The antibodies used for western blotting were: Phospho-Smad1/5 [Cell Signaling Technology (CST), 9516; RRID: AB_491015], Phospho-Smad2/3 (CST, 3108; RRID: AB_490941), Smad1 (CST, 9743; RRID: AB_2107780), Smad2/3 (CST, 5339; RRID: AB_10626777), Eomes (Abcam, ab23345; RRID:AB_778267), T (rabbit polyclonal anti-Brachyury, custom), Tcf3 (Santa Cruz Biotechnology, sc-166411), GAPDH (CST, 5174), H3 (Abcam, ab1791; RRID:AB_302613), active Ctnnb1 (CST, 8814; RRID: AB_11127203). The antibodies used for immunoprecipitation were: Tcf3 (Santa Cruz Biotechnology, sc-166411; RRID: AB_2302942), normal mouse IgG (Santa Cruz Biotechnology, sc-2025; RRID: AB_737182). The antibodies used for ChIP were: Phospho-Smad1/5/9 (CST, 13820; RRID: AB_2493181), Phospho-Smad2 (CST, 18338; RRID: AB_2798798), Eomes (Abcam, ab23345), T (Santa Cruz Biotechnology, sc-17743; RRID:AB_634980). The antibodies used for immunofluorescence and flow cytometry were: Myl7 (Proteintech, 17283-1-AP; RRID: AB_2250998; 1:250 dilution), Actc1 (Proteintech, 66125-1-Ig; RRID: AB_2881524; 1:200), Tnnt2 (Abcam, ab209813; 1:200), donkey anti-mouse IgG, Alexa Fluor 488 (Thermo Fisher Scientific, A-21202; RRID: AB_141607; 1:500), donkey anti-rabbit IgG, Alexa Fluor 488 (Thermo Fisher Scientific, A-21206; RRID: AB_2535792; 1:500).
Eomes
Manufactured by Abcam
Sourced in United States
Eomes is a transcription factor that plays a key role in the development and function of several cell types, including T cells, natural killer cells, and trophoblast cells. It functions as a regulator of gene expression, controlling the expression of genes involved in cell differentiation and lineage specification.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (3 mentions)
Sox17, by R&D Systems (2 mentions)
Nestin, by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Podocalyxin, by R&D Systems (1 mentions)
Laminin, by Merck Group (1 mentions)
P mlc, by Cell Signaling Technology (1 mentions)
Nanog, by Abcam (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Goat serum, by Agilent Technologies (1 mentions)
Vimentin, by Merck Group (1 mentions)
Donkey serum, by Merck Group (1 mentions)
Gata4, by Santa Cruz Biotechnology (1 mentions)
βiii tubulin, by Merck Group (1 mentions)
Alexa fluor dye, by Thermo Fisher Scientific (1 mentions)
Tra 1 60, by Santa Cruz Biotechnology (1 mentions)
Brachyury, by R&D Systems (1 mentions)
Oct3 4, by Santa Cruz Biotechnology (1 mentions)
10 protocols using eomes
1
Antibody Selection for Pluripotency and Lineage Analysis
2
Immunofluorescence Analysis of Embryo Development
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Pre-implantation embryos were fixed and stained as previously described (Bedzhov et al., 2012 ). Peri-implantation embryos were fixed with 4% PFA/PBS for 20 min. Cellular permeabilization was carried out for 10 min in 0.1M Glycin, 0.3% Triton X-100/PBS. The embryos were in incubated in primary antibody in 10% FCS/PBT for 2h to overnight at room temperature. Secondary antibodies were applied subsequently for 2h. Embryos were stained with DAPI (Invitrogen) and mounted in PBS droplets covered with mineral oil in glass bottom petri dishes. Confocal microscopy was performed using a Leica SP5 microscope and the images were processed using Imaris software (Bitplane). Antibodies: cleaved Caspase-3 (1:200, Cell signaling, 9664), Oct4 (1:200, Santa Cruz, sc-5279), Sox2 (1:200, Santa Cruz, sc-17320), aPKC (1:200, Santa Cruz, sc-216), Par6 (1:200, Santa Cruz, sc-67393), E-cad (1:200, (Vestweber and Kemler, 1984 )), gm130 (1:200, Calbiochem, CB1008), Eomes (1:200, Abcam), Nanog (1:200, Abcam, ab80892), Laminin (1:500, Sigma, L9393), Sox17 (1:200, R&D systems, AF1924), pMLC (1:200,Cell signaling, 3674), podocalyxin (1:200, R&D systems, MAB1556), β1-integrin (1:200, BD Pharmigen, 555005).
