Rabbit anti dcx

Four mice in each group were humanely sacrificed at 24 h or 22 days after the BrdU injection. Mice were anesthetized with chloralic hydras and perfused transcardially after a thoracotomy with 100 ml of ice-cold saline, followed by 4% paraformaldehyde (Aspen, Wuhan, China) at 4 °C. The fixed brain was rapidly removed, postfixed in 4% paraformaldehyde, embedded, and sectioned for immuostaining. 4 mice per group and 15 sections per mouse were analyzed, during section procedure, we choose every sixth sections mounting on slide for further analysis. For staining of BrdU-positive cells, sections were incubated with primary antibody (rabbit anti-BrdU, 1:300, Abcam, UK) and then biotin-conjugated secondary antibody (goat anti-rabbit IgG, 1:200, Aspen, USA). In order to estimate the total numbers of immature neurons, sections were incubated with primary antibody (rabbit anti-DCX, 1:200, Abcam, UK) and then biotin-conjugated secondary antibody (goat anti-rabbit IgG, 1:200, Aspen, USA). For BrdU-NeuN co-labeling, sections were multi-stained by incubation with primary antibodies (rabbit anti-BrdU at 1:100, or mouse anti-NeuN at 1:100, Abcam, UK) and then secondary antibodies (goat anti- rabbit IgG at 1:50, goat anti-mouse IgG at 1:50, Aspen, USA). Images were acquired using a fluorescence microscope (Olympus, Tokyo, Japan) equipped with an imaging system.

To show the morphological evidence of the changes in neurons, astrocytes, and microglia in the hippocampus, immunohistochemical staining was conducted for NeuN, GFAP, and Iba-1, respectively, as previously described [20 (link),57 (link)]. In addition, proliferating cells and neuroblasts were visualized with the immunohistochemistry of Ki67 and DCX. Briefly, animals (n = 5 per group) were anesthetized with a mixture of 75 mg/kg alfaxalone and 10 mg/kg xylazine on the 56th day of diet feeding, and blood was obtained by cardiac puncture in the right ventricle. Thereafter, animals were perfused transcardially, and the brain was coronally sectioned with a 30 μm thickness between 2.0 and 2.7 mm caudal to the bregma based on gerbil stereotaxic coordinates [58 (link)]. Four sections located 150 μm apart were selected and incubated with each antibody; mouse anti-NeuN antibody (1:1000; Merck Millipore, Temecula, CA, USA), rabbit anti-GFAP antibody (1:1000; Merck Millipore), rabbit anti-Iba-1 (1:500; Wako, Osaka, Japan), rabbit anti-Ki67 (1:1000; Abcam, Cambridge, UK), or rabbit anti-DCX (1:2000; Abcam). Immunoreaction was visualized with 3,3′-diaminobenzidine tetrachloride (Sigma, St. Louis, MO, USA) in 0.1 M Tris-HCl buffer (pH 7.2). Sections were dehydrated and mounted on gelatin-coated slides in Canada balsam (Kanto Chemical, Tokyo, Japan).

The following primary antibodies were used: rabbit polyclonal anti-human NSun2 (1:1,000; Meta) (Frye & Watt, 2006 (link)), rabbit polyclonal CUK-1079-A anti-mouse NSun2 (1:1,000; Covalab) (Blanco et al, 2011 (link)), mouse monoclonal anti-NMP1 (1:500; Sigma, B0556, clone FC82291), mouse monoclonal anti-angiogenin (1:200; Abcam, ab10600, clone 14017.7), goat polyclonal anti-eIF4AI (1:200; N-19) (Santa Cruz, sc-14211), goat polyclonal anti-4ET (1:200; E-18) (Santa Cruz, sc-13454), goat polyclonal anti-SK1 (1:200; A-13) (Santa Cruz, sc-17991), goat polyclonal anti-eIF3η (1:200; A-20) (Santa Cruz, sc-16378), rabbit polyclonal anti-cleaved caspase-3 (1:100; Cell Signaling, #9664), mouse anti-PSD95 (1:200; Thermo Scientific), rabbit antisynapsin (1:1,000; Synaptic Systems), mouse anti-GFAP (1:200; Millipore), rabbit anti-Tbr1 (1:500, Abcam), rabbit anti-Dcx (1:100, Abcam).
NaAsO₂ was used at 200 μM (Sigma). The angiogenin small-molecule inhibitor N65828 (8-amino-5-(4′-hydroxybiphenyl-4-ylazo)naphthalene-2-sulphonate) was obtained from the National Cancer Institute (http://dtp.cancer.gov). NAC was purchased from Sigma and O-propargyl-puromycin (OP-puromycin) from Medchem Source LLP.

Cells were lysed in lysis buffer containing 1% protease and 1% phosphatase inhibitor (Cell Signaling Technology) followed by centrifuging for 15 min at 15,000 rpm, 4°C to remove cell debris. Protein concentrations were determined using BCA assay (Thermo-Fisher). For each sample, 30 μg of total protein was separated by SDS-PAGE and transferred to nitrocellulose membrane. Membrane was blocked and incubated with an appropriate primary antibody and a secondary antibody. The following antibodies were used: mouse anti-β-actin (1: 5,000, Abcam), rabbit anti-Wnt1 (1: 1,000; Abcam), rabbit anti-β-catenin (1: 1,000; Abcam), rabbit anti-Cyclin D1 (1: 1,000, Abcam), rabbit anti-PROX1 (1: 1,000, Abcam), rabbit anti-NeuroD1 (1: 1,000, Abcam), rabbit anti-DCX (1: 1,000, Abcam), and mouse anti-glial antigen 2 (NG2, 1: 1,000, Abcam). Proteins were visualized by enhanced chemiluminescence (Thermo-Fisher).

The 30 μm coronal cryosections were applied to glass slides and allowed to dry at room temperature overnight. Tissue sections were washed in phosphate-buffered saline (PBS) for 15 min and then blocked in 5% donkey serum, 1% BSA, and 0.2% Triton X-100 for 1 h at room temperature and incubated with primary antibody at 4 °C overnight. Mouse anti-pY99 (1:1000, Santa Cruz), rabbit anti-MAP2 (1:200, Protein Tech Co.), rabbit anti-pY416 (1:200, Cell Signaling), rabbit anti-pan Src antibody (1:200, Santa Cruz), rabbit anti-Dcx (1:200, [17 (link)], rabbit anti-Tbr1 (1:200, Abcam), rabbit anti-GAD67 (1:200, Millipore), and mouse anti-GM130 (1:200, BD) were used as primary antibodies. Donkey anti-species IgG conjugated with Alexa 488, Alexa 555 or Alexa 647, and Alexa 555-conjugated phalloidin were used as secondary antibodies. Hoechst 33342 (2 μg/ml, Molecular Probes) was used to visualize individual cell nuclei. Images were collected with a Zeiss LSM780 laser scanning confocal microscope (Advanced Fluorescence Imaging Core, SUNY Upstate Medical University). To compare the tyrosine phosphorylation response after different treatment, the average pY99 signal intensity was collected and normalized to the MAP2 immunosignal intensity and then compared among different treated group.