24 °C). After washing the sections with PB (3 × 10 min), they were incubated with the corresponding secondary antibodies, which were all diluted 1:200 in PB with 0.3% Triton X-100 for 2 h at room temperature. Following additional washes PARP inhibitor (3 × 10 min), the sections were incubated with the avidin–biotin-peroxidase complex (ABC Elite kit, Vector Labs., Burlingame, CA, USA) for 2 h at room temperature. Labeling was developed with 0.05% diaminobenzidine tetrahydrochloride (DAB) and 0.03% (final concentration) hydrogen peroxide in PB. To confirm the specificity of the antibodies,

a separate set of sections from each group was incubated only with the secondary antibodies, a condition in which no staining was present. After the staining procedure, the sections

were PD-1/PD-L1 inhibitor mounted on glass slides and the staining was intensified with 0.05% osmium tetroxide in water. They were then dehydrated and coverslipped using Permount (Fisher, Pittsburg, PA, USA). The region of interest was identified based on a stereotaxic atlas (Paxinos and Watson, 2005) using a 20× objective on a Nikon E1000 microscope (Melville, NY, USA). Images were captured using a Nikon DMX1200 digital camera, encompassing an area of 54,000 μm2 of the dorsal hippocampus, between 3 and 4 mm behind the bregma (5–7 sections/brain) (Image J, NIH/USA). The animals (8 animals per group) were decapitated and their hippocampi quickly collected, frozen in liquid nitrogen and stored at − 70 °C until use. The tissue was then homogenized at 4 °C in extraction buffer (Tris, pH 7.4,

100 mM; EDTA 10 mM; PMSF 2 mM; aprotinin 0.01 mg/ml). The homogenates were centrifuged at 12,000 rpm (15294 g) (Eppendorf Centrifuge 5804R — Westbury, NY, USA) at 4 °C for 20 min, and the protein concentration of the supernatant was determined using Orotidine 5′-phosphate decarboxylase a protein assay kit (Bio-Rad, Hercules, CA, USA) (Bradford, 1976). The material was stored in a sample buffer (Tris/HCl 125 mM, pH 6.8; 2.5% (p/v) SDS; 2.5% 2-mercaptoethanol, 4 mM EDTA and 0.05% bromofenol blue) (Laemmli, 1970) at − 70 °C until starting the assays. Samples containing 75–100 μg of total proteins in Laemmli buffer were boiled for 5 min and separated by 6.5%, 8% and 12% acrylamide SDS gels (Bio-Rad, Hercules, CA, USA) at 25 mA (Laemmli, 1970) and electrophoretically transferred to nitrocellulose membranes (Millipore, Temecula, CA, USA) at 100 V for 80 min using a Trans-Blot cell system (Bio-Rad, Hercules, CA, USA). A sample of 800 ng of recombinant human BDNF (rhBDNF) (Sigma, St. Louis, MO, USA) reconstituted with 0.2 μm-filtered PBS/0.1% BSA to a concentration of 50 mg/ml was also applied to the 12% gels as a control for BDNF ( Das et al., 2001). The membranes were then blocked for 2 h at room temperature with PBS containing 0.