5 live confocal microscope

72 hours after injury, GIN mice were anesthetized by isoflurane inhalation to effect (lack of tail pinch response) and perfused transcardially with 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH=7.4) while anesthetized. Brains were then removed and placed in fixative overnight, equilibrated in 30% sucrose for 48 hours, and sectioned at 30 μm on a cryostat (-22°C). 1-in-6 serial section series (180 μm between mounted sections) containing the hippocampus were mounted in Vectashield with DAPI counterstain (Vector Labs; Burlingame, CA) and images were taken on a Zeiss 5 Live confocal microscope (Zeiss; Oberkochen, Germany). Hilar area was measured using ImageJ software to trace along the inner surface of the upper and lower blades of the dentate gyrus and in a line from the tip of each blade of the dentate gyrus to the proximal most point of CA3 pyramidal cell layer. Cell counts from each section were divided by this hilar area measure to give enhanced green fluorescent protein (eGFP) cell density measures for each section. On average, 15 serial sections were taken from each animal and cell density measurements were then averaged for each animal, divided into dorsal, medial, and ventral thirds of hippocampus. Each area covered ∼900 μm. The investigator was blinded to animal treatment for all cell counts.