Cells were treated with lysis buffer containing 150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 0.5% Triton X-100 (v/v) and a protease-inhibitor cocktail (Roche). The lysates were loaded onto 10% acrylamide/bis gel and transferred to a PVDF membrane after electrophoresis. Following blocking with 5% nonfat milk for 1 h, membranes were incubated at 4°C overnight with primary antibodies: Lsh (rabbit-polyclonal) [66 ]; Actin (Sigma, A2228); Foxa2 (Santa Cruz, sc-20692x); HNF4a (Santa Cruz, sc-8987x). After incubation with HRP-conjugated secondary antibodies for 1 h at room temperature, the ECL western blotting analysis system was used for protein detection.
Foxa2
Manufactured by Santa Cruz Biotechnology
Sourced in United States
FOXA2 is a transcription factor that plays a critical role in the regulation of genes involved in embryonic development and cell differentiation. It functions as a pioneer factor, opening chromatin structure and facilitating the binding of other transcription factors. FOXA2 is essential for the development of the endoderm, pancreas, liver, and other organs. It is commonly used in research related to stem cell biology, tissue engineering, and the study of developmental processes.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (5 mentions)
Lmx1a, by Merck Group (5 mentions)
Hnf4a, by Santa Cruz Biotechnology (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Pyrvinium, by Merck Group (2 mentions)
Survivin, by Cell Signaling Technology (2 mentions)
Brachyury, by Santa Cruz Biotechnology (2 mentions)
Ix51 inverted microscope, by Olympus (2 mentions)
Mouse igg2a, by R&D Systems (2 mentions)
Cellomics arrayscan vti, by Thermo Fisher Scientific (2 mentions)
Donkey anti rabbit af488, by Thermo Fisher Scientific (2 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (2 mentions)
Hoechst, by Thermo Fisher Scientific (2 mentions)
Donkey anti mouse af488, by Thermo Fisher Scientific (2 mentions)
Donkey anti rabbit af555, by Thermo Fisher Scientific (2 mentions)
C06sbz, by Merck Group (2 mentions)
Donkey anti chicken af647, by Thermo Fisher Scientific (2 mentions)
Donkey anti mouse af555, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Axin2, by Abcam (1 mentions)
24 protocols using foxa2
1
Protein Expression Analysis Protocol
2
Antibody-based Protein Detection Assays
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Antibodies used for immunohistochemistry (IHC) and/or Western blot were as follows: actin (Sigma-Aldrich, St Louis, MO, USA), Axin 2 and c-Myc (Abcam, Cambridge, MA, USA), FoxA2 (Santa Cruz Biotechnology, Dallas, TX, USA), Ki-67 (DAKO/Agilent; Dako, Denmark), survivin and cleaved caspase 3 (Cell Signaling Technology, Boston, MA, USA). Pyrvinium was purchased from Sigma-Aldrich (#P0027).
3
Immunofluorescence Staining of Neural Progenitor Cells
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Cells were rinsed in PBS and fixed in 4%PFA for 15 mins. Cells were then permeabilised via three washes in PBS containing 0.3% Triton-X-100 (PBST) and then blocked in PBST containing 1% BSA and 3% normal donkey serum. Primary antibodies were added in blocking solution for 2 hours at ambient temperature or overnight at 4 °C. The cells were washed in PBST three times before being incubated for 1 hour in the dark in Alexa-Fluor secondary antibodies, 1:200 (Invitrogen). Three PBST washes were then performed that included one with DAPI, 1:1000 (Molecular Probes). The primary antibodies used in this study were: FolR1(sheep, R&D), TH (rabbit, PelFreez), Pitx3 (rabbit, gift of M. Smidt, University of Amsterdam), Foxa2 (goat, Santa Cruz), Foxa2 (Rabbit, Abcam), Lmx1a (rabbit, gift of M. German, UCSF), Nestin (mouse, BD Pharmagen), GFAP (rabbit, Dako), GABA(rabbit, Sigma), 5HT(mouse, Abcam), Isl1(mouse, DSHB), Lim1/2(mouse, DSHB), Pax6(mouse, DSHB), Nkx6.1(mouse, DSHB), Dmrt5(rabbit, custom made). Images were taken on a Leica TCS SP5 confocal microscope. Quantification of markers was carried out manually by examining randomly selected fields from at least 3 independent experiments and presented as means ± sem.
4
Cardiac and Liver Marker Assay
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Troponin I (Santa Cruz Biotechnology), MLC2v (Proteintech Europe), Tuj1 (Santa-Cruz), FOXA2 (Santa Cruz), Alpha foeto protein (Sigma-Aldrich), Albumin (Cedarlane).
5
Immunohistochemistry Analysis of Transcription Factors
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Hematoxylin and eosin staining and immunohistochemistry were performed as previously described13 (link),42 (link). For immunohistochemistry, paraformaldehyde-fixed, paraffin-embedded sections were incubated with the following primary antibodies: ESR1 (sc-8005), PGR (sc-538), CEBPB (sc-150), FOXA2 (sc-6554), αSMA (sc-53142, Santa Cruz, Santa Cruz, CA, USA). The sections were stained with the Vectastain ABC Kit (Vector Laboratories, Burlingame, CA, USA). Immunofluorescence analysis was performed with Alexa Fluor protein-conjugated secondary antibodies (Thermo Fisher Scientific, Waltham, MA, USA) and counterstained with Hoechst 33342 (Sigma).
For EdU-immunostaining, mice were injected with EdU at 50 mg/kg body weight. One hour after the injection, animals were euthanized and tissues collected. EdU-incorporated cells were detected using Click-iT EdU Imaging Kits (Themo Fisher Scientific) as described in the manufacture’s protocol. In some samples, ESR1 (sc-8005) was detected with Alexa Fluor protein-conjugated secondary antibodies (Thermo Fisher Scientific) and immunofluorescent imaging. More than 3 animals were analyzed, and representative pictures are shown.
