Cells were fixed for 10 min at 4°C in 4% paraformaldehyde in PBS, permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO) for 10 min at room temperature, and blocked for 1 hour in PBS with 1% horse serum (Sigma-Aldrich, St. Louis, MO). The primary antibodies used were as follows: anti-NANOG (1:400; cat. no. 4903, Cell Signaling), anti-SOX17 (1:100; cat. no. 81778, Cell Signaling), anti-FOXA2 (1:100; cat. no. 685802, BioLegend), anti-SMAD2 (1:100; cat. no. 3122, Cell Signaling), anti-SMAD3 (1:100; cat. no. 9523, Cell Signaling), anti–p-SMAD2 (1:100; cat. no. 3108, Cell Signaling), and anti–p-SMAD3 (1:100; cat. no. 9520, Cell Signaling). The treated cells were subjected to three washes with PBS and further incubation with Alexa Fluor secondary antibodies (1:500; Jackson ImmunoResearch) for 1 hour at room temperature in the dark. The cells were then washed three times with PBS, with 4′,6-diamidino-2-phenylindole (Sigma-Aldrich, St. Louis, MO) added to the first wash to stain the nuclei. Images were acquired using a Confocal Zeiss LSM880. Intensity analysis was performed using ImageJ 1.51j8 (National Institutes of Health, MD, USA). For time-lapse imaging, cells were placed on an inverted microscope (Nikon, Ti-U inverted microscope system) and recorded every 2 hours for 72 hours in an environmental chamber maintained at 37°C with 5% CO2.
Anti foxa2
Manufactured by Cell Signaling Technology
Sourced in Canada, China, United States, Japan
Anti-FOXA2 is a primary antibody that recognizes the FOXA2 protein. FOXA2 is a transcription factor that plays a role in the regulation of gene expression during embryonic development and in adult tissues. The Anti-FOXA2 antibody can be used for applications such as Western blotting, immunohistochemistry, and immunocytochemistry to detect and analyze the FOXA2 protein.
Automatically generated - may contain errors
Lab products found in correlation
Anti nanog, by Abcam (4 mentions)
Anti tuj1, by Merck Group (4 mentions)
Anti sox2, by Santa Cruz Biotechnology (4 mentions)
Hoechst 33342, by Thermo Fisher Scientific (4 mentions)
Anti sma, by Merck Group (3 mentions)
Anti lap2, by BD (3 mentions)
Anti ki67, by ZSGB-BIO (3 mentions)
Anti oct4, by Santa Cruz Biotechnology (3 mentions)
Anti lamin b, by Santa Cruz Biotechnology (2 mentions)
Anti gapdh, by Santa Cruz Biotechnology (2 mentions)
Anti lamin a c, by Santa Cruz Biotechnology (2 mentions)
Anti β actin, by Cell Signaling Technology (2 mentions)
Anti sox17, by R&D Systems (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Anti sox17, by Cell Signaling Technology (1 mentions)
Alexa fluor secondary antibody, by Jackson ImmunoResearch (1 mentions)
Ti u inverted microscope system, by Nikon (1 mentions)
Horse serum, by Merck Group (1 mentions)
Anti smad3, by Cell Signaling Technology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
13 protocols using anti foxa2
1
Immunofluorescence Imaging and Time-Lapse Analysis of Stem Cell Markers
2
Stem Cell Marker Expression Analysis
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The primary antibodies used were as follows (company, catalogue number): anti-ERCC6 (Abcam, ab96098), anti-NANOG (Abcam, ab21624), anti-SOX2 (Santa Cruz, sc-17320), anti-OCT4 (Santa Cruz, sc-5279), anti-SMA (Sigma, A5228), anti-TUJ1 (Sigma, T2200), anti-FOXA2 (Cell Signaling Technology, 8186S), anti-CD90-FITC (BD Bioscience, 555595), anti-CD73-PE (BD Bioscience, 550257), anti-CD105-APC (BD Bioscience, 17-1057-42), anti-IgG-FITC (BD Biosciences, 555748), anti-IgG-PE (BD Biosciences, 555749), anti-IgG-APC (BD Biosciences, 555751), anti-Lamin B (Santa Cruz, sc-6217), anti-LAP2 (BD Bioscience, 611000), anti-Ki67 (ZSGB-BIO, ZM0166), anti-P16 (BD Bioscience, 550834), anti-γ-H2AX (Millipore, 05-636), anti-Nestin (Millipore, MAB5326), anti-PAX6 (Covance, PRB-278P), anti-CPD (Cosmo Bio, TMD-2), anti-cleaved PARP (Cell Signaling Technology, 9541), anti-β-Actin (Santa Cruz, sc69879), anti-GAPDH (Santa Cruz, sc-25778), and anti-hCD31 (BD Bioscience, 555445).
