Pregnant mice were humanely killed with CO2 and e18.5 embryos were extracted. Brains were dissected under stereomicroscope and snap-frozen in liquid nitrogen before processing for RNA extraction. To prepare brain sections for immunohistochemistry (IHC), dissected brains were rinsed in PBS before fixing in 4% PFA in PBS overnight at 4 °C. Brains were cryoprotected in 30% sucrose in PBS overnight before embedding in OCT. Coronal sections (20 μm) were obtained using Leica Cryostat CM1520. IHC was carried out similarly to that in adult brains. The following antibodies were used: Anti-Tbr1 (1:100, Abcam, Cambridge, UK), Ctip2 (1:100, Abcam, Cambridge, UK), Mfsd2a (1:100, in house), Glut1 (1:200, Abcam, Cambridge, UK), and Nes (1:100, Abcam, Cambridge, UK). Images were obtained using LSM710 Confocal Microscope (Carl Zeiss, Oberkochen, Germany).
Anti tbr1
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-Tbr1 is a laboratory antibody product used for the detection and analysis of the T-box brain protein 1 (Tbr1) in various biological samples. Tbr1 is a transcription factor involved in the regulation of neuronal development and differentiation. The antibody can be utilized in techniques such as Western blotting, immunohistochemistry, and immunocytochemistry to study the expression and localization of Tbr1 in research settings.
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Lab products found in correlation
Anti ctip2, by Abcam (5 mentions)
Anti map2, by Merck Group (4 mentions)
Anti satb2, by Abcam (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Anti flag, by Merck Group (2 mentions)
Anti ssea 4, by Developmental Studies Hybridoma Bank (2 mentions)
Anti tra 1 81, by Merck Group (2 mentions)
Tuj1 anti tubulin beta 3 isoform, by Merck Group (2 mentions)
Anti musashi, by Merck Group (2 mentions)
At8 anti p tau, by Thermo Fisher Scientific (2 mentions)
Anti sma, by Agilent Technologies (2 mentions)
Anti nestin, by R&D Systems (2 mentions)
Anti afp, by Agilent Technologies (2 mentions)
Anti nanog, by R&D Systems (2 mentions)
Anti lc3b, by Cell Signaling Technology (2 mentions)
Anti sox2, by Merck Group (2 mentions)
Anti oct4, by Santa Cruz Biotechnology (2 mentions)
Anti gfap, by Agilent Technologies (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
12 protocols using anti tbr1
1
Embryonic Mouse Brain Immunohistochemistry
2
Protein Expression Analysis by Western Blot and Immunofluorescence
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Western blotting and immunofluorescence analyses were performed as published23 (link) with the following antibodies: anti-OCT4 (Abcam, Cambridge, UK), anti-Tra-1-60 (Merck Millipore, Darmstadt, Germany), anti-NANOG (Stemgent, Lexington, USA), anti-SSEA-4 (Merck Millipore, Darmstadt, Germany), anti-TUJ1 (Covance Inc., Princeton, USA), anti-Tbr1 (Abcam, Cambridge, UK), anti-Brn2 (Santa Cruz), anti-ε-sarcoglycan (esg2-1355, published antibody against the brain-specific isoform of the protein)24 (link), anti-FLAG (Sigma Aldrich, St. Louis, USA), anti-Flotilin-1 (Cell Signaling Technology, Danvers, USA), and anti-β-actin (Sigma Aldrich, St. Louis, USA).
3
Developmental Profiling of Murine Brains
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P1 brains, representing pups from 4–5 independent litters, were fixed in 4% PFA, cryopreserved through incubation in 10%-20%-30% sucrose/1 X PBS gradient over 72 hours at 4°C, mounted in O.C.T. compound and stored at -20°C. Brains were cryo-sectioned coronally at 20 μm over a series of 7 slides, such that each slide contained representative non-consecutive brain sections. Slides were re-hydrated in 1X PBS and incubated in blocking solution containing 2% goat serum/0.01% Triton-X in 1X PBS for 1 hour at room temperature. Antibodies were dissolved in blocking solution and applied over night at 4°C as follows: anti-PECAM1 (rat, 1:50; Thermo Fisher Scientific); anti-PH3 (rabbit, 1:1000; Millipore-Sigma) anti-Satb2 (mouse, 1:250; Abcam); anti-Tbr1 (rabbit, 1:250; Abcam). DAPI (1:4000; Millipore-Sigma) was used to detect cell nuclei. The following secondary antibodies were used: goat anti-rabbit CY3 (1:250; Jackson Immunoresearch); goat anti-mouse Alexa488 (1:1000; Jackson Immunoresearch); goat anti-rat CY3 (1:250; Jackson Immunoresearch).
