Mouse anti gfap

Immunostaining was carried out using previously established protocols^{32 (link)}. Briefly, the brain sections were incubated in a blocking solution with 5% normal goat serum (Cat #005–000-121, Jackson ImmunoResearch, West Grove, PA, USA), 2% bovine serum albumin (Cat #A6059, Sigma-Aldrich) and 0.1% Triton X-100 (Cat #0694, VWR, Avantor, Radnor, PA, USA) for 1 h at room temperature (RT). Tissue samples were then incubated overnight at RT with primary antibody for rabbit-anti-Iba1 (Cat #019–19741, 1:1000; Wako, Osaka, Japan) and mouse-anti-GFAP (Cat #MAB360, 1:2000; Millipore, Darmstadt, Germany) or NeuN (Cat #MAB377, 1:1000; Millipore). After being rinsed in 0.1 M PBS, the samples were incubated for 1 h at RT with a mixture of Cy3- or FITC-conjugated secondary antibodies (Cat #111–165-003 and #115–095-020, 1:200; Jackson ImmunoResearch) and mounted with VectaShield medium (Cat #H-1500 Vector Labs, Burlingame, CA, USA). Fluorescence images were obtained using a confocal microscope (LSM800; Carl Zeiss, Oberkochen, Germany) and fluorescence intensity were automatically analyzed by Zen software (Carl Zeiss, Oberkochen, Germany).

Paraffin embedded sections of 7μm thickness were obtained and fluorescent immunohistochemistry was performed as described in [17 (link)], with the exceptions that anti-SLC38A9 was diluted 1:25, together with mouse anti-NeuN (1:400, Millipore, Germany), mouse anti-GAD67 (1:400, Millipore, Germany), mouse anti-GFAP (1:500, Millipore, Germany), mouse anti-glutaminase (1:100, Abcam, United Kingdom). Sections were analyzed using a fluorescence microscope (Zeiss Axioplan2 imaging) connected to a camera (AxioCam HRm) with the Axiovison 4.7 software.

Free floating tissue sections were triple washed in PBS, then incubated for 1 hour in blocking solution (12.5% normal donkey serum and 0.25% triton in PBS), followed by incubation with one or more of the following primary antibodies diluted in blocking solution: goat anti-MCM2 1:100 (Santa Cruz Biotechnology, Cat# sc-9839), mouse anti-GFAP 1:500 (Millipore, Cat# MAB3402), rabbit anti-Kusabira Orange 1:500 (MBL International, Cat# PM051), rat anti-BrdU 1:2500 (Accurate Chemical, Cat# OBT0030G), mouse anti-NeuN 1:1000 (Millipore, Cat# MAB377). Before incubation in blocking solution, tissue being stained with anti-BrdU antibody underwent antigen retrieval by pre-treatment with 2N hydrochloric acid (HCl) at 37 °C for 30 minutes, followed by 0.1M boric acid at room temperature for 10 minutes and three PBS washes. Tissue was incubated with primary antibodies at 4 °C overnight or for 40 hours with anti-BrdU. Following primary antibody incubation, tissue was triple washed in PBS and incubated for one hour with donkey fluorescent secondary antibodies (Jackson ImmunoResearch) diluted 1:400 in PBS with Hoechst 1:10,000 (ThermoFisher Scientific, Cat# 33342). After secondary antibody incubation, tissue was triple washed in PBS, mounted onto slides and coverslipped with Aqua-Poly/Mount (VWR, Cat# 87001-902).

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 fixed with 4% paraformaldehyde (PFA) in PBS, permeabilized and blocked with 0.1% Triton-X100 plus and 5% normal goat serum in PBS. Then, they were incubated overnight at 4°C with the following primary antibodies: mouse anti-DLX2, 1:500 (Santa Cruz), rabbit anti-GABA, 1:1000 (Sigma), rabbit anti-GAD65/67, 1:250 (Millipore), mouse anti-GFAP, 1:200 (Millipore), mouse anti-MAP2, 1:200 (Chemicon), rabbit anti-MAP2, 1:1000 (Millipore), mouse anti-nestin, 1:200 (Chemicon), mouse anti-NeuN, 1:500 (Millipore), rabbit anti-neurofilament M, 1:200 (Millipore), mouse anti-NKX2.1, 1:1000 (Millipore), rabbit anti-OLIG2, 1:1000 (a gift from Dr. Charles Stiles, Harvard Medical School), mouse anti-PAX6, 1:250 (Millipore), mouse anti-PSD95, 1:500 (Millipore), rabbit anti-S100, 1:250 (Dako), mouse anti-SOX2, 1:500 (Millipore), rabbit anti-synaptophysin, 1:250 (Sigma), rabbit anti-TuJ1, 1:1000 (BioLegend) and mouse anti-vGAT, 1:200 (Synaptic Systems). After washing with PBS, cells were incubated with the secondary antibodies, Alexa Fluor® 555 anti-mouse IgG (Molecular Probes) and Alexa Fluor® 488 anti-mouse IgG (Molecular Probes). Cells were then counter-stained with 4,6-diamidino-2-phenylindole (DAPI) (Santa Cruz). The images were captured using a confocal laser-scanning microscope (LSM700; Zeiss) and digital inverted fluorescence microscope (DM5000B; Leica).