IPSC lines were rinsed with 1× PBS, fixed with 4% paraformaldehyde, and stored in PBS for staining. Cells were permeabilized with PBST (PBS supplemented with 0.1% Triton X-100) for 10 min at room temperature. Non-specific antigens were then blocked by incubating the cells with PBST+BSA at room temperature for 1 hr. Cells were incubated overnight at 4°C in PBS-BSA containing 10% donkey serum and the following specific primary antibodies: 1:500 anti-human OCT4 (Cell Signaling Technologies), 1:500 anti-human NANOG (Cell Signaling Technologies), 1:500 anti-human SSEA4 (Cell Signaling Technologies),), 1:500 anti-TRA-1–60 (Cell Signaling Technologies), 1:500 anti-SOX2 (Cell Signaling Technologies), and 1:200 anti-Lin28 (R&D Systems). Following 3 washes with PBS, cells were incubated with appropriate Alexa Fluor conjugated secondary antibodies at 1:200 dilution. After 3 washes with PBS, the samples were incubated for 10 min with DAPI (1 µg/mL) in PBS, followed by a final wash in PBS. Fluorescence images were captured at 10× or 20× on a Zeiss LSM 710/780 confocal microscope and processed using ImageJ.
Anti human nanog
Manufactured by Cell Signaling Technology
Anti-human NANOG is a laboratory reagent used to detect the NANOG protein, which is a key transcription factor involved in the regulation of embryonic stem cell self-renewal and pluripotency. This antibody can be used in various immunoassay techniques to identify and quantify the NANOG protein in human samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti human oct4, by Cell Signaling Technology (2 mentions)
Anti human ssea4, by Cell Signaling Technology (2 mentions)
Anti lin28, by R&D Systems (2 mentions)
Lsm 710 780 confocal microscope, by Zeiss (2 mentions)
Anti tra 1 60, by Cell Signaling Technology (2 mentions)
Anti sox2, by Cell Signaling Technology (2 mentions)
Donkey serum, by Jackson ImmunoResearch (1 mentions)
Anti human ssea4, by Abcam (1 mentions)
Eclipse te2000 u, by Olympus (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Anti tra 1 60, by Merck Group (1 mentions)
Biorevo bz 9000 fluorescence microscope, by Keyence (1 mentions)
Anti ssea4, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Dojindo Laboratories (1 mentions)
Anti human smooth muscle actin, by Agilent Technologies (1 mentions)
Fitc labeling kit nh2, by Dojindo Laboratories (1 mentions)
Anti tra 1 81, by Merck Group (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Anti oct3 4, by Santa Cruz Biotechnology (1 mentions)
Anti α fetoprotein, by R&D Systems (1 mentions)
4 protocols using anti human nanog
1
Characterization of Induced Pluripotent Stem Cells
2
Characterization of Induced Pluripotent Stem Cells
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IPSC lines were rinsed with 1× PBS, fixed with 4% paraformaldehyde, and stored in PBS for staining. Cells were permeabilized with PBST (PBS supplemented with 0.1% Triton X-100) for 10 min at room temperature. Non-specific antigens were then blocked by incubating the cells with PBST+BSA at room temperature for 1 hr. Cells were incubated overnight at 4°C in PBS-BSA containing 10% donkey serum and the following specific primary antibodies: 1:500 anti-human OCT4 (Cell Signaling Technologies), 1:500 anti-human NANOG (Cell Signaling Technologies), 1:500 anti-human SSEA4 (Cell Signaling Technologies),), 1:500 anti-TRA-1–60 (Cell Signaling Technologies), 1:500 anti-SOX2 (Cell Signaling Technologies), and 1:200 anti-Lin28 (R&D Systems). Following 3 washes with PBS, cells were incubated with appropriate Alexa Fluor conjugated secondary antibodies at 1:200 dilution. After 3 washes with PBS, the samples were incubated for 10 min with DAPI (1 µg/mL) in PBS, followed by a final wash in PBS. Fluorescence images were captured at 10× or 20× on a Zeiss LSM 710/780 confocal microscope and processed using ImageJ.
3
Immunofluorescence Staining of Stem Cells
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Cell lines were rinsed with 1X PBS, fixed with 4 % paraformaldehyde (Santa Cruz, #sc-281692) in PBS for 5 min at room temperature. Nonspecific binding sites were blocked by incubation with PBST (PBS supplemented with 0.1 % Triton X-100) containing 10 % donkey serum (Jackson Labs, #017-000-1210) for 30 min at room temperature. Cells were subsequently incubated overnight at 4 °C in PBST containing 10 % donkey serum and specific primary antibodies: 1:500 anti-Human OCT4 (Stemgent, #09-0023), 1:100 anti-human NANOG (Cell Signaling Technologies, #4903), 1:250 anti-human SSEA4 (Abcam, #ab16287), 1:500 anti-TRA-1-60 (Life Technologies), 1:200 anti cTnT, 1:200 anti-ACTIN, 1:200 anti-SOX1, 1:200 anti-SOX2, 1:200 anti-NESTIN, 8 ng/ml anti-SOX17, 10 ng/ml anti-ALB, 1:500 anti-Sendai Vector (MBL, #PD029). Following a 3 times wash with PBS, cells were incubated with one of the following secondary antibodies: Alexa Fluor® 488 donkey anti-Mouse (#A-21202; 1:1000 dilution), Alexa Fluor® 555 donkey anti-goat IgG and Alexa Fluor® 555 donkey anti-rabbit IgG (#A-21428; 1:1000 dilution). After washing 3 times with PBS, the samples were incubated for 10 min with Hoechst (1 μg/ml) in PBS, followed by a final wash in PBS. Fluorescence images were captured with the Celigo, Nikon Eclipse TE 2000-U or Olympus BX41 fluorescent microscopes.
4
Immunocytochemical Characterization of Stem Cells
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Immunocytochemical analysis was performed as described previously [16 (link)–18 (link)]. The following primary antibodies were used in this study: anti-SSEA4 (1:300 dilution; Millipore), anti-TRA-1-60 (1:300 dilution; Millipore), anti-TRA-1-81 (1:300 dilution; Millipore), anti-Oct-3/4 (1:300 dilution; Santa Cruz Biotechnology), anti-human Nanog (1:800 dilution; Cell Signaling Technology), anti-Class IIIβ-tubulin (TUJ1; 1:500; Covance), anti-human smooth muscle actin (SMA; 1:50; DAKO), and anti-α-fetoprotein (1:100; R&D systems). Cells were incubated with a primary antibody diluted in 1% bovine serum albumin (BSA) and 5% serum containing PBS at 4°C overnight. Secondary staining was performed with an appropriate secondary antibody-conjugated to Alexa Fluor 488 or Alexa Fluor 594 (1:300; Life technologies) for 1 h at room temperature. Lectin staining was performed as described previously [19 (link),20 (link)]. Briefly, rBC2LCN (Wako) was fluorescent-labeled using FITC Labeling Kit-NH2 (Dojindo) according to the manufacturer’s instruction. Next, 10 μg/mL of FITC-conjugated rBC2LCN in 1% BSA containing PBS was used for staining of 4% paraformaldehyde-fixed cells for 1 h at room temperature. Cells were counterstained with DAPI (Dojindo). Images were collected with a BIOREVO BZ-9000 fluorescence microscope (Keyence).
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