4
Immunohistochemical Profiling of Neuronal Markers
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Cryosections were incubated with primary antibodies diluted in PBS/Triton X-100 overnight at 4°C, rinsed with PBS, and incubated with secondary antibodies for 1 hour. Sections were counterstained with DAPI (1 µg/ml, Sigma) and mounted in SlowFade Gold (Invitrogen) or Vectashield H-1000 (Vector Labs). Antigen retrieval was performed prior to overnight incubation. Antibodies used were anti-TBR1 (1:200, Abcam), anti-CTIP2 (1:500, Abcam), anti-BRN2 (1:50, Santa Cruz), anti-TBR2 (1:200, Abcam), anti-SATB2 (1:200, Abcam), and anti-γ-tubulin (1:200, Sigma). Secondary antibodies used were goat-anti-rabbit Alexa 594 and goat-anti-mouse Alexa 488 (1:800, Invitrogen).
5
Immunocytochemical and Western Blot Analysis
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Immunocytochemistry and Western blot analysis were performed as we described before.27 , 28 The following primary antibodies were used: anti‐OCT4 (1:200; Santa Cruz), anti‐SOX2 (1:200; Millipore), anti‐NANOG (1:200; R&D Systems), anti‐SSEA‐4 (1:100; Developmental Studies Hybridoma Bank), anti‐TRA‐1‐81 (1:100; Chemicon), Tuj1 anti‐tubulin beta III isoform (1:200; Millipore), anti‐SMA (1:100; DAKO); anti‐AFP (1:100; DAKO), anti‐Nestin (1:200; R&D Systems), anti‐Musashi (1:200; Millipore), anti‐Map2 (1:200; Millipore), anti‐TBR1 (1:100; Abcam), anti‐CTIP2 (1:100; Abcam), Aβ42 anti‐β‐Amyloid42 (1:500; Calbiochem), AT8 anti‐p‐Tau (1:1000; Thermo Fisher Scientific) and anti‐LC3B (1:500; Cell Signaling), Tau5 anti‐tau (1:1000; Thermo Fisher Scientific), anti‐Mfn1 (1:1000; Abcam), anti‐Mfn2 (1:1000; Cell Signaling), anti‐DRP1 (1:1000; Cell Signaling), anti‐Fis1 (1:1000; Santa Cruz), anti‐Ub (1:4000; Santa Cruz), anti‐LAMP2 (1:1000; Santa Cruz), anti‐Beclin1 (1:1000; Cell Signaling), p62 anti‐SQSTM1 (1:1000; Santa Cruz) and anti‐β‐actin (1:10 000; Santa Cruz).
6
Molecular Markers for Neuronal Lineages
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Plasmid pCAG-dsRed was from Addgene (Plasmid #11151). Neurod1-Cre was provided by Dr. Franck Polleux (Columbia University). Pak3K297R was gifted by Dr. Rick Horwitz (University of Virginia). The PAK3 mutant was subcloned into the pCMVmyc vector.
The following primary antibodies were used: anti-EZH2 1:500 (BD Biosciences, San Jose, USA, Cat. No. 612666), anti-EZH2 1:1000 (Cell Signaling, Danvers, USA, Cat. No. #5246), anti-GAPDH 1:5000 (Sigma-Aldrich, St. Louis, USA, Cat. No. G8795), anti-GFP 1:1200 (Thermo Fisher Scientific, Waltham, USA, Cat. No. A10262), anti-TUJ1 1:1000 (Biolegend, San Diego, USA, Cat. No. 801201), anti-H3K27me3 1:2000 (Millipore, Burlington, USA, Cat. No. 07449), anti-H3 1:1000 (Cell Signaling, Cat. No. #4499), anti-dsRED 1:1000 (Clontech, Mountain View, USA, Cat. No. 632496), anti-Tbr1 1:1000 (Abcam, Waltham, USA, Cat. No. ab31940), anti-Ctip2 1:500 (Abcam, Cat. No. ab18465), anti-Foxp1 1:1000 (Abcam, Cat. No. ab16645), and anti-Cre 1:1000 (Millipore, Cat. No. MAB3120).
