Rabbit anti dcx

Immunodetection of proliferation, progenitor and neuronal markers, and BrdU was carried out as previously described^{7 (link),93 (link)}. Primary antibodies used were rabbit anti-DCX (1:750, Cell Signaling Technology Inc.), goat anti-DCX (1:250, Santa Cruz Biotechnology), rabbit anti-Ki67 (1:250, Abcam), mouse anti-NeuN (1:300, Millipore), mouse anti-GFAP (1:8000, Sigma), goat anti-Sox2 (1:2000, R&D Systems), rat anti-BrdU (1:300, Abcam). As secondary antibodies Alexa (1:500, Molecular Probes) and DyLight (1:500, Abcam) conjugated antibodies were used. NucBlue (Life Technologies) was used as nuclear dye. Slices were mounted on gelatin-coated slides with Fluoromont-G (Electron Microscopy Sciences).

After the VLIUS stimulation, the mice were sacrificed and perfused with 10 % formaldehyde/PBS. The brain was then harvested and fixed with 10% formaldehyde/PBS at room temperature. Samples were embedded in paraffin and serial 7 μm transverse sections were mounted on slides. The samples were deparaffinized, rehydrated, antigen retrieved (100℃, 20 min) and washed in PBST. Slices were blocked with 10% newborn calf serum (NCS) and 1% BSA in PBST for 1 hr, incubated with primary antibody overnight at 4℃. After washing with PBST, the samples were incubated with the secondary antibody for 1 hr at room temperature, washed with PBST and mounted with EverBrite Hardset Mounting Medium containing DAPI to label the nuclei (Biotium). Slides were viewed, and images were captured with LSM780 confocal microscope (Zeiss, Jena, Germany). The primary antibodies used for immunostaining and their dilutions were as follows: rabbit anti-DCX (1:200, Cell signaling), mouse anti-MAP2 (1:200, Thermo). The secondary antibodies used were Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:100, Thermo) and Alexa Fluor 555-conjugated goat anti-mouse IgG (1:100, Thermo).

Slides were incubated with primary antibodies diluted in blocking solution overnight at 4 °C, rinsed, and incubated with the secondary antibodies for 1 h at room temperature. Primary antibodies were the following: rabbit anti-CD31 (1:100, BD Biosciences), rabbit anti- VEGFR2 (1:200, Abcam), rabbit anti-DCX (1:200, Cell Signaling), mouse anti-GFAP (1:200, Abcam), mouse anti-PSA-NCAM (1:200, Millipore), mouse anti-α smooth muscle actin (1:500, Sigma), mouse anti-acetylated α tubulin (1:1000, Sigma), rabbit anti-β catenin (1:500 Abcam), rabbit anti-phosphorylated EGFR (1:200, Cell Signaling), and rabbit anti-Ki67 (1:200, Abcam). For nuclear staining 4′,6-diamidino-2-phenylindole (DAPI) 500 ng/ml (Sigma) was used. Secondary antibodies were Alexa Fluor dye-conjugated goat anti-mouse or goat anti-rabbit IgG or donkey anti-goat IgG (diluted 1:200 in blocking solution, Jackson ImmunoResearch or Invitrogen). Confocal images were taken on LSM 510 META NLO (Carl Zeiss MicroImaging, Inc., Thornwood, NY, USA). The following filter setting is applied: FITC Ch2 band pass (BP) 500–550 IR Texas Red Ch3: long pass (LP) 595 DAPI Ch2: BP 435–485 IR. For low magnification fluorescence micrographs Nikon E800 and 80i upright microscopes with Spot RT cooled CCD cameras were used.

Mice were overdosed with chloral hydrate (400 mg/kg, i.p.) and then intra-cardially perfused with PBS and then PFA 4%; brains were then isolated, post-fixed in 4% paraformaldehyde for further 24 h and cryopreserved with 30% sucrose and snap frozen. Immunofluorescence staining procedures were conducted on cryostat sections (10 μm) containing the hippocampal DG starting at −1.46 mm and ending at −2.80 mm from the bregma (for the analysis we collected one section every 240 μm). The whole region of interest was covered by 20× scanslide. The same number of sections for mouse (at least six) was incubated for 1 h in 3% goat serum and 0.3% Triton X-100, in PBS 0.1 M at RT. Sections were then incubated in rabbit anti DCX (Cell Signaling, USA) diluted in 1% goat serum 0.1% Triton X-100, in PBS 0.1 M, for 24 h at 4 °C. After washing in PBS, the sections were incubated in secondary antibody (donkey anti rabbit, Alexafluor, Invitrogen) for 1 h a RT, washed in PBS and then stained with Hoechst (Invitrogen) for 5 min, washed again and mounted on a microscope slide for the analysis of fluorescence (Eclipse, Nikon). DCX⁺ cells in the SGZ and the granule cell layer of the DG were counted exhaustively. In the same slices analyzed for neurogenesis, DG hippocampal area was calculated with MetaMorph software and the counting of DCX⁺ cells were normalized to DG area.

Free-floating sections of hippocampal tissues were washed in PBS, then were sequentially treated with 3% hydrogen peroxide (H₂O₂) in 0.01 M PBS for 15 min and 5% bovine serum albumin (BSA, Sigma, St. Louis, MO, USA) in 0.01 M PBS containing 0.3% Triton X-100 for 1 h. Then, they were incubated with rabbit anti-Iba-1 (1:1,000, Wako, Japan) and rabbit anti-DCX (1:1000, Cell Signaling Technology, Danvers, MA, USA) overnight. Then the sections were washed in PBS, subsequently exposed to corresponding biotinylated secondary antibody (1:200, Vector Laboratories, Burlingame, CA, USA). After 1 h incubation with the avidin-biotin complex reagents (ABC Elite Kit, Vector Laboratories, Burlingame, CA, USA), immunoreaction product was visualized by DAB kit (Beijing ZhongshanJinqiao Biological Technology Co., Ltd., China). Finally, the floating sections were mounted, dehydrated, cleared and coverslipped in permount TM mounting medium. The photographs were captured under a microscope (Nikon, Tokyo, Japan) and analyzed. Based on the Iba1 staining, the percentage of activated microglia in dentate gyrus was counted and analyzed as described by Tang et al. (2017 (link)).

GPCs and astrocytes were fixed with 4% paraformaldehyde for 15 min at 4 °C. Fixed cells were washed before 30-min incubation at room temperature with blocking solution: 0.1% Triton X-100 and 3% normal horse serum in PBS. Cells were incubated with primary antibodies in blocking solution at 4 °C overnight. Primary antibodies used were rat anti-CD44 (1:500; BD Pharmingen 550538, San Diego, CA), rabbit anti-NF1-A (1:250; Novus Biologicals NBP1-81406, Centennial, CO), mouse IgM anti-A2B5 (1:100; EMD Millipore MAB312, Burlington, MA), rabbit anti-S100β (1:1000; Dako GA504, Santa Clara, CA), goat anti-Vimentin (1:500; EMD Millipore AB1620), rabbit anti-DCX (1:1000; Cell Signaling 14802S, Danvers, MA), chicken anti-MAP2ab (1:1000; Abcam ab5392, United Kingdom). Cells were washed before incubation with secondary antibodies in blocking solution at 4 °C for 1 h at room temperature. Nuclei were visualized with DAPI and slides were coverslipped with polyvinyl alcohol DABCO mounting medium (Sigma Aldrich, St Louis, MO).