Rabbit anti tbr1

Immunostaining and in situ hybridization were performed as previously described [47 (link)]. The Fezf2 cRNA probe corresponds to nucleotides 644 to 1,374 of mouse Fezf2 (GenBank: NC_000080). LacZ staining was preformed according to standard protocols. Primary antibodies used: Chicken anti β-gal (Abcam, 1:500), Rabbit anti BLBP (Millipore, 1:500), Rat anti CTIP2 (Abcam, 1:1,000), Guinea Pig anti GFAP (Advaned Immuno Chemical, 1:100), Rabbit anti PAX6 (Covance, 1:100), PHH3 (Cell Signaling, 1:100), Rabbit anti SATB2 (Abcam, 1:1,000), Goat anti SOX2 (Santa Cruz Biotech, 1:500), Rabbit anti SOX5 (Abcam, 1:500), Rabbit anti TBR1 (Abcam, 1:1,000), Mouse anti Tuj1 (Covance, 1:1,000). Primary antibodies were detected using AlexaFluor-conjugated secondary antibodies (Invitrogen, 1:1,000). DNA was visualized with DAPI (1:50,000).

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.

Embryos were dissected, and the brains were fixed in 4% paraformaldehyde (PFA) for 1–3.5 hr. For postnatal stage, brains were fixed with 4%PFA overnight. Following 30% sucrose replacement, fixed brains were embedded in optimum cutting temperature (OCT) compound, and 20 micrometer slices were cut on a cryostat. The antibodies used were, rat anti-GFP (1∶500; nakalai tesk), rabbit anti-GFP (1∶200; IBL), rabbit anti-DsRed (1∶500; Invitrogen), goat anti-Unc5D (1∶100; R&D), rabbit anti-Tbr2 (1∶300; abcam), goat anti-NeuroD1 (1∶100; Santa Cruz), mouse anti-PCNA (1∶100; Cell Signaling), mouse anti-Tuj1 (1∶500; SIGMA), rabbit anti-Tbr1 (1∶100; abcam), mouse anti-RORb (1∶100; PERSEUS PROTEOMICS), rat anti-Ctip2 (1∶300; abcam), goat anti-Brn2 (1∶100; Santa Cruz), and mouse anti-Prdm8 [24] (link). Alexa Fluor-conjugated secondary antibodies (Invitrogen) were also used. EdU labeling (intraperitoneal injection of 12.5 mg/kg EdU) and staining were performed according to manufacturer's instructions (Invitrogen). Stainings were examined with Zeiss LSM 710 or Olympus IX81, and the images were finally processed with Adobe Photoshop.

The cells were fixed with 4% paraformaldehyde for 15 min. After several washes, they were blocked with 0.01M phosphate-buffered saline supplemented with 1% BSA and 0.3% Triton X-100 for 30 min. Primary antibodies (mouse anti-LAP2α, 1:500, Abcam, UK; mouse anti-betaIII tubulin, 1:500, Abcam; mouse anti-NeuN, 1:1000, Abcam; rabbit anti-NeuN, 1:200, Abways; rabbit anti-Tbr1, 1:500, Abcam; mouse anti-γH2AX, 1:250, Millipore, USA; mouse anti-Lamin A/C, 1:200, Abcam; rabbit anti-H3K9me3, 1:4000, Abcam; mouse anti-6E10, 1:200, Abcam) were diluted with blocking buffer and applied overnight at 4° C. The cells were stained with suitable Alexa Fluor–labeled secondary antibodies (Invitrogen) in blocking buffer at a concentration of 1:500 for 1.5 h at room temperature. 4', 6-Diamidino-2-phenylindole (Thermo Fisher, IL, USA) was applied to counterstain cells for visualizing nuclei at 1:1000 in Milli Q water (Biocel, Millipore, USA). The images were obtained with an Olympus IX71 microscope using a Hamamatsu ORCA CCD camera and Leica TCS SP5.

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.

Transcardial perfusion with 4% paraformaldehyde (PFA) was performed on the mice for fixation. The brains of E17.5, P0, P2, P4, P7, P14, P30 mice were dissected and post-fixed at 4°C with 4% PFA for 2h to overnight, depending on the size of brains. The brains were dehydrated in 30% sucrose and embedded in OCT compound. Cryosections were cut at 14-μm, or 80-μm thickness with a Cryostat (HM505E, Microm, Germany). Immunostaining was performed with standard protocols: brain sections were incubated overnight with primary antibodies at 4°C and incubated with appropriate fluorescent secondary antibodies for 2h at room temperature. The following primary antibodies were used: Goat anti-GFP (1:1000, Novus Biologicals); Mouse anti-FOXP1 (1:125, Abcam); Rabbit anti-Tbr2 (1:300, Abcam); Rabbit anti-Tbr1 (1:200, Abcam); Mouse anti-Satb2 (1:100, Santa Cruz); Mouse anti-Nestin (1:200, Abcam); Rabbit anti-CDP (1:50, Santa Cruz); Rat anti-Ctip2 (1:500, Abcam); Mouse anti-phospho Histone H3(1:300, Abcam); Mouse anti-β-III-tubulin (1:500, Promega).
Immunofluorescence images were obtained using either Olympus BX41 or Zeiss LSM 710 confocal microscope. The morphology of neurons in the cortex or culture was traced and analyzed using Neurolucida software (MBF Bioscience).

Immunolabelling techniques were used on free-floating sections, and all the washes and antibody incubations were performed in blocking solution containing 1% horse serum and 0.2% triton in phosphate buffer saline (PBST). The following primary antibodies were applied for 24 h (or 48 h for anti-cFos antibodies) at 4 °C: mouse anti-APC/CC1 Ab-7 (Millipore, OP80, 1:100), chicken anti-mCherry (Abcam, 205402, 1:1000), rat anti-Ctip2 monoclonal (Abcam, ab18465, 1:500), rabbit anti-cFos (Synaptic System 226003, 1:1000) for P9 MS/SFR pups, rabbit anti-cFos antiserum (Abcam, 190289, 1:1000) for DREADDs experiments, mouse anti-NeuN (US Biological, 1:600), rabbit anti-Olig2 (Millipore Ab9610, 1:1000), rat anti-PDGFRα (BD Bioscience, 558774, 1:600) and rabbit anti-Tbr1 (Abcam, 1:1000). The corresponding fluorescent donkey antisera secondary (Jackson ImmunoResearch, 1:200) were applied for 2 h at room temperature. Sections were subsequently mounted using the SlowFade (Molecular Probes 536939).