2.5. Western Blot Analysis of Hippocampus Tissue

After completion of the behavioral tests, mice were sacrificed by dislocation and the brains were dissected on ice to obtain the hippocampus. The tissue was frozen with liquid nitrogen and stored at −80 °C until protein analysis by Western blot, as previously described [41], with some modifications. Hippocampi (right hippocampus samples) were suspended in 15 volumes of ice-cold RIPA buffer (1% IGEPAL, 0.5% sodium deoxycholate, 0.1% SDS in PBS) supplemented with 1 mM orthovanadate, 5 mM sodium fluoride and Complete Protease Inhibitor Cocktail (#11697498001; Roche, Mannheim, Germany). Samples were sonicated and, after centrifuging (13,000× g, 10 min, 4 °C), supernatants were collected. The protein concentration of cell lysates was determined using the Bradford protein assay (#5000002; Bio-Rad, Munich, Germany), and equal quantities of proteins (20 μg) were denatured by boiling at 95 °C for 5 min in loading buffer (2% SDS, 10% glycerol, 0.05% bromophenol blue and 50 mM DTT in 50 mM Tris-HCl buffer pH 6.8) and separated by SDS-PAGE at 100 V for 2 h. Polyacrylamide gels were prepared with 30% Acrylamide/Bis Solution 29:1 (#1610156, Bio-Rad) at 10% or 15% according to the target protein size, Tris-HCl/SDS, 10% (w/v) ammonium persulfate and TEMED [42]. Electrophoresed proteins in the gels were transferred to 0.45 μm PVDF membranes (Immobilon-P, Millipore, Burlington, MA, USA) at 200 mA for 1 h 30 min. The different membranes were blocked for 1 h at room temperature with TBS-T buffer containing 5% Blotting-Grade Blocker (#170-6404; Bio-Rad). Subsequently, the membranes were incubated overnight at 4 °C with primary antibodies diluted 1:1000. Total tau clone HT7 mouse monoclonal antibody (#MN1000; Pierce Endogen, ThermoFisher Scientific, Waltham, MA, USA), p-tau clone AT8 mouse monoclonal antibody (#MN1020; ThermoFisher Scientific), and p-tau Ser396 rabbit polyclonal antibody (#44752G; Life Technologies, Carlsbad, CA, USA) were used for immunodetection of tau-related changes. Mouse monoclonal antibody against amyloid β clone 4G8 (SIG-39220; BioLegend, San Diego, CA, USA) were used to detect increased amyloid pathology. Ionized calcium-binding adapter molecule 1 (Iba1) (#019-19741; Wako; Richmond, VA, Canada) were used to immunodetect activated microglia. Furthermore, monoclonal 8C10 antibody obtained as described above was used to detect mCRP. However, the Western blot technique will not discern between CRP isoforms because the pentameric form will dissociate to monomers in the presence of reducing and denaturing reagents of the blotting buffers [29]. After washing, the corresponding secondary antibody (1:2000) was prepared for 1.5 h incubation at room temperature. Antibodies used for loading control were rabbit polyclonal actin (20–33) (#A5060; Sigma-Aldrich) and monoclonal β-tubulin (#T4026; Sigma-Aldrich) at 1:10,000. Secondary antibodies were peroxidase-conjugated. Antibodies were diluted in the West Vision Block and Diluent SP-7000 (Vector Labs Inc., Burlingame, CA, USA). Proteins were visualized using enhanced chemiluminescence (ECL) detection (Chemidoc™ Imaging System, Bio-Rad, Hercules, CA, USA) and the semi-quantitative fold differences were identified using Image Lab software (v3.0.1; Bio-Rad). Proteins were normalized to actin, to β-tubulin or to the total form for phosphorylated proteins, always analyzed in the same membrane. When p-tau and total tau could not be analyzed in the same membrane, both proteins were previously normalized to actin or to β-tubulin. All membranes contained samples from the control group and the other experimental groups. Normalized densitometry value for each sample was calculated relative to the mean of the values of the control sample in each membrane. To increase the reliability of the results in the mouse Western blots, we analyzed some of the samples in duplicate membranes. In this case, we used the mean value obtained from each hippocampus sample as a value for statistical analysis. Cell culture samples were more readily available, and each well extract was analyzed once.