3
Immunofluorescence and Western Blot Analyses
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The following antibodies were used for immunofluorescence staining or western blot analyses: anti-FoxA2 (used at 1:400, Cell Signaling), anti-HNF4α (used at 1:500, Cell Signaling), anti-AFP (used at 1:1000, Sigma-Aldrich), anti-ALB (used at 1:1000, Cedarlane, Burlington, Canada), anti-ZO-1 (used at 1:1000, Thermo Fisher), anti-E cadherin (used at 1:500, Cell Signaling), anti-SR-BI (used at 1:100, Novus Biologicals), anti-BCRP (used at 1:100, Millipore), anti-MRP2 EAG548 (link) (used at 1:200, a kind gift from Anne Nies, IKP Stuttgart48 (link)), anti-Apo-CIII (used at 1:500, Abcam), anti-CYP8B1 (used at 1:100, Abcam), anti-ORF2 (used at 1:400, a kind gift from Suzanne U. Emerson, NIH) and anti-HAV capsid (used at 1:1000, a kind gift from Stanley M. Lemon, UNC School of Medicine). Alexa Fluor 488 and 549 anti-mouse (used at 1:1000) and Alexa Fluor 488 and 549 anti-rabbit (used at 1:1000) antibodies were purchased from ThermoFisher. Alexa 594-conjugated transferrin was purchased from ThermoFisher. Tenofovir, Tenofovir disoproxil fumarate, and Emtricitabine were obtained through the AIDS Reagent Program, Division of AIDS, NIAID, NIH. Elvitegravir and Cobicistat were purchased from SelleckChem. Sofosbuvir was purchased from Acme Bioscience. BX795, oleic acid and lomitapide were purchased from Sigma-Aldrich.
4
Nurr1-Foxa2 Interaction Analysis
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Interaction between Nurr1 and Foxa2 (in mouse VM tissue at 10 weeks of age) was assessed by IP. Tissues were lysed in IP lysis buffer (Thermo Scientific, Waltham, MA) supplemented with protease inhibitors. Lysates were incubated with anti-Nurr1 (1:1,000, mouse, R&D Systems) or anti-Foxa2 (1:1,000, goat, Santa Cruz) for 18–24 h at 4°C. The mixtures were shaken with magnetic beads (Life Technologies) for 1–2 h at room temperature. After washing, immunoprecipitated proteins were eluted in sample buffer and subjected to Western blot analysis with anti-Foxa2 (1:1,000, goat, Cell Signaling) or anti-Nurr1 (1:500, mouse, R&D Systems).
5
Immunofluorescence Profiling of Osteogenic Markers
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Cells were cultured in a 12-well plate, and FOXA2, RUNX2, COL1A1, t-ERK and p-ERK were detected using a fluorescence microscope (EU5888; Leica, Wetzlar, Germany). Briefly, cells were fixed in 4% paraformaldehyde (Sigma) for 15 min at room temperature, permeabilized and blocked for 30 min in 0.05% Triton X-100 and 5% bovine serum albumin (BSA). Fixed cells were washed three times with PBS and incubated at 4 °C overnight with anti-FOXA2 (1:400; Cell Signalling Technology), RUNX2 (1:1600; Cell Signalling Technology), COL1A1 (1:500; Abcam, Shanghai, China), t-ERK (1:800; Cell Signalling Technology) or p-ERK (1:200; Cell Signalling Technology). Cells were incubated with a fluorescence-conjugated secondary antibody (Beyotime) at room temperature for 2 h and nuclei were stained with 4′,6-diamidino-2-phenylindole (KeyGen Biotech, Nanjing, China) for 4 min. Samples were then observed and photographed under a fluorescence microscope (Leica).
6
Immunocytochemistry for Pluripotency and Lineage Markers
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Cells were fixed in 4% formaldehyde for 30 min, permeabilized in 0.4% Triton X-100 in PBS for 30 min and incubated with blocking buffer (10% donkey serum in PBS) for 30 min. Samples were incubated with primary antibody overnight in 4 °C and with secondary antibody for 1 h at room temperature. Cell images were taken using confocal microscopy. Nuclear DNA was stained by Hoechst 33342 (Invitrogen, H3570). Antibodies used were as follows: anti-NANOG (Abcam, ab21624, 1:200), anti-SOX2 (Santa Cruz, sc-17320, 1:100), anti-OCT4 (Santa Cruz, sc-5279, 1:100), anti-TUJ1 (Sigma, T2200, 1:100), anti-αSMA (Sigma, A5228, 1:100), anti-FOXA2 (Cell Signaling Technology, 8186S, 1:100), anti-CD31-FITC (BD Biosciences, 555445, 1:50), anti-vWF (Dako, A0082, 1:200), Dil-Ac-LDL (Molecular probes, 1:400), anti-SM22 (Abcam, ab14106, 1:200), anti-Calponin (BD Biosciences, 2017-03, 1:200) and anti-Ki67 (Vector Labs, VP-RM04, 1:1,000) anti-RelA (Cell Signaling Technology, 8242, 1:200).
