Immunohistochemical studies were performed using cryocut hippocampal sections (coronal, 16 µm) as previously described [28 (link)], with slight changes in blocking. Sections were blocked with either normal chicken serum (Nptx2, Vector S3000, Vector Laboratories, Burlingame, CA, USA) or normal goal serum (Npas4, Vector S1000, Vector Laboratories) for one hour at room temperature. Sections were incubated with different primary antibody concentrations in antibody solution (2.5% BSA in PBS) at 4 °C overnight (Nptx2, 1:100, Abcam, Cambridge, England; Npas4, 1:200, Abcam). Following primary antibody incubation, slides were washed in 1xPBS three times for five minutes each at room temperature. Slides were then incubated with appropriate fluorescent secondary antibody (1:500, Alexa-488 and 594, Invitrogen, Carlsbad, CA, USA) in antibody solution for 1.5 h at room temperature. The slides were then rinsed in 1× PBS three times for five minutes each and cover slipped using VectaMount (Vector Laboratories). Sections were viewed and images were captured using a Nikon Eclipse Ni microscope equipped with DS-Qi1 monochrome, cooled digital camera and NIS-AR 4.20 Elements imaging software. CEPO + IGF-1 and vehicle-treated sections were captured using identical exposure sections. Sections = −3.30 mm from Bregma.
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