Rabbit anti arc

After treatments, neurons were washed twice with 37°C 4% sucrose/1X phosphate-buffered-saline (PBS; 10X: 1.4 M NaCl, 26.8 mM KCl, 62 mM Na₂HPO₄, 35.3 mM KH₂PO₄, pH 7.4), then fixed for 15 min with 4% sucrose/4% formaldehyde (Thermo Fisher Scientific) in 1X PBS. Neurons were washed 3 × 5 min with 1X PBS, permeabilized for 10 min with 0.2% Triton X-100 (Amresco, Solon, OH) in 1X PBS, and blocked for 30 min in 5% normal donkey serum (Jackson ImmunoResearch, West Grove, PA) in 1X PBS. Neurons were then incubated in primary antibody diluted in block for 1 h at RT, washed 3 × 5 min in 1X PBS, and incubated in secondary antibody diluted in block for 1 h at RT. Neurons on coverslips were mounted on glass slides in Fluoromount (Thermo Fisher Scientific) and dried overnight at RT. Primary antibodies used were: rabbit anti-Arc (1:1000; custom-made; ProteinTech, Rosemont, IL); rabbit anti-Arc (1:1000; Synaptic Systems, Goettingen, Germany); chicken anti-MAP2 (1:5000; ab5392; Abcam); mouse anti-Rab5 (1:1000; BD Biosciences, San Jose, CA); DAPI nuclear stain (Molecular Probes, Thermo Fisher Scientific). Secondary antibodies used were: Alexa Fluor 405, 488, 555, or 647 for the appropriate animal host (1:750; Thermo Fisher Scientific or Jackson ImmunoResearch).

Ninety minutes after the CFC test, mice were deeply anesthetized with ketamine/xylazine (150/15 mg/kg) and transcardially perfused with 1× phosphate buffered saline (PBS), followed by 4% paraformaldehyde (PFA) in 1× PBS. Brains were extracted and post-fixed overnight at 4°C in 4% PFA and then transferred to a 30% sucrose in 1× PBS at 4°C for two days. Thirty-five μm coronal sections were collected on a cryostat and stored in cryoprotectant at −20°C.
For immunohistochemistry, sections were washed in 1× PBS and blocked at room temperature (RT) for 2 h in 10% normal donkey serum (NDS) in 1× PBS with 0.5% Triton-X (PBS-T). Sections were incubated with primary antibodies (1:2,000 rabbit anti-Arc (Synaptic Systems); 1:500 chicken anti-GFP (Abcam); 1:1000 rabbit anti-c-Fos (Millipore)) diluted in 5% NDS in 1× PBS-T overnight at 4°C. Sections were rinsed in 1× PBS-T and incubated in secondary antibodies (Jackson ImmunoResearch; 1:500 donkey anti-rabbit Cy3; 1:500 biotinylated donkey anti-chicken) in 1× PBS-T for 2 h at RT. Sections were rinsed in 1× PBS-T and incubated in tertiary antibody (1:250 avidin Cy-2 (Jackson ImmunoResearch)) and 1:1000 DAPI for 1 h at RT. Sections were washed in 1× PBS, mounted onto slides, and coverslipped with ProLong Gold (Invitrogen). See the Life Sciences Reporting Summary for additional information.