7
Immunofluorescence Staining of Diverse Cellular Markers
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5 × 105 cells were seeded on coverslips (Thermo Fisher Scientific), washed with PBS and fixed with 4% paraformaldehyde (PFA) for 30 min and permeabilized with 0.4% Triton X-100 in PBS for 30 min at RT. After washing with PBS three times, the cells were blocked with 10% donkey serum (Jackson ImmunoResearch) in PBS for 1 hr at RT. The cells were then incubated with primary antibodies in 10% donkey serum at 4°C overnight. Afterwards, cells were washed with PBS and stained with secondary antibodies and Hoechst 33342 (Thermo Scientific) for 1 hr at RT. A Leica SP5 confocal system was used for imaging. Antibodies used in this study were as follows: anti-Ki67 (ZSGB-BIO, ZM0166), anti-53BP1 (Bethyl Laboratories, A300-273A), anti-FOXA2 (Cell Signaling Technology, 8186S), anti-SMA (Sigma, A5228), anti-TuJ1 (Sigma, T2220), anti-H3K9me3 (Abcam, Ab8898), anti-NANOG (Abcam, Ab21624), anti-OCT3/4 (Santa Cruz, sc-5279), anti-SOX2 (Santa Cruz, sc-17320), and anti-LAP2 (BD Bioscience, 611000), anti-HP1α (Cell Signaling Technology, #2616S), and anti-Lamin A/C (Santa Cruz, sc-376248).
8
Immunoblotting of Cell Signaling Proteins
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Lysis from whole-cell extracts of cultured murine and human cell lines was prepared in diluted RIPA buffer (10× concentration; Cell Signaling 9806). Phosphatase and protease inhibitors were added to the RIPA buffer prior to extraction. Cells were dissociated by mechanical homogenization followed by repeated passaging through successive small gauge needles. Protein concentration was determined using Pierce BCA protein assay kit (Thermo Fisher Scientific 23227). Immunoblotting was performed using standard protocols. The following antibodies were used in this study: anti-β-ACTIN (1:2000; Cell Signaling 4970), anti-GAPDH (1:2000; Santa Cruz Biotechnology sc-32233), anti-NEDD8 (1:1000; Cell Signaling 2745), anti-RBX1 (1:1000; Cell Signaling 11922), anti-INSM1 (1:1000; Santa Cruz Biotechnology sc-271408), anti-MASH1 (ASCL1; 1:1000; BD 24B72D11.1), anti-FOXA2 (1:1000; Cell Signaling 8186), anti-BRN2 (POU3F2; 1:1000; Proteintech 18998-1-AP), anti-NRF2 (1:1000; Cell Signaling 12721), anti-P21 (1:1000; Santa Cruz Biotechnology sc-6246), anti-CSN4 (COPS4; 1:1000; Bethyl Laboratories A300-013A), anti-PARP (1:1000; Cell Signaling 9542), anti-Caspase 7 (1:1000; Cell Signaling 12827), anti-IKKα (1:1000; Cell Signaling 2682), anti-MIOS (1:1000; Bethyl Laboratories A304-699A), and anti-IRAK1 (1:1000; Santa Cruz Biotechnology sc-5288).
9
Protein Extraction and Western Blot Analysis
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All cells were washed with PBS for twice and lysed in IP lysis buffer (Beyotime, China) supplemented with protease and phosphatase inhibitor on ice for 30 min. After that, cell lysates were centrifuged for 30 min (12000 g, 4 °C). Then the protein sample was collected and the protein concentration was measured by using BCA protein concentration assay kit (Beyotime, China). Protein samples were diluted to a certain concentration (5 ug/ul) with the lysis buffer. The proteins (50 μg) were separated by 12%SDS-PAGE and transferred to PVDF membranes (Millipore). After being blocked with skimmed milk, the blots were incubated overnight at 4°C with rabbit Anti-Foxa2 and anti-β-actin. The membranes were then washed and incubated with goat anti-rabbit secondary antibodies for 60 min, and then developed with BeyoECL Plus kit (Beyotime). The antibodies were purchased from the following sources: Anti-Foxa2 (1:1000, Cell Signaling Technology #3143, USA), anti-β-actin (1:1200, Cell Signaling Technology #8457, USA) and goat anti-rabbit secondary antibodies (1:1200, Santa Cruz, sc2030, USA).
10
Immunofluorescence Staining of Stem Cell Markers
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Cells were fixed with 4% paraformaldehyde for 25 min, permeabilized with Triton X-100 (0.3% in PBS) for 25 min, incubated with blocking buffer (10% donkey serum in PBS) for 1 h at RT, and stained with primary antibodies overnight at 4 °C. Then, the cells were incubated with secondary antibodies for 1 h at RT. Hoechst 33342 (Invitrogen) was used to stain nuclear DNA. The primary antibodies used in immunofluorescence assays were as follows: anti-NANOG (Abcam, ab21624), anti-SOX2 (Santa Cruz, sc-17320), anti-OCT4 (Santa Cruz, sc-5279,), anti-SMA (Sigma, A5228), anti-TUJ1 (Sigma, T2200), anti-FOXA2 (Cell Signaling Technology, 8186S), anti-BIP (Cell Signaling Technology, 3177), anti-LaminA/C (Santa Cruz, sc-6215), anti-Calreticulin (Abcam, ab2907), anti-LaminB (Santa Cruz, sc-6217), anti-LAP2 (BD Bioscience, 611000), and anti-Ki67 (ZSGB-BIO, ZM0166).
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