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Rabbit anti nanog

Manufactured by Santa Cruz Biotechnology

Rabbit anti-NANOG is a primary antibody that specifically recognizes the NANOG protein, a transcription factor that plays a crucial role in the maintenance of embryonic stem cell pluripotency and self-renewal. This antibody can be used in various immunoassay techniques to detect and quantify NANOG expression in samples.

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10 protocols using rabbit anti nanog

1

Immunofluorescent Staining of iPSC Markers

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Immunofluorescent staining was used to examine stem cell markers in iPSC colonies [50 (link)]. Briefly, cells were fixed by 4% paraformaldehyde/PBS for 10–15 min and rinsed with PBS, then permeabilized and blocked with 0.1% Triton X-100/PBS containing 3% BSA for 30 min. After incubation with primary antibodies for 1 h at room temperature or overnight at 4C, the samples were washed three times with PBS and then incubated with secondary antibody for 1 h. The following antibodies were used in the immunostaining: rabbit anti-NANOG (1:100 dilution, Santa Cruz) and rabbit anti-OCT4 (1:100 dilution, Millipore). The cell samples were subsequently incubated with Cy3- or Alexa Fluor 488-labeled secondary antibodies for 1 h. After washing three times with PBS, samples were counterstained with Hoechst 33258 (Invitrogen). Alternatively, the pluripotency of stem cells was examined by Fluorescent Mouse ES/iPS Cell Characterization kit (Cat.#SCR077, Millipore, MA) following the protocol provided by the manufacturer. Fluorescence images were acquired with a Zeiss AxioCam Camera.
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2

iPSC Pluripotency Marker Immunostaining

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Immunofluorescent staining was used to examine stem cell markers in iPSC colonies15 (link). Cells were briefly rinsed by freshly prepared PBS, fixed by freshly made 4% paraformaldehyde for 10min at RT, washed three times in PBS for 5 min each, permeabilized with freshly made 0.5% v/v Triton X-100/PBS on ice for 5min, then blocked in 1% w/v BSA for 30min at RT. After incubation with primary antibodies diluted in 1% BSA (2ug/ml 1:500) for 1-3h at RT, the samples were washed three times in PBS for 5 min each, incubated with secondary antibody for 1h at RT. The following antibodies were used in the immunostaining: rabbit anti-NANOG (1:100 dilution, Santa Cruz). The cell samples were subsequently incubated with Cy3 or Alexa Fluor 488 labeled secondary antibodies for 1 hour. After washing three times with PBS, samples were counterstained with Hoechst 33258 (Invitrogen). Alternatively, the pluripotency of stem cells was examined by Fluorescent Mouse ES/iPS Cell Characterization kit (Cat.#SCR077, Millipore, MA) following the protocol provided by the manufacturer. Fluorescence images were acquired with a Zeiss AxioCam Camera.
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3

Immunocytochemistry and Western Blot Protocols

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Primary antibodies used for immunocytochemistry or Western blot include mouse anti-GAPDH (Millipore), mouse anti-Nestin (Millipore), mouse anti-Tuj1/βIII-tubulin (Millipore), mouse anti-Tuj1/βIII-tubulin (Santa Cruz Biotenchnology), mouse anti-TRA-1-60 (Millipore), mouse anti-TRA-1-81 (Millipore), goat anti-Lin28a (R&D Systems), rabbit anti-Nanog (Santa Cruz Biotechnology), rabbit anti-Oct3/4 (Santa Cruz Biotechnology), rabbit anti-DCX44 (link), goat anti-DCX (Santa Cruz Biotechnology), goat anti-SOX2 (Santa Cruz Biotechnology), rabbit anti-PAX6 (Covance), mouse anti-MAP2 (Millipore), mouse anti-BLBP (Abcam), rabbit anti-GFAP (Sigma), rabbit anti-OLIG2 (Abcam), mouse anti-β-Catenin (BD), rabbit anti-Arp3 (Santa Cruz Biotechnology), mouse anti-β-actin (Sigma), rabbit anti-EIF3D (Bethyl Labs), rat anti-αN-catenin obtained from the NIH NICHD Developmental Studies Hybridoma Bank (Catalog: NCAT2, deposited by Masatoshi Takeichi and Shinji Hirano). Fluorophore conjugated secondary antibodies were purchased from Invitrogen, and immunohistochemistry was performed with ABC Elite peroxidase staining kit, using ImmpactDAB as a substrate.
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4

Immunofluorescence Staining of Human iPSCs

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Human iPSC colonies grown in Matrigel-coated 8-well chamber glasses (Thermo Scientific) were fixed using 4% paraformaldehyde (PFA) and permeabilized with 0.5% Triton X-100. After blocking samples with 5% goat serum in PBST (PBS with 0.1% Tween20), cells were stained with mouse anti-SSEA4 (R&D systems), rabbit anti-OCT3/4 (Santa Cruz Biotechnology), rabbit anti-NANOG (Santa Cruz Biotechnology), and mouse anti-SOX2 (R&D systems). Cells were then incubated with Alexa Fluor-conjugated secondary antibodies (Life Technologies) and Hoechst 33342 (Life Technologies) to visualize the specific stains. Image acquisition was performed on an Eclipse 80i fluorescence microscope (Nikon Instruments).
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5