For EdU-immunostaining, mice were injected with EdU at 50 mg/kg body weight. One hour after the injection, animals were euthanized and tissues collected. EdU-incorporated cells were detected using Click-iT EdU Imaging Kits (Themo Fisher Scientific) as described in the manufacture’s protocol. In some samples, ESR1 (sc-8005) was detected with Alexa Fluor protein-conjugated secondary antibodies (Thermo Fisher Scientific) and immunofluorescent imaging. More than 3 animals were analyzed, and representative pictures are shown.
6
Immunofluorescence analysis of mESC differentiation
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After fixation for 20 min with 4% paraformaldehyde at room temperature, mESCs were permeabilized with 0.0125% Triton X-100. Then, the mESCs were incubated with 1% BSA and incubated overnight with a primary antibody specific to Tuj1 (1:500; Sigma, St. Louis, MO, USA), Map2 (1:500; Cell Signaling Technology, USA), Brachyury (1:500; Santa Cruz, CA, USA), Foxa2 (1:500; Santa Cruz, CA, USA), Oct4 (1:500; Santa Cruz, CA, USA), or Nanog (1:500; Bethyl Lab, Montgomery, TX, USA). After washing with PBS, mESCs were incubated at room temperature for 90 min with specific secondary antibodies (1:1000; Invitrogen, Carlsbad, CA, USA). Counterstaining was performed with DAPI (5 mg/ml, Sigma, St. Louis, MO, USA). Cells ere imaged with a Nikon eclipse Ti. The three-color images were saved as a tif file format and merged with Adobe Photoshop software.
7
Trilineage Differentiation of hiPSCs
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For trilineage differentiation, a StemMACS™ Trilineage Differentiation Kit, human (Miltenyi Biotec) was used. Putative hiPSCs were cultivated for seven days in three different chemically defined media, driving differentiation into three germ layers. The three germ layers differentiated were fixed with 4% paraformaldehyde and stained with the following antibodies: PAX6 (1:20, Santa Cruz Biotechnology), SM22A (1:40, Santa Cruz Biotechnology), and FOXA2 (1:50, Santa Cruz Biotechnology).
8
Immunostaining and FACS Analysis for Neural Stem Cells
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OTX2 (Abcam cat# ab21990 1:500), LMX1 (Millipore cat# AB10533 1:3000), FOXA2 (Santa Cruz cat# 6554 1:500), TH (Pelfreeze cat# P40101–1 1:1000), GIRK2 (Alomone cat# APC-006 1:100), and MAP2 (Abcam cat# ab11267 1:500) were used for immunostaining following fixation. CORIN (R&D cat# MAB2209, 1:250), CD166 (BD cat# 559263, 1:100), and CXCR4 (BD cat# 555976, 1:200), BCAM (R&D cat# FAB1481P, 1:100), CD63 (Abcam cat# ab18235, 1:100), CD47 (Abcam cat# ab134484, 1:100), SORT1 (Bioss cat# bs-6329R-Cy3, 1:100) were used for FACS.
9
Immunocytochemical Analysis of Dopaminergic Neuron Differentiation
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Cells were fixed with 4% formaldehyde for 20 min and washed three times in PBS. Blocking buffer (0.1% Triton X-100, 2% goat serum or donkey serum in PBS) was added to fixed cells for 30 min. Primary antibodies TH (1:1000, rabbit, Millipore), β-III tubulin (1:1000, mouse IgG2a, R&D), LMX1A (1:2000, rabbit, Millipore), FOXA2 (1:100, goat, Santa Cruz), EN1 (1:50, rabbit, GeneTex), and CORIN (1:1000, rat, R&D) were incubated with fixed cells overnight at 4°C and then washed three times in PBS with 0.1% Triton X-100. Secondary antibodies (Thermo Fisher Scientific) in blocking buffer were incubated with cells for 2 h in the dark at room temperature. They were washed three times in PBS with Triton X-100 and incubated with DAPI (10 μg/ml, Thermo Fisher Scientific) before imaging on an Olympus IX51 inverted microscope. Quantification of TH and β-III tubulin immunostaining was performed with Fiji software (Schindelin et al., 2012 (link)). Briefly, RGB images were converted to 8-bit grayscale, and then manually thresholded during conversion to binary images. Efficiency of DA neuronal differentiation was estimated by calculating the ratio of total TH to total β-III tubulin immunostaining for each image.
10
Immunofluorescence Characterization of Neuronal Cells
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The following primary antibodies were used: AADC (Millipore/AB1569), ASCL1 (Millipore/AB5696), DAT (Abcam/AB5990), FOXA2 (Santa Cruz/SC-6554), FOXG1 (Abcam/AB18259), GIRK2 (Abcam/AB30738), KI67 (DAKO/M7240), LMX1A (Millipore/AB10533), NESTIN (Abcam/AB22035), NGN2 (R&D Systems/MAB3314), NURR1 (Santa Cruz/SC-990), SOX1 (Millipore/AB15766), SOX2 (R&D Systems/MAB2018), TH (Millipore/MAB5280), TH (Millipore/AB152), TUJ1 (Biolegend/801202), VMAT2 (Millipore/AB1598P). Fluorescent imaging was performed on a Nikon Eclipse TE2000U microscope with an Optronics Microfire camera or Photometrics Evolve EMCCD camera. Phase contrast images were taken on the Nikon Eclipse TS100 microscope with a Nikon Digital Sight DS-U1 camera. For cell quantification experiments, the investigator was blinded to the condition during image acquisition and counting.
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