Sc 488

Immunohistochemistry for NeuN (a neuronal marker) and ID1 was performed using a published procedure (28 (link)). The sections were incubated in mouse anti-NeuN (1:800; MAB377; EMD Millipore, Billerica, MA, USA) or rabbit anti-ID1 (1:200; sc-488; Santa Cruz Biotechnology, Inc.) as primary antibodies, and in biotinylated horse anti-mouse (1:200; BA-2001; Vector Laboratories, Inc., Burlingame, CA, USA or rabbit anti-goat immunoglobulin G (1:200; BA-1100; Vector Laboratories, Inc.) and streptavidin peroxidase complex (VECTASTAIN^® Elite ABC kit 1:200; Vector Laboratories, Inc.) as secondary antibodies. The antibodies were finally visualized with 3,3′-diaminobenzidine tetrahydrochloride. A negative control test was performed to establish the specificity of the immunostaining using pre-blocking serum (S-1000; Vector Laboratories, Inc.) instead of primary antibody. The negative control test showed no immunoreactivity in structures observed.

ID1 levels in the mouse hippocampus (n=7 at each time point) were analyzed using a previously-published method (28 (link)). Hippocampal tissues were homogenized and ID1 levels were determined using a micro bicinchoninic acid (BCA) protein assay kit (Pierce; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Nitrocellulose transfer membranes (Pall Life Sciences, Port Washington, NY, USA) were incubated with rabbit anti-ID1 (1:1,000; sc-488; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), β-actin (1:2,000, sc-47778; Santa Cruz Biotechnology, Inc.) and exposed to peroxidase-conjugated goat anti-rabbit immunoglobulin G (1:2,000; sc-2004; Santa Cruz Biotechnology, Inc.) for 2 h at 4°C and an enhanced luminol-based chemiluminescence kit (Pierce; Thermo Fisher Scientific, Inc.). The results of this analysis were scanned and densitometric analysis, as the relative optical density (ROD), was used for quantification of the bands using Scion Image software, version 2.0 (Scion Corporation, Frederick, MD, USA). A ratio of the ROD was calibrated as a percentage, with control mice designated as 100%.