Anti dcx

The day after behavioural assessments, the mice were deeply anaesthetised with isoflurane and then perfused with phosphate-buffered saline (0.01 M PBS; pH 7.4) followed by 4% paraformaldehyde (PFA). The brains were extracted and stored in 4% PFA overnight and transferred into 30% sucrose until they sank. Coronal sections (1-in-6 series, 30-μm thick) were prepared using a vibratome (Leica Biosystems, Nußloch, Germany). The brain slices were preserved in cryoprotectant solution (30% glycerol and 30% ethylene glycol) at 4 °C. Antigens were retrieved by citric acid buffer (pH 6.0) at 95 °C for 10 min. The sections were incubated with primary antibody anti-Ki-67 (1:1000; abcam, Cambridge, UK) or anti-DCX (1:200; Santa Cruz Biotechnology, Dallas, TX, USA) overnight at room temperature, followed by biotinylated goat anti-rabbit IgG or goat anti-mouse IgG (1:200; Vector Laboratories, Burlingame, CA, USA) for 2 h. Finally, the sections were visualised using the peroxidase method with commercially available DAB kits (Vector Laboratories, Burlingame, CA, USA).

The following primary antibodies, and the dilutions at which they were used in this study were: anti-GFAP (rabbit polyclonal, 1:1000, DAKO, #Z0334), anti-Ki67 (mouse monoclonal, 1:200, BD Pharmingen Biosciences, #556003), conjugated anti-SOX2 Alexa Fluor 488 (rat monoclonal, 1:400, eBioscience, #53-9811), anti-DCX (goat polyclonal, 1:200, Santa Cruz Biotechnology, #sc-8066), anti-TBR2 (rabbit polyclonal, 1:200, Abcam, #ab23345), anti-NeuN (mouse monoclonal, 1:400, Millipore, #MAB377).

We used the following primary antibodies: mouse monoclonal anti-PV (1:2000, Sigma-Aldrich), CB (1:2000, Sigma-Aldrich), calretinin (CR; 1:10000, Millipore, Billerica, MA, USA), and glutamate decarboxylase 67 (GAD67) (1:10,000, Millipore); rat monoclonal anti-BrdU (1:100; Abcam, Cambridge, MA, USA); rabbit polyclonal anti-gamma-aminobutyric acid (GABA; 1:1000, Sigma-Aldrich) and anti-Ki67 (1: 10, Ylem, Rome, Italy), and neuropeptide Y (NPY, 1: 2000, Sigma-Aldrich); and goat polyclonal anti-DCX (1:200, Santa Cruz Biotechnology, Dallas, TX, USA). We also used the following secondary antibodies: Alexa Fluor 488 and 594 goat anti-mouse IgG (both 1:200, Life Technologies, Carlsbad, CA, USA), Cy3 goat anti-mouse IgM (1:200, Millipore), and Alexa Fluor 594 goat anti-rabbit IgG and anti-rat IgG (both 1:200, Life Technologies). Biotinylated Wisteria floribunda agglutinin (1:200, Sigma-Aldrich), followed by Alexa Fluor 488 conjugated to streptavidin (10 μg/ml), was used to label PNNs using the method described above [12 (link)].

Subjects were anesthetized to a surgical depth with sodium pentothal and transcardially perfused with saline and formalin [21 (link)]. The brains were removed and postfixed in 4% paraformaldehyde for immunohistochemical staining and analysis. The anterior left hippocampus was cut into 40 μm sections and every 40th section was immunostained to detect cells labeled with the immature neuronal marker doublecortin (DCX) using the monoclonal primary antibody anti-DCX (1 : 3000; Santa Cruz) (see Figure 1). The secondary antibody was biotinylated horse anti-mouse or rabbit anti-goat IgG (1 : 200; Vector Laboratories) visualized with avidin-biotin complex solution (Vector) and diaminobenzidine (DAB; Sigma). For each animal, the density of DCX-labeled cells per mm³ of the subgranular zone (SGZ) was measured microscopically. Two independent raters, masked to treatment condition (interrater reliability > 0.95) counted all unambiguously DCX-labeled cells in the SGZ of the dentate gyrus (defined as a two-cell-body-wide zone on either side of the border of the granule cell layer) using a 40x objective.

The following primary antibodies were used: anti-nestin (mouse, 1:1000, BD Pharmingen), anti-laminin (rabbit, 1:1000, Sigma), anti-Ki67 (rabbit, 1:100, Abcam), anti-vimentin (chicken, 1:1000, Millipore), anti-Sox2 (goat, 1:200, Santa Cruz), anti-DCX (goat, 1:100, Santa Cruz), anti-Tuj1 (mouse, 1:1000, Covance) and anti-heparan sulfate (mouse, 1:500, US Biological).
The following secondary antibodies were used: goat anti-mouse CY3 (Amersham), donkey anti-mouse 488 (Molecular Probes), goat anti-rabbit 488 (Molecular Probes), donkey anti-rabbit 488 (Molecular Probes), rabbit anti-chicken CY3 (Chemicon), donkey anti-goat 546 (Molecular Probe). Nuclei were stained with the nuclear marker TO-PRO3 (Invitrogen).

Mice were anesthetized and perfused with cold PBS. The whole brain was fixed with 4% paraformaldehyde solution for 24 h at 4 °C. Next, the fixed brain was incubated in 30% sucrose solution for 72 h at 4 °C. Brain tissue was cut to a 30-μm thickness using a cryostat (Leica). Brain sections were washed in PBS and then incubated with 70% formic acid solution for antigen retrieval. The sections were immersed in a solution with BSA, 0.3% Triton X-100, and 5% horse serum overnight at room temperature to block nonspecific binding and enhance antibody permeability. Next, the sections were incubated with primary antibodies against the target proteins overnight at 4 °C. The primary antibodies included anti-Iba-1 (Wako; 1:1000), anti-GFAP (Invitrogen, 1:1000), anti-SOX2 (Abcam, 1:100), anti-Calbindin (Cell Signaling, 1:200), anti-4G8 (Biolegend, 1:500), anti-DCX (Santa Cruz, 1:200), and anti-synaptoporin (SYSY, 1:250). After reaction with primary antibodies, the sections were incubated with secondary antibodies (Invitrogen; 1:400) for 1 h at room temperature. Finally, the sections were incubated with DAPI (0.4 μg/ml) to stain nuclei. Mounted brain sections were imaged by an LSM700 confocal laser scanning microscope (Carl Zeiss), and images were analyzed by ImageJ software.