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.