Cells were fixed in phosphate buffered saline (PBS) containing 4% paraformaldehyde for 15 min at room temperature, washed with PBS, and treated with PBS containing 0.1% Triton X-100 and 5% goat or donkey serum for 30 min at room temperature. Thereafter, cells were stained with the following primary antibodies for 1 h at room temperature or overnight at 4°C: anti-NANOG (1:100, R&D Systems), anti-TRA-1-81 (1:100, Millipore), anti-cardiac troponin T (cTnT; 1:200, Thermo Scientific), and anti-dystrophin (1:100, MANDRA1, Sigma-Aldrich). Next, cells were stained with the following secondary antibodies for 1 h at room temperature: Cy3-conjugated donkey anti-mouse IgM (1:500, Jackson ImmunoResearch), Alexa Fluor 488-conjugated donkey anti-goat IgG (1:500, Invitrogen), Alexa Fluor 568-conjugated donkey anti-mouse IgG (1:500, Invitrogen) and a Zenon Alexa Fluor 488 labelling kit (Invitrogen). Nuclei were counterstained with 10 μg/mL Hoechst 33342 (Invitrogen). Images were acquired using a fluorescence microscope (BZ-X700 or BZ-X710, Keyence). Confocal images were captured by TCS SP8 confocal microscope (Leica Microsystems).
Anti nanog
Manufactured by R&D Systems
Sourced in United States
Anti-NANOG is a polyclonal antibody that recognizes the NANOG protein. NANOG is a transcription factor that plays a critical role in the self-renewal of undifferentiated embryonic stem cells. The Anti-NANOG antibody can be used for the detection and analysis of NANOG expression in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti oct4, by Santa Cruz Biotechnology (5 mentions)
Anti tra 1 81, by Merck Group (5 mentions)
Anti ssea 4, by Developmental Studies Hybridoma Bank (3 mentions)
Tuj1 anti tubulin beta 3 isoform, by Merck Group (3 mentions)
Anti musashi, by Merck Group (3 mentions)
Anti sma, by Agilent Technologies (3 mentions)
Anti nestin, by R&D Systems (3 mentions)
Anti afp, by Agilent Technologies (3 mentions)
Anti map2, by Merck Group (3 mentions)
Anti sox2, by Merck Group (3 mentions)
Anti sox2, by Cell Signaling Technology (2 mentions)
Ab100938, by Merck Group (2 mentions)
Anti ubh2a, by Merck Group (2 mentions)
Anti ubh2b, by Merck Group (2 mentions)
Anti h3, by Abcam (2 mentions)
Anti gapdh, by Merck Group (2 mentions)
Anti ssea4, by Merck Group (2 mentions)
Anti tbr1, by Abcam (2 mentions)
Anti ctip2, by Abcam (2 mentions)
At8 anti p tau, by Thermo Fisher Scientific (2 mentions)
10 protocols using anti nanog
1
Immunofluorescence Staining of Stem Cells
2
Isolation and Characterization of Embryonic Stem Cells from Usp16 Mutant Mice
3
Immunophenotyping of Pluripotent Stem Cells
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The cells were fixed with 4% paraformaldehyde, and treated with PBS containing 5% normal goat or donkey serum, 1% BSA, and 0.2% TritonX-100. The following antibodies were used: SSEA3 (1:10), TRA-1-81 (1:50), TRA-1-60 (1:50) (these antibodies were used at Kyoto University and were kind gifts from Dr. Peter W. Andrews), anti-SSEA4, anti-TRA-1-81, anti-TRA-1-60 (1:500, all contained in the ES Cell Characterization Kit from Merck Millipore; these antibodies were used at Gifu University), anti-NANOG (1:20, R&D Systems), anti-OCT3/4 (1:1000, Santa Cruz Biotechnology), anti-βIII-tubulin (1:200, Cell Signal Technology), anti-βIII-tubulin (1:2000, Covance), anti-α-SMA (1:500, DAKO), and anti-AFP (1:100, R&D). Nuclei were stained using 1 μg/mL Hoechst33342 (Life Technologies).
4
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).
5
Immunophenotyping of ECFCs and iPSCs
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Cells (ECFCs, ECFC-derived iPSCs) were fixed in 4% PFA/1X PBS (Electron Microscopy Sciences, ref 15714–5) for 10min at room temperature (RT) and washed with 1X PBS. For intracellular staining, cells were permeabilized with 0.2% Triton X100/PBS for 10min at RT. Cells were incubated overnight at +4°C with the following antibodies: anti-Phosphatase Alkaline (PA) (1:40, R&D Systems), anti-OCT3/4 (1:40, R&D Systems), anti-NANOG (1:40, R&D Systems), anti-SSEA4 (1:40, R&D Systems), anti-Tra-160/Tra1-81 (1:200, Abcam), anti-SOX2 (1:40, R&D Systems,), anti-CD31 (1:20, BD Pharmingen, clone WM59) or anti-von Willebrand factor (vWF) (1:300, Dako Cytomation A0082), diluted in 3% BSA/PBS and labeled with Alexa Fluor 488 and 546 Donkey Anti-Goat IgG or Alexa Fluor 488 and 546 Goat anti-Mouse IgG secondary antibodies (Invitrogen, Cergy-Pontoise, France). Cells were then stained with 40,6-diamidino-2-phenylindole (DAPI) and examined with a DMR fluorescence microscope (Leica, Rueil Malmaison, France) equipped with a CoolSnap HQ2 camera (Photometrics, Tucson, AZ) controlled by MetaVue Analyzing Software (Molecular Devices LLC, Sunnyvale, CA).
