‐fixed in 4% formaldehyde overnight at 4°C, and then placed into 20% sucrose in phosphate‐buffered saline (PBS) with 0.01% sodium azide for 2 days prior to freezing. A total of 40‐μm sections were cut on a cryostat and collected into PBS‐azide for storage prior to staining.

c‐Fos immunohistochemistry was carried out on free‐floating sections. All incubations and rinses took place at room temperature on a shaker. Sections were rinsed in PBS three times between steps. Sections were first placed into 0.3% H₂O₂ for 15 minutes, followed by blocking in 2% normal donkey serum in PBS with 0.3% triton‐X (PBST) for 1 hour and incubation in primary antibody (1:20 000 rabbit anti‐cFos, EMD‐Millipore, cat# PC38) diluted in blocking solution overnight. Sections were then incubated in 1:1000 biotinylated donkey anti‐rabbit (Jackson Immuno Research) for 1 hour and 1:1000 ABC (Vector Elite Kit, Vector Labs) for 45 minutes. The reaction was visualized via incubation for 10 minutes in 0.025% 3,3′‐diaminobenzidine (DAB), 0.05% nickel ammonium sulfate, and 0.015% H₂O₂. Sections were mounted onto Superfrost Plus slides, dried, and coverslipped using Permount.