Cells were treated with 0.25% Trypsin and diluted with 10% KOSR before passing through 40 μm cell strainer. Single cells were then washed three times with DPBS and fixed with 4% paraformaldehyde on ice for 20 min. The cells were blocked on ice for 30 min with 10% KOSR in DPBS with or without 0.1% Triton-X for intracellular or surface marker staining respectively. For surface marker staining, the cells were incubated with EPCAM-PE (Biolegend, 1:100) for 30 min and proceeded with blocking with 10% KOSR in DPBS supplemented with 0.1% Triton-X for another 30 min. The cells were then co-stained with SOX17 (R&D Systems, 1:100) before secondary antibody staining with Alexa Fluor® 488 Donkey anti-goat IgG (ThermoFisher Scientific, 1:500) for 1 h at 4°C for each condition. For co-staining of SOX17 (R&D System, 1:100) and EOMES (Abcam, 1:100), cells were incubated with primary antibodies on ice for 1 h before secondary antibody staining. For controls, cells were stained with Mouse IgG PE, Mouse IgG2a APC (BD Pharmingen) and Alexa Fluor® 488 (ThermoFisher Scientific). Cells were washed three times and diluted with DPBS for flow cytometry (LSR II, BD Biosciences). Flow cytometry data was analyzed using FlowJo_V10. Primary antibodies used are listed in Table S7 .
Sox17
Manufactured by R&D Systems
Sourced in United States, Denmark, United Kingdom, Japan
SOX17 is a recombinant protein that functions as a transcription factor involved in the regulation of gene expression. It plays a role in embryonic development and cell differentiation. This product is suitable for use in various research applications.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (12 mentions)
Tra 1 60, by Merck Group (9 mentions)
Oct3 4, by Santa Cruz Biotechnology (7 mentions)
Ssea 4, by Merck Group (7 mentions)
Hoechst 33342, by Thermo Fisher Scientific (7 mentions)
α sma, by Agilent Technologies (6 mentions)
Nanog, by R&D Systems (6 mentions)
Brachyury, by R&D Systems (6 mentions)
Triton x 100, by Merck Group (5 mentions)
In cell analyzer 6000, by GE Healthcare (5 mentions)
Gata4, by Santa Cruz Biotechnology (4 mentions)
Nanog, by Abcam (4 mentions)
Lsm 700, by Zeiss (4 mentions)
Lsm 510 meta, by Zeiss (4 mentions)
Cleaved caspase 3, by Cell Signaling Technology (3 mentions)
Hnf4a, by Cell Signaling Technology (3 mentions)
Foxa2, by Abcam (3 mentions)
Glial fibrillary acidic protein (gfap), by Agilent Technologies (3 mentions)
Nestin, by STEMCELL (3 mentions)
Nanog, by ReproCELL (3 mentions)
62 protocols using sox17
1
Flow Cytometry Analysis of Pluripotency Markers
2
Protein Expression Analysis Using Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed in buffer containing RIPA and PSMF (TransGen Biotech, China). Proteins were quantified using a BCA protein assay kit (Beyotime Biotechnology) according to the manufacturer’s specifications. After denaturation, proteins were separated on 10% polyacrylamide gels and then transfered to polyvinylidene fluoride (PVDF) membranes (Millipore, CA, USA). After being blocked, the membranes were incubated overnight with primary antibodies for E-cadherin, Vimentin, Gata4, Sox2, Foxa2, Desmin (Cell Signalling Technology), Sox17 (R&D Systems), and Gadph (ProteinTech) at 4 °C and then with secondary antibodies on the next day. Finally, the protein bands were visualised with hypersensitive chemiluminescence (Beyotime Biotechnology).
3
Blastocyst Immunostaining for Cell Lineage
4
Immunofluorescence Analysis of Embryo Development
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Pre-implantation embryos were fixed and stained as previously described (Bedzhov et al., 2012 ). Peri-implantation embryos were fixed with 4% PFA/PBS for 20 min. