Unfixed tumors were embedded in OCT (Tissue Tek). Frozen sections were cut to a thickness of 10μm on a Leica cryostat and mounted on SuperFrost Plus slides (Fisher). Slides were air-dried for 10min, then fixed for 10min with 4% formaldehyde, rinsed with PBS, permeabilized with 0.5% Triton X-100 in PBS for 15min, then blocked for 1h (5% normal donkey serum, 1% BSA, 0.3% Triton X-100 in PBS) and incubated in primary antibody diluted in blocking buffer at 4°C overnight. After washing with PBS, secondary antibodies, conjugated to Alexa 488, Alexa FITC, Alexa 568, DyLight 649, and Hoechst 33342 (83218, AnaSpec) were diluted in blocking buffer and incubated with the slides for 1hr at room temperature (RT). After washing, slides were mounted in ProLong Gold (Invitrogen). Imaging was performed on a Nikon Eclipse TiE Microscope or a Zeiss LSM780 Confocal Microscope. Images have been analyzed in NIS-Elements, Zen software or ImageJ. Antibodies for immunofluorescence were CD49f (1:200; Biolegend, 313618), SOX2 (1:1000; Abcam, Ab92494), pKi67 (1:1000; Novocastra, NCL-Ki67p), PITX1 (1:500; Novus, NBP1–88644), KLF4 (1:1000; R&D, AF3158), phospho Histone H3 (ser19) (1:1000; Millipore, 06–570), Filaggrin (1:1000; BioLegend, 905801) and Nidogen (1:1000; Santa Cruz, sc-33706).
Af3158
Manufactured by R&D Systems
Sourced in United States
AF3158 is a laboratory reagent produced by R&D Systems. It is a recombinant human protein.
Automatically generated - may contain errors
Lab products found in correlation
Mab2018, by R&D Systems (5 mentions)
Af2729, by R&D Systems (5 mentions)
Mbs462020, by MyBioSource (4 mentions)
Mab1759, by R&D Systems (4 mentions)
Mab2155, by R&D Systems (4 mentions)
Mab2736, by R&D Systems (4 mentions)
Lsm 780, by Zeiss (4 mentions)
Lsm 780 confocal microscope, by Zeiss (3 mentions)
Af3640, by R&D Systems (2 mentions)
Nbp1 49537, by Novus Biologicals (2 mentions)
Dia 310, by Cell Signaling Technology (2 mentions)
Mab293, by R&D Systems (2 mentions)
Mab1536, by R&D Systems (2 mentions)
Mab1259, by R&D Systems (2 mentions)
Ab130244, by Abcam (2 mentions)
Ab52903, by Cell Signaling Technology (2 mentions)
Dia 310, by Merck Group (2 mentions)
Pa1 1748a, by R&D Systems (2 mentions)
Filaggrin, by BioLegend (1 mentions)
Cd49f, by BioLegend (1 mentions)
13 protocols using af3158
1
Immunofluorescence Staining of Frozen Tumor Sections
2
Characterizing Hypoxia-Induced Stemness Markers
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Sorted cells were cultured under hypoxia for 48 h to detect the expression of CD133, SOX-2, OCT-4, Lin-28A, KLF-4, Nanog, CD15 and NESTIN. The sorted cell lines were exposed to hypoxia for 48 h, and 4% paraformaldehyde was used to fix the cells at 4 °C for 10 min. The cells were washed with PBS, and 10% serum in PBS containing 0.5% Triton X-100 was used to block the cells. The cells were then incubated for 24 h at 4 °C with primary antibodies against CD133 (1 : 150, MBS462020, MyBiosource, San Diego, CA, USA), SOX-2 (1 : 100, MAB2018, R&D Systems), KLF-4 (1 : 100, Human: AF3640; Mouse: AF3158; R&D Systems), OCT-4 (1 : 100, MAB1759 R&D Systems), Nanog (1 : 100, Human: AF1997; Mouse: AF2729 R&D Systems), Lin-28A (1 : 100, NBP1–49537, Novus Biologicals), CD15 (1 : 100, MAB2155, R&D Systems) or NESTIN (1 : 100, MAB2736, R&D Systems). The cells were then washed with PBS three times. Appropriate fluorophore-labeled secondary antibodies were added to the cells and incubated at 37 °C for 1 h. Images were acquired with a laser scanning confocal microscope (LSM780, ZEISS, Jena, Thuringia, Germany).
3
Immunoprecipitation and Fractionation Protocol
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For immunoprecipitation, cells were lysed on ice in TNE buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.5% Tergitol-type NP-40, 1 mM EDTA, and protease inhibitors). Anti-FLAG M2 magnetic beads (M8823, Sigma) were incubated overnight with cell lysate fractions. Samples were then washed 6 times with TBS. Western blotting was performed following standard principles and immunofluorescence was assessed using a Leica TCS SP2 spectral confocal microscope. Nuclear-cytoplasmic fractionation was performed following standard protocol. The following primary antibodies were used: anti-FLAG (F7425, Sigma), anti-NCoR (ABE251, Millipore), anti-HA (H6908, Sigma), anti-SMRT (ab24551, Abcam), anti-HDAC7 (ab12174, Abcam), anti-E-cadherin (BD Biosciences), anti-SSEA-1 (MC480, Cell Signaling), anti-SOX2 (MAB2018, R&D Systems), anti-OCT4 (sc-8628, Santa Cruz), anti-KLF4 (AF3158, R&D Systems), anti-MYC (AF3696, R&D Systems), anti-histone H3 (ab1791, Abcam), anti-ACTIN (A5316, Sigma), and anti-GAPDH (G8795, Sigma). DAPI was purchased from Sigma (D9542).
