Ma5 12023

MVNs were fixed with 4% PFA (Thermo Scientific, 11490570) for 1 h at RT, permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, T8787) for 10 min at RT and blocked with 10% Donkey Serum (PAN-Biotech, PANP30-0101). MVNs were then stained with primary antibodies diluted 1:100-1:200 in blocking buffer, overnight at 4 °C. We used anti-CD31 (Abcam, ab24590), anti-PDGFRβ (Abcam, ab32570), anti-S100b (Sigma, S2532), anti-desmin (ab15200), anti-GFAP (Invitrogen, MA5-12023), anti-CLDN5 (Invitrogen, 341600), anti-ZO-1 (Thermo Fisher, 339100), anti-VE-cadherin (Abcam, ab33168), anti-laminin (Abcam, ab7463) and anti-collagen type IV (Sigma, MAB1910) antibodies. Devices were then washed 5 times with 1× PBS for >5 min, and stained with corresponding secondary antibodies Alexa Fluor 488 donkey anti-rabbit (Invitrogen, A-21206) or Alexa Fluor 647 donkey anti-mouse (Invitrogen, A-31571) diluted 1:250 in blocking buffer, overnight at 4 °C. Devices were washed again 5 times with 1X PBS for >5 min, stained with lectin Ulex europaeus agglutinin I (UEA I) Rhodamine (Vector Laboratories, RL-1062-2) or phalloidin (Invitrogen, R37112) and DAPI (Invitrogen, R37606), and washed overnight at 4 °C.

At the end of all experiments, mice were intraperitoneally injected with CNO or vehicle for 1 h, and then deeply anesthetized with sodium pentobarbital. After perfusion with .01 M phosphate buffer saline (PBS), the brain tissues were removed and post-fixed with 4% paraformaldehyde then placed in 20% and 30% sucrose solutions for dehydration at 4°C. The brain tissue was sliced and mounted on a slide. The slices were washed with PBS and incubated with .7% TritonX100 in a 37°C-incubator for 1 h. The slices were then incubated with mouse-anti-GFAP antibody (1:1,000, MA5-12023, Invitrogen) or mouse-anti-NeuN antibody (1:1,000, MAB377, Millipore) at 37°C for 24 h. Then, the slices were incubated with donkey-anti-mouse-Alexa Fluor 594 (1:1,000, 715–585-151, Jackson) at 37°C for 2 h after 15 min PBS washes.

Paraffin embedded slices were deparaffined and antigen retrieval carried out by incubating the samples in a 10 mM sodium citrate solution at pH 6 + 0.05% Tween20 in distilled water, where they were boiled 3 times, maintained at 95–98 °C for 20 min and finally cooled at room temperature. After blocking in 5% bovine serum albumin buffer, brain samples were incubated overnight with the following primary antibodies: mouse anti-GFAP (1:100, MA5-12023, Thermo Fisher) to label radial-glia/neural stem cells and then assess neurogenic activity close to the ventricular wall; mouse anti-DCX (doublecortin,1:50, sc-271390, Santa Cruz Biotechnology) to identify young neurons/neuroblasts; and mouse anti-Ki67 (1:50, STJ96966, St Johns Labs, UK) to label dividing cells/proliferation. To study the proliferation of neural stem/progenitor cells, we performed a double immunohistochemistry with Ki67 and Sox2 (1:100, Santa Cruz Biotechnology) for cell colocalization. The day after, brain samples were incubated with Alexa Fluor 488 and/or Texas Red secondary antibodies (both 1:300, Thermo Fisher) for 1 h at room temperature in the dark, and, after several washes, slices were mounted (F4680, Sigma). Negative controls received identical treatment except for omission of primary antibodies and showed no specific staining.