3
Immunofluorescence Staining Protocol for Cell Analysis
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Cells were fixed in 4% (wt/vol) paraformaldehyde for 10 min, permeabilized with 0.2% Triton X‐100 for 5 min and blocked in 3% (vol/vol) goat serum (Dako) or donkey serum (Sigma) for 45 min. They were then incubated in primary antibodies for 45 min followed by secondary antibodies for 30 min (Alexa Fluor dyes, 1:1000, Invitrogen). All antibodies were diluted in the blocking buffer. Nuclei were counterstained with DAPI (Sigma) for 5 min and coverslips were mounted on slides with FluorSave (Merck). All procedures were performed at room temperature. Primary antibodies used in this study were Vimentin (1:100, Millipore), NFIA (1:250, abcam), GFAP (1:500, Dako), GFAP (1:500, Sigma), S100B (1:500, Dako), βIII‐tubulin (1:1000, Sigma), TDP‐43 (1:250, Abnova), NANOG (1:250, R&D Systems), SOX2 (1:250, Millipore), TRA‐1‐60 (1:250, Santa Cruz), OCT3/4 (1:250, Santa Cruz), SOX1 (1:100, R&D Systems), Nestin (1:1000, Millipore), Brachyury (1:100, R&D Systems), EOMES (1:600, abcam), FOXA2 (1:100, R&D Systems), GATA‐4 (1:100, Santa Cruz), SMI32 (1:250, Covance), and Caspase‐3 (1:500, Abcam).
Fluorescent imaging was performed on fields of view containing uniform DAPI staining using either an Axio Observer.Z1 (Zeiss) epifluorescence microscope or an LSM710 confocal microscope (Carl Zeiss). Images were processed and blindly analyzed by using the ImageJ64 (v 1.47) software.
Fluorescent imaging was performed on fields of view containing uniform DAPI staining using either an Axio Observer.Z1 (Zeiss) epifluorescence microscope or an LSM710 confocal microscope (Carl Zeiss). Images were processed and blindly analyzed by using the ImageJ64 (v 1.47) software.
4
Flow Cytometry Analysis of Pluripotency Markers
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Cells were treated with 0.25% Trypsin and diluted with 10% KOSR before passing through 40 μm cell strainer. Single cells were then washed three times with DPBS and fixed with 4% paraformaldehyde on ice for 20 min. The cells were blocked on ice for 30 min with 10% KOSR in DPBS with or without 0.1% Triton-X for intracellular or surface marker staining respectively. For surface marker staining, the cells were incubated with EPCAM-PE (Biolegend, 1:100) for 30 min and proceeded with blocking with 10% KOSR in DPBS supplemented with 0.1% Triton-X for another 30 min. The cells were then co-stained with SOX17 (R&D Systems, 1:100) before secondary antibody staining with Alexa Fluor® 488 Donkey anti-goat IgG (ThermoFisher Scientific, 1:500) for 1 h at 4°C for each condition. For co-staining of SOX17 (R&D System, 1:100) and EOMES (Abcam, 1:100), cells were incubated with primary antibodies on ice for 1 h before secondary antibody staining. For controls, cells were stained with Mouse IgG PE, Mouse IgG2a APC (BD Pharmingen) and Alexa Fluor® 488 (ThermoFisher Scientific). Cells were washed three times and diluted with DPBS for flow cytometry (LSR II, BD Biosciences). Flow cytometry data was analyzed using FlowJo_V10. Primary antibodies used are listed in Table S7 .
