Npas4

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.

Western immunoblotting was carried out as previously described (Silasi et al., 2004 (link); Kovalchuk et al., 2016a (link),b (link),c (link)). In brief, hippocampal tissues were sonicated in ice-cold 1% SDS and immediately boiled. Protein concentrations were determined using the Bradford assay (BioRad, Hercules, CA). Equal amounts of protein (10–30 μg) were separated by SDS-PAGE into slab gels of 10–15% polyacrylamide and transferred to polyvinylidene difluoride membranes (Amersham Biosciences, Baie d'Urfé, Quebec). Eight membranes were prepared. The membranes were incubated with primary antibodies against 4-HNE, AKT 1, NPAS4 (1:1,000, Abcam), ERK1/2, FOSB, PCNA (1:1,000, Cell Signaling), and actin (1:2,000, Abcam) overnight at 4°C. Primary antibody binding was detected using horseradish peroxidase-conjugated secondary antibodies and the Enhanced Chemiluminescence Plus System (Amersham Biosciences, Baie d'Urfé, Quebec). Chemiluminescence was detected using a FluorChem HD2 camera with FluorChem software (Cell Biosciences). The membranes were stained with Coomassie blue (BioRad, Hercules, CA) to confirm equal protein loading. Signals were quantified using NIH Image J64 software and normalized relative to actin or Coomassie staining.