Immunofluorescence Staining of Cultured Cells

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The cultured cells were fixed in 4% paraformaldehyde for 20 min at room temperature, subsequently washed with PBS and treated with 0.3% Triton X-100(Sigma) in PBS for 15 min. Then the samples were incubated with PBS containing 2% bovine serum albumin (BSA) at 37°C for 10 min. Incubation with the primary antibodies(mouse anti-c-kit, Santa Cruz, 1:200; Rabbit anti-vimentin, Abcam, 1:100; Rabbit anti-CD34, Bioss, 1:300; Rabbit anti-Nanog, Santa Cruz, 1:100; Rabbit anti-sca-1, Millipore,1:200) was performed at 4°C overnight. The samples were subsequently incubated FITC- or Cy3-conjugated secondary antibodies (Beyotime). The nuclei were counterstained with 1μg /ml DAPI (Roche). Negative controls were obtained by following all the same protocol but the primary antibodies. All experiments were performed in triplicate.
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6

Immunocytochemical Characterization of Pluripotent Stem Cells

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Human ESC and iPSC colonies grown in Matrigel-coated 8-well chamber glasses (Thermo Scientific) were fixed using 4% paraformaldehyde (PFA) and permeabilized with 0.5% Triton X-100. After blocking samples with 5% goat serum in PBST (PBS with 0.1% Tween20), cells were stained with mouse anti-SSEA4 (R&D systems), rabbit anti-OCT3/4 (Santa Cruz Biotechnology), rabbit anti-NANOG (Santa Cruz Biotechnology), and mouse anti-TRA1-60 (EMD Millipore) antibodies. Cells were then incubated with AlexaFluor-conjugated secondary antibodies (Life Technologies) and Hoechst 33342 (Life Technologies) to visualize the specific stains. Image acquisition was performed on an Eclipse 80i fluorescent microscope (Nikon Instruments).
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7

Pluripotency Marker Immunofluorescence in hESCs

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hESCs were fixed with 4% paraformaldehyde for 20 minutes and then permeabilized with 0.1% Triton X-100 in PBS for 5 min. After a treatment with 5% normal goat serum for 30 min, the cells were incubated with primary antibodies against one of the pluripotency markers mouse anti-Oct-4 (Santa Cruz Biotechnology Inc., CA, 1:250) and rabbit anti-Nanog (Santa Cruz Biotechnology Inc, 1:250), for 18 h at 4°C. The cells were then washed 3 times with PBS and incubated with Alexa Fluor 488-conjugated secondary antibodies (Invitrogen, NY, 1:1000) for 1 h. After washing 3 times with PBS, DAPI (Invitrogen) staining was performed for 5 minutes. The samples were immediately imaged using epi-fluorescence microscopes (Nikon TE 2000-U, Nikon, Japan).
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8

Immunofluorescence Staining of Human iPSCs

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Human iPSC colonies grown in Matrigel-coated 8-well chamber glasses (Thermo Scientific) were fixed using 4% paraformaldehyde (PFA) and permeabilized with 0.5% Triton X-100. After blocking samples with 5% goat serum in PBST (PBS with 0.1% Tween20), cells were stained with mouse anti-SSEA4 (R&D systems), rabbit anti-OCT3/4 (Santa Cruz Biotechnology), rabbit anti-NANOG (Santa Cruz Biotechnology), and mouse anti-SOX2 (R&D systems). Cells were then incubated with Alexa Fluor-conjugated secondary antibodies (Life Technologies) and Hoechst 33342 (Life Technologies) to visualize the specific stains. Image acquisition was performed on an Eclipse 80i fluorescence microscope (Nikon Instruments).
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9

Immunocytochemistry and Western Blot Protocols

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Primary antibodies used for immunocytochemistry or Western blot include mouse anti-GAPDH (Millipore), mouse anti-Nestin (Millipore), mouse anti-Tuj1/βIII-tubulin (Millipore), mouse anti-Tuj1/βIII-tubulin (Santa Cruz Biotenchnology), mouse anti-TRA-1-60 (Millipore), mouse anti-TRA-1-81 (Millipore), goat anti-Lin28a (R&D Systems), rabbit anti-Nanog (Santa Cruz Biotechnology), rabbit anti-Oct3/4 (Santa Cruz Biotechnology), rabbit anti-DCX44 (link), goat anti-DCX (Santa Cruz Biotechnology), goat anti-SOX2 (Santa Cruz Biotechnology), rabbit anti-PAX6 (Covance), mouse anti-MAP2 (Millipore), mouse anti-BLBP (Abcam), rabbit anti-GFAP (Sigma), rabbit anti-OLIG2 (Abcam), mouse anti-β-Catenin (BD), rabbit anti-Arp3 (Santa Cruz Biotechnology), mouse anti-β-actin (Sigma), rabbit anti-EIF3D (Bethyl Labs), rat anti-αN-catenin obtained from the NIH NICHD Developmental Studies Hybridoma Bank (Catalog: NCAT2, deposited by Masatoshi Takeichi and Shinji Hirano). Fluorophore conjugated secondary antibodies were purchased from Invitrogen, and immunohistochemistry was performed with ABC Elite peroxidase staining kit, using ImmpactDAB as a substrate.
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10

Immunocytochemical Characterization of Pluripotent Stem Cells

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Human ESC and iPSC colonies grown in Matrigel-coated 8-well chamber glasses (Thermo Scientific) were fixed using 4% paraformaldehyde (PFA) and permeabilized with 0.5% Triton X-100. After blocking samples with 5% goat serum in PBST (PBS with 0.1% Tween20), cells were stained with mouse anti-SSEA4 (R&D systems), rabbit anti-OCT3/4 (Santa Cruz Biotechnology), rabbit anti-NANOG (Santa Cruz Biotechnology), and mouse anti-TRA1-60 (EMD Millipore) antibodies. Cells were then incubated with AlexaFluor-conjugated secondary antibodies (Life Technologies) and Hoechst 33342 (Life Technologies) to visualize the specific stains. Image acquisition was performed on an Eclipse 80i fluorescent microscope (Nikon Instruments).
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