Quantifying Neurogenesis in Aging Mice

Immunohistochemistry was conducted as previously described [31 (link)]. Briefly, 14- months old mice were overdosed with pentobarbital and transcardially perfused with 0.9% saline solution. Brains were removed and hemispheres were separated. Hemi-brains were placed in 4% paraformaldehyde overnight prior to cryoprotection in increasing sucrose gradients (10%, 20% and 30% each day). Hemi-brains were sectioned horizontally on a freezing microtome at a thickness of 25 µm and stored in phosphate-buffered saline containing 0.02% NaN₃ at 4 °C until staining. Six sections containing the hippocampus were selected for staining. Immunofluorescent staining was completed using anti-BrdU (1:200; AbD Serotec) and anti-NeuN (1:100; Millipore) primary antibodies with Alexa Fluor 594 and 633 secondaries (1:1000; Invitrogen). Sections were then mounted in glass slides and allowed to dry overnight, followed by cover-slipping the next day. Slides were scanned into digital images using a Zeiss AxioScan.Z1 slide scanner. Positive staining was determined visually based on morphology and co-localization of BrdU with NeuN and counted by hand for both the dentate gyrus and subventricular zones.