4
Hypoxia-Induced Stem Cell Marker Analysis
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Proteins were collected from sorted cells after hypoxia exposure for 0, 12, 24, 48 and 72 h. We then subjected the proteins to SDS-PAGE and transferred them to nitrocellulose membranes, which were blocked with 5% non-fat milk and incubated with primary antibodies against CD133 (1 : 1000, MBS462020, MyBiosource, San Diego, CA, USA), SOX-2 (1 : 1000, MAB2018, R&D Systems, Minneapolis, MN, USA), KLF-4 (1 : 1000, Human: AF3640; Mouse: AF3158 R&D Systems), OCT-4 (1 : 1000, MAB1759, R&D Systems), Nanog (1 : 1000, Human: AF1997; Mouse: AF2729 R&D Systems), Lin-28A (1 : 1000, NBP1–49537, Novus Biologicals, Littleton, CO, USA), CD15 (1 : 1000, MAB2155, R&D Systems) or NESTIN (1 : 1000, MAB2736, R&D Systems). β-Actin was used as an internal control.
5
Western Blot and Immunoprecipitation of Stem Cell Markers
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Protein lysates were run on 4%–20% Mini Protean TGX gels (Bio-Rad), blotted onto Immobilon-P membrane (EMD Millipore), and incubated with anti-SUMO2 antibody ab3742 (Abcam) and anti-RAN antibody 610341 (BD) for western blot analysis. We used anti-KLF4 antibody AF3158 (R&D Systems), anti-OCT4 antibody 11,263-1-AP (ProteinTech), and anti-SUMO2 antibody ab81371 (Abcam) for immunoprecipitation experiments.
6
Immunostaining of Stem Cell Markers
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Immunostaining was performed with standard protocols. Primary antibodies used were against Oct4 (sc-5279, Santa Cruz Biotechnology; 1:200), Nanog (AF2729, R&D; 1:100), Klf4 (AF3158, R&D; 1:100), nestin (2Q178, Santa Cruz Biotechnology, 1:100), GATA4 (G4, Santa Cruz Biotechnology, 1:100) and myosin (MF-20, Developmental Studies Hybridoma Bank, 1:50).
7
Immunohistochemical Analysis of Tissue Samples
8
Immunophenotyping of Hypoxic Neurospheres
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Newly formed neurospheres and differentiated cells after 48 h of hypoxia were fixed with 4% paraformaldehyde for 10 min at 4°C, washed with PBS and blocked with 10% normal serum for 20 min in PBS that contained 0.5% Triton X-100. The cells were incubated for 24 h at 4°C with primary antibodies against SOX-2 (1:100, MAB2018, R&D Systems, USA), OCT-4 (1:100, MAB1759, R&D Systems, USA), Nanog (1:100, Human: AF1997; Mouse: AF2729, R&D Systems, USA), KLF-4 (1:100, Human: AF3640; Mouse: AF3158, R&D Systems, USA), CD133 (1:150, MBS462020, MyBiosource, USA), CD15 (1:100, MAB2155, R&D Systems, USA), NESTIN (1:100, Human: MAB1259; Mouse: MAB2736, R&D Systems, USA), ABCG2 (1:100, ab130244, Abcam, USA), VEGF (1:100, MAB293, R&D Systems, USA) and HIF1α (1:100, MAB1536, R&D Systems, USA). Neurospheres and cells were washed three times with PBS for 5 min and then incubated at 37°C for 1 h with appropriate fluorophore-labeled secondary antibodies. Images were acquired with a laser scanning confocal microscope (LSM780, ZEISS, Germany).
9
KLF4 Protein Expression Analysis
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Proteins were collected from mESCs and KLF4-KO cells using RIPA buffer supplemented with a protease inhibitor cocktail. Sample loading was based on the results of BCA assay (Bicinchoninic acid). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore), which were then blocked and incubated with KLF4 antibody (R&D Systems, AF3158, lot: WRR061703) overnight at 4°C, followed by secondary antibodies (Abcam) for 1 h at room temperature. Bands were detected by Image Quant LAS 4000 with an Enhanced Chemiluminescence Kit (Thermo Pierce).
10
Immunohistochemical Analysis of Tissue Samples
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Tissue samples were fixed in 4% formaldehyde overnight, dehydrated in 100% ethanol, and embedded in paraffin. 5 μm thick sections were used for hematoxylin & eosin and immunohistochemistry staining. The following antibodies were used for immunostaining: rat anti-PECAM (1:20, Histo Bio Tech DIA-310), rabbit anti-pMLC2 (1:200, Cell Signaling 3674S), goat anti-KLF4 (1:100, R&D AF3158), and rabbit anti-RFP (1:50, Rockland 600-401-379). Littermate control and experimental animal sections were placed on the same slide and immunostained at the same time under identical conditions. Images were taken at the same time using the same exposure times and color channels, and were subsequently overlaid using ImageJ.
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