The following primary antibodies were used: anti-EZH2 1:500 (BD Biosciences, San Jose, USA, Cat. No. 612666), anti-EZH2 1:1000 (Cell Signaling, Danvers, USA, Cat. No. #5246), anti-GAPDH 1:5000 (Sigma-Aldrich, St. Louis, USA, Cat. No. G8795), anti-GFP 1:1200 (Thermo Fisher Scientific, Waltham, USA, Cat. No. A10262), anti-TUJ1 1:1000 (Biolegend, San Diego, USA, Cat. No. 801201), anti-H3K27me3 1:2000 (Millipore, Burlington, USA, Cat. No. 07449), anti-H3 1:1000 (Cell Signaling, Cat. No. #4499), anti-dsRED 1:1000 (Clontech, Mountain View, USA, Cat. No. 632496), anti-Tbr1 1:1000 (Abcam, Waltham, USA, Cat. No. ab31940), anti-Ctip2 1:500 (Abcam, Cat. No. ab18465), anti-Foxp1 1:1000 (Abcam, Cat. No. ab16645), and anti-Cre 1:1000 (Millipore, Cat. No. MAB3120).
7
Comprehensive Neuronal Cell Marker Profiling
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The following primary antibodies were used for indirect immunofluorescence staining: anti-Cux1 (sc-13024, Santa Cruz Biotechnology); anti-Ctip2 (ab18465, Abcam); anti- GFP (GFP-1010, Aves Labs); anti-Pax6 (PD022, MBL); anti-bromodeoxyuridine (BrdU; catalog #555627, BD Biosciences); anti-phospho-histone-H3 (Ser10; catalog #09-797, Millipore); anti-Rapgef2 (Wei et al., 2007 (link)); anti-Rapgef6 (Yoshikawa et al., 2007 (link)); anti-β-catenin (catalog #610153, BD Biosciences); anti-N-cadherin (catalog #610920, BD Biosciences); anti-E-cadherin (catalog #610181, BD Biosciences); anti-afadin (catalog #ab90809, Abcam); anti-ZO-1 (catalog #61-7300, Life Technologies); anti-nestin (catalog #556309, BD Biosciences); anti-Tbr1 (catalog #ab31940, Abcam); and anti-c-Myc (catalog #23941-54, Nacalai Tesque). To detect immunoreactive signals, the following secondary antibodies were used: Alexa Fluor 488-conjugated anti-chicken Ig Y (catalog #703-545-155, Jackson ImmunoResearch); Alexa Fluor 647-conjugated anti-rabbit IgG (catalog #A21244, Life Technologies); CF647-conjugated anti-mouse IgG (catalog #20281, Biotium); CF488A-conjugated anti-IgG (catalog #20302, #20015, #20027, Biotium); CF488A anti-mouse IgG (Biotium); and CF555-conjugated anti-IgG (catalog #20231, #20038, #20233, Biotium). An Alexa Fluor 647-conjugated anti-Tbr2 antibody (catalog #51-4875, eBioscience) was also used.
