Evaluation of ventral Hippocampal Cell Proliferation and brain-derived neurotrophic factor- (BDNF) induction
Different free-floating sections were incubated for antigenical retrieval at 65 °C during 90 min in saline sodium citrate buffer (pH 6; 10 nM sodium citrate). After this procedure to remove endogenous peroxidases, the slices were treated 30 min in 0.6% H2O2. Then, a set of slices were incubated at RT overnight with a primary antibody mouse anti-PCNA (1:1500, P8825, Sigma, St. Louis, MO, USA) or a different one with mouse anti-BDNF (Abcam, ab205067, 1:500) in 2.5% donkey serum. After several washes with PBS, the slices were incubated with a secondary antibody for 90 min (biotinylated anti-mouse IgG, 1:200, B8520, Sigma, St. Louis, MO, USA). Then, ExtrAvidin peroxidase (1:100, Sigma, St. Louis, MO, USA) was used to amplify the specific signal for one hour at room temperature in darkness. Detection was performed with 0.05% diaminobenzidine (DAB; Sigma) and 0.03% H2O2 in PBS. After several washes, slices were mounted on gelatin-coated slides, dehydrated in graded alcohols, and cover-slipped with DePeX mounting medium (Merck Life Science S.L.U., Darmstadt, Germany). PCNA and BDNF-labeled cells were studied using the optical fractionator method in unbiased stereological microscopy (Olympus BX51 Microscope, Olympus, Denmark), as described above.