The animals were deeply anesthetized with phenobarbital and perfusion-fixed with 5% formaldehyde in 0.1 M phosphate-buffered saline (PBS), followed by immersion fixation in the same fixative for 24 h at 4°C. After dehydration with graded ethanol and xylene, the brains were paraffin-embedded, serial cut in 5 μm coronal sections and mounted on glass slides. Every 100th section (typically 12 sections) was stained for MAP2. Every 50th section from the hippocampus level (typically six sections) and every 25th section from the striatum level (for the SVZ, typically six sections) were stained for BrdU or phospho-histone H3. Antigen retrieval was performed by boiling the sections in 10 mM citrate buffer (pH 6.0) for 10 min. Sections were incubated for 30 min in 4% horse or donkey or goat serum in PBS in order to block non-specific binding. Monoclonal mouse anti-MAP2 (1 : 1000, clone HM-2, Sigma, St Louis, MO, USA), monoclonal rat anti-BrdU (1 : 100, 5 μg/mL; clone: BU1/75, Oxford Biotechnology Ltd. Oxfordshire, UK), rabbit anti-phospho-histone H3 (ser10) (1 : 500, 2 μg/mL, Upstate, Temecula, CA, USA), goat anti-IL-18 (sc-6179, 4 μg/mL, Santa Cruz Biotechnology, Santa Cruz, CA, USA) primary antibody was applied and incubated at 20°C for 60 min, followed by biotinylated horse anti-mouse (1 : 200, Vector Laboratories, Burlingame, CA, USA), donkey anti-rat IgG (H + L) (1 : 200, 5.5 μg/mL; Jackson ImmunoResearch Lab. PA, USA), goat anti-rabbit (1 : 200, Vector Laboratories, Burlingame, CA, USA) or biotinylated horse anti-goat IgG (2 μg/mL) secondary antibody for 60 min at 20°C. Endogenous peroxidase activity was blocked with 3% H2O2 in PBS for 10 min. Visualization was performed using Vectastain ABC Elite (Vector Laboratories, Burlingame, CA, USA) with 0.5 mg/mL 3,3′-diaminobenzidine enhanced with 15 mg/mL ammonium nickel sulfate, 2 mg/mL beta-d glucose, 0.4 mg/mL ammonium chloride, and 0.01 mg/mL beta-glucose oxidase (all from Sigma).
The phenotype of BrdU-labeled cells was determined using antibodies against NeuN and Iba1 to detect mature neurons and microglia, respectively. Antigen recovery, was performed as above, followed by incubation with rat anti-BrdU (1 : 100, 5 μg/mL; clone: BU1/75, Oxford Biotechnology Ltd. Oxfordshire, UK) together with either mouse anti-NeuN monoclonal antibody (1 : 200, 5 μg/mL; clone: MAB377, Chemicon, Temecula, CA, USA) or rabbit anti-Iba1 antibody (1 : 1000, 0.5 μg/mL; Wako, Osaka, Japan) in PBS at 20°C for 60 min. After washing, the sections were incubated with secondary antibodies, Alexa Fluor 488 donkey anti-rat IgG (H + L), combined with either Alexa Fluor 555 donkey anti-mouse IgG (H + L) or Alexa Fluor 555 donkey anti-rabbit IgG (H + L) at 20°C for 60 min. All secondary antibodies were from Jackson ImmunoResearch Lab, and were diluted 1 : 1000. After washing, the sections were mounted using Vectashield mounting medium.