8
Immunofluorescence Assay for Pluripotency and Neural Markers
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The cells were fixed with 4% paraformaldehyde (PFA) for 15 min at room temperature (RT). Fixed cells were permeabilized and washed with TPBS containing 0.1% TritonX-100 (Sigma) in phosphate-buffered saline (PBS) and blocked with 5% normal horse serum (Vector Labs) for 30 min at RT. Afterwards, they were incubated with primary antibodies in blocking solution overnight with gentle rocking at 4℃. After washing three times with TPBS, they were incubated with secondary antibodies (ThermoFisher) for 90 min at RT, and then were counterstained using DAPI for 15 min. All fluorescence images were captured by confocal microscopy (TCSSP5II, Leica). The following primary antibodies were used: anti-OCT4 (1:200, Santa Cruz), anti-SOX2 (1:200, Millipore), anti-NANOG (1:200, R&D Systems), anti-SSEA-4 (1:100, Developmental Studies Hybridoma Bank)), anti-TRA-1-81 (1:100, CHEMICON), Tuj1 anti-tubulin beta III isoform (1:200, Millipore), anti-SMA (1:100, DAKO); anti-AFP (1:100, DAKO), anti-Nestin (1:200, R&D Systems), anti-Musashi (1:200, Millipore), anti-Map2 (1:200, Millipore), anti-TBR1 (1:100, Abcam), anti-CTIP2 (1:100, Abcam), 6E10 anti-Amyloid β (1:500, Covance), AT8 anti-p-tau (1:1000, ThermoFisher), anti-ChAT (1:200, Millipore) and anti-LC3B (1:500, Cell Signaling).
9
Immunostaining and Western Blotting Antibody Panel
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The following primary antibodies and dilutions were used for Immunostaining and Western blotting anti‐Arid1a (Sigma‐Aldrich, HPA005456); anti‐biotinylated IsolectinB4 (Vector Laboratories, B‐1205); anti‐GFAP (Dako, Z0334); anti‐GFAP (Sigma, G6171); anti‐SOX2 (Cell Signalling Technology, 3728s); anti‐Tbr1 (Abcam; ab31940); anti‐GLAST (Proteintech, 20785‐1‐AP); anti‐S100𝛽 (Abcam, ab52642); anti‐MAP2 (Millipore, MAB3418); anti‐BLBP (Abcam, ab32423); anti‐PDGFR𝛽 (Abcam, ab32570); anti‐CTIP2 (Abcam, ab18465); anti‐SATB2 (Abcam, ab51502); anti‐TUJ1 (Sigma, T2200); anti‐NeuN (Abcam; ab177487); anti‐𝛽‐Actin (Proteintech, 20536‐1‐AP); anti‐𝛽‐Actin (Proteintech; 60008‐1‐Ig); anti‐CD31(BD Biosciences, 553370); anti‐IgG (Bioss; bs‐0295p); anti‐Flag (Sigma, F1804), anti‐HA (Cell Signaling Technology); anti‐Claudin 5 (Invitrogen, 35‐2500). The following florescence secondary antibodies were used: anti‐rabbit Cy3 (Jackson ImmunoResearch), anti‐rat Cy3 (Jackson ImmunoResearch), anti‐mouse Cy3 (Jackson ImmunoResearch), anti‐rat Alexa Fluor 488 (Jackson ImmunoResearch), anti‐rabbit Alexa Fluor 488 (Jackson ImmunoResearch), anti‐goat Alexa Fluor 488 (Jackson ImmunoResearch).
10
Brain Lysates Preparation and Immunoblotting
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To prepare brain lysates, pregnant mice were sacrificed by cervical dislocation, and the cerebral cortices were harvested from the embryo heads. Each cortex was homogenised, and proteins were extracted in RIPA buffer [25 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1% (w/v) NP-40, 1% (w/v) sodium deoxycholate, 0.1% (w/v) SDS] with cOmplete protease inhibitors (Roche Diagnostics, Indianapolis, IN, USA) as described20 (link). To prepare cell lysates, the cells were washed with PBS and harvested using RIPA buffer with cOmplete protease inhibitor. Cell debris was removed by centrifugation. The protein concentrations in the lysates were determined using the BCA Protein Assay kit (Thermo Fisher Scientific). Proteins (10 μg per sample) were separated by SDS-PAGE (10% [w/v] polyacrylamide gel) and transferred to a PVDF membrane (Merck Millipore, Billerica, MA, USA). The following primary antibodies and dilutions were used: anti-DCX (dilution, 1:1000; Santa Cruz Biotechnology, Dallas, TX, USA; sc-8066), anti-Tbr1 (1:1000; Abcam, Cambridge, UK; ab31940), and anti-γ1-actin (1:10,000; Wako, Osaka, Japan; 013-24553).
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