5
Modulation of γδ T Cell Epigenetics
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1 × 106 freshly isolated γδ T cells were cultured with HDMAPP (1 nM), rIL-2 (50 IU/ml), and with or without HDAC inhibitors at the given concentrations VPA (2, 1, 0.5 mM), TSA (100, 50, 25 nM), and SAHA (1, 0.5, 0.25 µM) at 37°C. After 72 h of treatment, cells were harvested and whole cell lysates were prepared with SDS lysis buffer (1 M Tris–HCl pH 6.8, 10%w/v SDS, glycerol, β-mercaptoethanol, 1M DTT, and bromophenol blue). 10% Polyacrylamide gels were used to resolve the protein samples and transferred to Hybond-ECL nitrocellulose membrane (Amersham Pharmacia Biotech, Piscataway, NJ, USA). The primary antibodies to T-bet (1:1,000) (Cell Signalling Technology), p21 (1:1,000) (Abcam), Eomes (1:1,000) (Abcam), p53 (1:500) (Santa Cruz), NF-κB (1:1,000) (Abcam), total H3 (Abcam) (1:1,000), total H4 (Abcam) (1:1,000), acetyl H3 (Abcam) (1:1,000), acetyl H4 (Abcam) (1:1,000), and β-actin (1:4,000) (Sigma-Aldrich) as loading control were added at different dilution. Immunostaining was performed using appropriate secondary antibody at a dilution of 1:10,000 and developed with ECL plus Western blot detection system (Amersham Pharmacia).
6
Western Blot Analysis of Cellular Proteins
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Experiments were performed as described (42 (link)). Briefly, cells were lysed in lysis buffer and cell lysate containing 30 μg protein was used for Western blots. Membranes were incubated with the primary antibody against EOMES (Cat No. ab23345, Abcam, 1:1000), FAM46B (Cat No. 23149-1-AP, Proteintech, 1:1000), GAPDH (Cat No. sc-47724, Santa Cruz Biotechnology, 1:5000), β-Tubulin (Cat No. 30303ES50, YEASEN, 1:1000) and Laimin B1 (Cat No. 16048, Abcam, 1:1000). The membranes were then incubated with IRDye 680LT Donkey anti-Rabbit IgG (Cat No. 926-68023, Li-COR Biosciences) or IRDye 800CW Donkey anti-Mouse IgG (Cat No. 926-32212, Li-COR Biosciences) as secondary antibodies and visualized on an Odyssey Infrared Imager (Li-COR Biosciences).
7
Immunofluorescence Staining of Neural Markers
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Antibodies were used at the following dilutions: rabbit anti-Pax6 (Biolegend, polyclonal, 1:300); Rat anti-CTIP2 (Abcam, clone 25B6, 1:300); Rat anti-tbr2 (Invitrogen, Eomes clone Dan11mag, 1:100); mouse anti-Satb2 (Abcam, clone SATBA4B10,1:50); Mouse anti-Neun (Millipore, clone A60, 1:200); and Rabbit anti-Tbr1 (Abcam, polyclonal, 1:100). Anti-Rabbit, Mouse, or Rat conjugated with Alexa Fluor 488 or Alexa Fluor 647 fluorophore secondary antibodies (Life Technologies) were used at 1:500 in PBTA solution.
8
Immunoblot Analysis of Cell Signaling
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Immunoblots were performed as described previously38 (link),77 (link). Briefly, cells were harvested and pellets were lysed and prepared via boiling for 15 min in 1X loading dye (50 mM Tris [pH 6.8], 100 mM DTT, 2% SDS, 0.1% bromophenol blue, 10% glycerol). Lysates were separated by SDS-PAGE on 10% Bis-Tris Plus Bolt gels (ThermoFisher) and transferred onto 0.45 µm nitrocellulose membrane. Membranes were blocked with 2% nonfat dry milk in 1X TBST (10 mM Tris [pH 8], 150 mM NaCl, 0.05% Tween-20), and detection of indicated proteins was carried out using the following antibodies: Aiolos (39293, Active Motif, 1:20,000), Bcl-6 (clone K112, BD Biosciences, 1:500), pSTAT5(Y694/9) (clone 47 BD Biosciences, 1:5000), STAT5 (clone (D206Y, Cell Signaling, 1:5000), β-Actin (GenScript, 1:15,000), Eomes (Abcam, 1:5,000) goat anti-mouse:HRP (Jackson Immunoresearch, 1:5,000-1:30,000), mouse anti-rabbit:HRP (Santa Cruz, 1:5000-1:20,000).
