Tissues from two offspring/litter and three to four litters/treatment group (n = 6–8 pups/treatment group) were used for all in vivo immunohistochemistry experiments. Tissue was coronally sectioned using a Leica cryostat at 30 μm thickness and serially collected. Animal numbers were randomized for blinded counting. The primary antibodies used for staining of select cortical markers for E18.5 samples were anti-GFP 1:1000 (Aves Lab, Tigard, OR, CAT#GFP-1020), anti-Ki67 (Abcam, Cambridge, MA, CAT#ab15580), anti-Sox2 1:1000 (Abcam, Cambridge, MA, CAT#ab97959), anti-Tbr2 1:1000 (Millipore, Billercia, MA CAT#AB15894), anti-RELN 1:1000 (Invitrogen, CAT#PA5–47537), anti-CUX1 1:100 (Atlas Antibodies, St. Louis, MO, CAT#HPA003277), anti-Ctip2 1:300 (Abcam, Cambridge, MA, CAT#ab187668), and anti-SATB2 1:1000 (Abcam, Cambridge, MA, CAT#ab92446). The primary antibody used for staining of neuronal populations of PND70 samples was anti-TH (Abcam, Cambridge, MA, CAT#ab76442). Primary antibody incubation occurred at 4° C overnight, following which sections were incubated with secondary antibodies (goat anti-chicken 488, CAT#A11039, goat anti-rabbit 568, CAT#A11011 and goat anti-mouse 568, CAT#AB175473). Sections were then washed and incubated with DAPI nuclear stain (Thermo-Fisher, CAT#D1306).
Anti satb2
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-SATB2 is a primary antibody that targets the SATB2 protein. SATB2 is a nuclear matrix-associated transcription factor that regulates gene expression. This antibody can be used to detect and study the SATB2 protein in various applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti ctip2, by Abcam (3 mentions)
Anti tbr1, by Abcam (3 mentions)
Anti sox2, by Abcam (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Anti flag, by OriGene (2 mentions)
Anti e cadherin, by Cell Signaling Technology (2 mentions)
Anti vimentin, by Cell Signaling Technology (2 mentions)
Anti h3k9me2, by Active Motif (2 mentions)
Anti map2, by Merck Group (2 mentions)
Anti gfap, by Merck Group (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Am4300, by Thermo Fisher Scientific (2 mentions)
Ecl western blotting protocol, by GE Healthcare (2 mentions)
Anti pde10a sab2700582, by Merck Group (2 mentions)
Mabs1753, by Merck Group (2 mentions)
Anti phospho pka c thr197, by Cell Signaling Technology (2 mentions)
Mab1598, by Merck Group (2 mentions)
Ab34735, by Abcam (2 mentions)
A5060, by Merck Group (2 mentions)
Cryostat, by Leica (1 mentions)
19 protocols using anti satb2
1
Immunohistochemical Analysis of Cortical Development
2
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).
3
Immunofluorescence Staining of Cells
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Cells were fixed in 4% paraformaldehyde first, then permeabilized with 0.1% Triton X-100 and blocked with 5% bovine serum albumin (Solarbio, China). Anti-Flag (1:1000; Origene, USA), anti-SATB2 (1:10; Abcam, USA) and anti-BrdU (1:1000; Invitrogen, USA) were used for overnight incubation at 4℃, and fluorescent secondary antibody (red) was used for 1 h incubation at room temperature. At last, the slides were mounted with mounting media containing DAPI (blue) to stain the nuclei before observed with a laser scanning confocal microscope (Zeiss, Germany). For BrdU assay, three independent samples were used for each group. Cell numbers of BrdU-positive cells and total cells were counted in 5 images per sample.
4
Western Blot Analysis of Exosomal Markers
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Total proteins were isolated with RIPA lysis buffer (Beyotime) and quantified with a BCA kit. An equal amount of protein lysate from each sample was separated on SDS-polyacrylamide gels, and the protein bands were transferred onto polyvinylidene difluoride membranes that were subsequently blocked with 5 % skimmed dry milk. Next, the membranes were incubated with the following antibodies: anti-HIF-1α (Hypoxia Inducible Factor 1 Subunit Alpha, Abcam, Cambridge, MA, USA), anti-CD63 (Abcam), anti-TSG101 (Tumor Susceptibility 101, Abcam), anti-GAPDH (glyceraldehyde 3-phosphate dehydrogenase)anti-Flotillin (Abcam), anti-SATB2 (Abcam); anti-phosphorylated (p)-MEK (Abcam), anti-MEK (Abcam), anti-phosphorylated (p)-ERK (Affinity Biosciences, Cincinnati, OH, USA), anti-ERK (Affinity); anti-E-cadherin (Cell Signaling Technology, Danvers, MA, USA), anti-N-cadherin (Cell Signaling Technology), anti-Vimentin (Cell Signaling Technology) at 4℃ overnight and then incubated with a secondary antibody for 1 h at room temperature. β-actin (Abcam) and Calnexin (Boster, Wuhan, China) were served as a loading control and a negative control for exosomal markers, respectively. The chemiluminescent signals were detected and the immunostaining intensity of each band was quantified using a Bio-Rad ChemiDoc XRS system (Bio-Rad, Hercules, CA, USA).
