NeuN Immunohistochemistry

NeuN protein is specific to neurons, where it localizes in the nuclei of postmitotic neurons and perikarya. NeuN IHC staining was performed in adult mouse brains (n=3 per group) after perfusion with chilled 0.9% NS and 4% paraformaldehyde. Briefly, brains were embedded in paraffin sections of 40-µm thickness and rehydrated for antigen retrieval with 100 mM citrate-phosphate buffer (pH 6.30). Sections were then washed with Tris-buffered saline containingTween-20 (TBST) and submerged in primary NeuN antibody (Chemicon International Inc., Temecula, CA, USA) at 1:100 dilution overnight. Slides were rinsed in TBST and incubated with donkey anti-mouse IgG secondary antibody (Cell Signaling Technology) for 3 h. Slides were rinsed again in TBST, coverslipped, and mounted. The density of NeuN-stained neurons was determined using laser scanning confocal microscopy (Nikon, Tokyo, Japan) as described previously.25–27 Brain sections containing NeuN-stained neurons were examined in two regions: the CA3 pyramidal cell layer of the hippocampus and retrosplenial cortical regions adjacent to the brain midline. Data are quantitatively expressed as raw neuronal density counts relative to the control.