9
Immunocytochemistry Protocol for Stem Cell Markers
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Cells were fixed in 4% paraformaldehyde (PFA; Sigma-Aldrich) for 15 min at room temperature. They were
permeabilized with 0.2% Triton X-100 (Sigma-Aldrich) for 10 min, blocked with 5% goat serum (Sigma-Aldrich) in
PBS and incubated overnight with primary antibodies at 4ºC. The primary antibodies were detected after
incubation with secondary antibodies for 45 min at room temperature. The primary antibodies used in this study
were against OCT-4 (Abcam, Cambridge, MA, USA; 1:200), SSEA-4 (Abcam; 1:200), TRA-1-60 (Chemicon, Temecula,
CA, USA; 1:100), TRA-1-81 (Chemicon; 1:100), Nestin (Chemicon; 1:200), Brachyury (Abcam; 1:200),
alpha-fetoprotein (AFP, Chemicon; 1:200), CDX2 (Abcam; 1:200) and EOMES (Abcam; 1:200). The secondary
antibodies used in this study were FITC conjugated- goat anti-rabbit (Abcam; 1:200), FITC conjugated- rabbit
anti-goat (Abcam; 1:200), Alexa Fluor 568 goat anti-rabbit (Life Technologies; 1:500) and Cy3 conjugated- goat
anti-mouse (Chemicon; 1:200). Cells were counterstained for nuclei with the 4'-6-diamidino-2-phenylindole
(DAPI; Sigma-Aldrich; 1:1000).
permeabilized with 0.2% Triton X-100 (Sigma-Aldrich) for 10 min, blocked with 5% goat serum (Sigma-Aldrich) in
PBS and incubated overnight with primary antibodies at 4ºC. The primary antibodies were detected after
incubation with secondary antibodies for 45 min at room temperature. The primary antibodies used in this study
were against OCT-4 (Abcam, Cambridge, MA, USA; 1:200), SSEA-4 (Abcam; 1:200), TRA-1-60 (Chemicon, Temecula,
CA, USA; 1:100), TRA-1-81 (Chemicon; 1:100), Nestin (Chemicon; 1:200), Brachyury (Abcam; 1:200),
alpha-fetoprotein (AFP, Chemicon; 1:200), CDX2 (Abcam; 1:200) and EOMES (Abcam; 1:200). The secondary
antibodies used in this study were FITC conjugated- goat anti-rabbit (Abcam; 1:200), FITC conjugated- rabbit
anti-goat (Abcam; 1:200), Alexa Fluor 568 goat anti-rabbit (Life Technologies; 1:500) and Cy3 conjugated- goat
anti-mouse (Chemicon; 1:200). Cells were counterstained for nuclei with the 4'-6-diamidino-2-phenylindole
(DAPI; Sigma-Aldrich; 1:1000).
10
Immunohistochemical Analysis of Organoid Cultures
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Organoids were randomly selected and fixed in 4% PFA in PBS for 2–4 hours. The organoids were then cryopreserved in 25% sucrose overnight, embedded in O.C.T. (Sakura), and frozen on dry ice before being stored at −80°C. Serial cryosections were obtained with a thickness of 12–16 µm. Immunostaining was performed by incubating the sections in blocking solution (PBS, 10% Donkey Serum, 1% Triton-100) for 1 hour, followed by incubation with primary antibodies (overnight, 4°C) and secondary antibodies (1–2 hours, from Jackson ImmunoResearch or ThermoFisher Scientific). The slides were then mounted with coverslips using VECTASHIELD (Vector Labs) and imaged on a Zeiss microscope equipped with an apotome module and ZEN 3.3 (ZEN pro) software. Three cell lines were used for immunocytochemical analyses, and a minimum of four organoids per line were analyzed. Images were acquired randomly to cover the entire extent of the organoid. Antibody list: FOXG1 (rabbit, 1:200, Takara) and PAX6 (mouse, 1:200, BD Bioscience), EOMES (rabbit, 1:1000, Abcam), FOXP2 (goat, 1:200, Santa Cruz), GAD1 (mouse, 1:200, Chemicon), HuC/D (mouse, 1:200, Invitrogen), SOX1 (goat, 1:100, R&D Systems), CTIP2 (rat, 1:500, Abcam).
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