5
Protein Extraction and Western Blot Analysis
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RIPA buffer mixed with protease inhibitor (Thermo Fisher Scientific, USA) was used to lyse cells for harvest of total protein. The protein was separated by 4–20% Precast-Gel (Solarbio, China) and transferred to a PVDF membrane (Millipore, USA). Then 5% bovine serum albumin was used to block the membrane for 1 h at room temperature, and incubated with primary antibody overnight at 4℃. The following primary antibodies were used: anti-Flag (Origene, USA), anti-SATB2 (Abcam, USA), anti-β-catenin, anti-active β-catenin (Cell Signaling Technology, USA), anti-β-actin (Zhongshanjinqiao, China), anti-RUNX2 (Biorbyt, England), anti-JHDM1D (Abcam, USA), anti-DKK1 (Santa Cruz, USA), anti-H3K9me2, anti-H3K27me2 (Abcam, USA). HRP-conjugated secondary antibody (1:10,000; Zhongshanjinqiao, China) was then used to incubate the membrane for 1 h at room temperature. At last, we used ECL and Super Signal detection reagents (Thermo Fisher Scientific, USA) to detect the membrane.
6
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).
7
Chromatin Immunoprecipitation from Cortical Tissue
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We thawed the cortices derived from a single brain and filtered the tissues into a single-cell suspension using gentle mechanical dissociation. We washed cells twice with PBS containing protease inhibitor cocktail (Cat# 78437; Thermo Scientific) and re-suspended them in medium (DMEM F-12 Cat# 11320-033; Invitrogen) containing 0.1 volume crosslinking solution [33 (link)]. We carried out chromatin immunoprecipitation reactions following the previously published protocol [34 (link)]. The specific antibodies used in this study include anti-H3K9me2 (Cat# 39239; Active Motif, RRID:AB_2793199), antiH3K9me3 (Cat# 05-1242; Millipore-Sigma, RRID:AB_1587136), antiH4K20me3 (Cat# 91107; Active Motif, RRID:AB_2793777), anti-SATB2 (Cat# ab34735; Abcam, RRID:AB_2301417) and the negative IgG control (Cat# SC-2027; Santa Cruz, RRID:AB_737197). We used antibodies for modified histones and the IgG control at a concentration of 1 µg/ChIP reaction and 4 µg/ChIP reaction for SATB2. We purified precipitated DNA using the QIAquick PCR Purification Kit (catalog # 28106, Qiagen) and assayed the enrichment of the indicated sequences using quantitative PCR. We performed qPCR using the Dynamo Flash supermix (Cat# F-415XL; Thermo Scientific) on a Bio-Rad CFX384 Touch PCR system. Primer sequences are listed in Additional file 1 .
8
Fetal Cortex Protein Extraction and Analysis
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We isolated protein from the GD17 fetal cortex using the AllPrep DNA/RNA/Protein Mini Kit (Catalog No. 80004; Qiagen) according to the manufacturer’s instructions. We then separated 5–10 µg of protein on 10–15% sodium dodecyl sulfate-polyacrylamide gels and transferred proteins to nitrocellulose membranes. The primary antibodies we used in this study are: anti-H3K9me2 (Cat# 39239; Active Motif, RRID:AB_2793199), anti-pan histone H3 (Cat# ab1791; Abcam, RRID:AB_302613), anti-SATB2 (Cat# ab34735; Abcam, RRID:AB_2301417) and anti-Tubulin (Cat# ab40742; Abcam, RRID:AB_880625). We visualized blots using secondary antibodies conjugated to horseradish peroxidase (catalog# sc-2004; RRID:AB_631746; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and an enhanced chemiluminescence detection system (Pierce, Rockford, IL, USA). We calculated relative levels of H3K9me2 as a ratio to total histone H3, and relative levels of SATB2 as a ratio to Tubulin. We quantified band intensities by densitometry using ImageJ (RRID:SCR_003070; National Institutes of Health, Bethesda, MD, USA). Each experimental group contains protein extracts derived from 5 control, 3 unaffected, 4 affected animals.
9
Immunohistochemical Characterization of Cells
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Anti-ACE2 (Santa Cruz Biotechnology, SC-390851, Dallas, TX, USA), anti-SATB2 (Abcam, ab51502, Eugene, OR, USA), anti-beta III Tubulin (TUJ-1) (Abcam, ab14545), anti-MAP2 (EMD Millipore, AB2290, Burlington, MA, USA), Anti-LaminA/C (Cell Signaling Technology, 2032, Danvers, MA, USA), and anti-SOX2 (Abcam, ab59776) antibodies were used as primary antibodies for immunohistochemistry, immunocytochemistry, and immunoblot assays. Goat anti-Rabbit IgG (H+L), TRITC (Thermo Fisher Scientific, A16101, Waltham, MA, USA), and goat anti-Mouse IgG (H+L), Alexa Fluor 488 (Thermo Fisher Scientific, A11001) were used as secondary antibodies for immunohistochemistry and immunocytochemistry. Goat anti-Rabbit IgG (H+L), HRP (Thermo Fisher Scientific, 31460) and goat anti-Mouse IgG (H+L), HRP (Thermo Fisher Scientific, 31430) were used as secondary antibodies for immunoblot assay.
10
Multimodal Immunohistochemistry Profiling
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Immunohistochemistry was performed according to previous reports by using following primary antibodies: anti-GFP (rat monoclonal, Nacarai Tesque, 1:1000; rabbit polyclonal, Millipore, 1:1000), anti-NeuN (rabbit polyclonal, Millipore, 1:1000), anti-S100 (rabbit polyclonal, abcam, 1:1000), anti-GFAP (mouse monoclonal, Sigma-Aldrich, 1:1000), anti-Satb2 (mouse monoclonal, abcam, 1:1000), and anti-Dcx (goat polyclonal, Santa Cruz, 1:1000) antibodies. After washing with tris-buffered saline with Tween-20 (TBST), the sections were incubated with secondary antibodies conjugated with Alexa-Fluor 488, 594 or 633 (Thermo Fisher Scientific, 1:500), and Hoechest 33258 (Thermo Fisher Scientific, 1:1000) for nuclear staining. Coverslipped samples were examined with a fluorescent microscope (BX51, Olympus) or a laser confocal microscope (FV1000D, Olympus).
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