6
Isolation and Characterization of Embryonic Stem Cells from Usp16 Mutant Mice
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Blastocysts were flushed out from the uterus of Usp16+/− or Usp162lox/+:CAGG-Cre E3.5 female mice with a 27.5 gauge needle connected to 1 ml syringe filled with 1 ml 2i medium45 (link). Blastocysts were then transferred to feeder cell plates with 200 μl sterile tip and cultured with 2i medium46 (link) or regular ESC medium for 5–8 days. ESC-like colonies formed from ICM outgrowths were handpicked using sterile pipette tips and dissociated by 0.25% trypsin and expanded in ESC medium. For proliferation rate assay, 1 × 104 ESCs were seeded in 12 well plates and cell numbers were counted and the same seeding procedure was repeated every other day. Western blot assay and qPCR were performed using standard protocol included in the method section47 (link). Antibodies include anti-Usp16 (1:1000)26 (link), anti-ubH2A (Millipore, 05-678, 1:1000), anti-ubH2B (Millipore, 05-1312, 1:1000), anti-H3 (Abcam, ab100938, 1:5000), anti-GAPDH (Sigma, G9545, 1:5000), Anti-Nanog (R&D Systems, AF2729, 1: 2000), Anti-Oct4 (Santa Cruz Biotechnology, SC9081, 1:2000), and anti-Sox2 (Cell signaling, 3579, 1:2000). Alkaline phosphatase substrate kit was from Vector laboratory (SK-5100).
7
Immunofluorescence Staining of Stem Cells
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Cells were fixed with 3.7% formaldehyde in phosphate-buffered saline (PBS). After permeablisation with 0.1% to 0.2% Triton X-100/PBS, cells were incubated with a primary antibody, followed by staining with a secondary antibody conjugated with AlexaFluor488 (1:500) or AlexaFluor555 (1:500). The following primary antibodies were used in this study: anti-SSEA4 (1:250; Merck Millipore, Darmstadt, Germany), anti-OCT4 (1:400; Abcam, Cambridge, UK), anti-NANOG (1:20; R&D Systems, Minneapolis, MN, USA), anti-TRA-1-60 (1:250; e-Bioscience, Santa Clara, CA, USA), anti-β-III Tubulin (1:1,000; BioLegend, San Diego, CA, USA), anti-SOX17 (1:200; Abcam), anti-smooth muscle actin (1:250; Dako, Santa Clara, CA, USA), anti-Desmin (1:200; Thermo Fisher Scientific), anti-MAP2 (1:500; Santa Cruz, Dallas, TX, USA), and anti-Synapsin I (1:500; Abcam). Nuclei were counterstained with DAPI using VECTASHIELD mounting medium with DAPI (Vector Laboratories, Burlingame, CA, USA).
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
Immunofluorescence Characterization of Stem Cells
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Immunofluorescence on coverslip cultures was performed according to previous reports (Yan et al., 2005 (link)). Cells were fixed in 4% paraformaldehyde in PBS for 20 min and blocked in blocking solution (PBS containing 10% normal donkey serum and 0.2% Triton X-100) for 1 h. The following primary antibodies were used: mouse anti-SSEA-3 (1:200), mouse anti-SSEA-4 (1:400), mouse anti-Nkx6.1 (1:100), mouse anti-En1 (1:100) (DSHB, Iowa City, USA), anti-TRA-1-60 (1:150), anti-TRA-1-81 (1:150), mouse anti-Synaptophysin (1:1000), rabbit anti-v-Glut1 (1:2000) (Millipore, Billerica, USA), anti-Nanog (1:150), goat anti-Otx2 (1:500) (R&D), mouse anti-Tuj1 (1:5000) (Abcam, Cambridge, UK), rabbit anti-MAP2 (1:1000), mouse anti-TH (1:1000) (Sigma), mouse anti-HB9 (1:50) and goat anti-FoxA2 (1:500) (Santa Cruz, California, USA). After an overnight incubation at 4°C, species-specific secondary antibodies conjugated with Alexa Fluor 488 or 594 or CY5 (1:1000) (Invitrogen) were applied for 1 h at room temperature. Cell nuclei were stained with DAPI (Sigma). Images were obtained using a Leica TCS SP8 confocal laser-scanning microscope (Leica, Wetzlar, Germany).
10
Immunostaining Protocols for Stem Cell Characterization
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All neurons were prepared by the methods described in a previous report [23 (link)]. The following primary antibodies were used: anti-OCT4 (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-SOX2 (1:200, Millipore, Burlington, MA, USA), anti-NANOG (1:200, R&D Systems), anti-SSEA-4 (1:100, Developmental Studies Hybridoma Bank, Iowa City, IA, USA), anti-TRA-1-81 (1:100, Thermo Fisher Scientific), Tuj1 anti-tubulin beta III isoform (1:200, Millipore), Tuj1 anti-tubulin beta III (1:200, Abcam, Cambridge, UK), anti-SMA (1:100, DAKO, Santa Clara, CA, USA), anti-AFP (1:100, DAKO), anti-Nestin (1:200, R&D Systems), anti-Musashi (1:200, Millipore), anti-MAP2 (1:200,Millipore), anti-TDP43 (1:200, Proteintech, Rosemont, IL, USA), anti-FUS/TLS (1:200, Santa Cruz Biotechnology), p-Tau (AT8), and anti-Caspase-3 (1:100, Millipore).
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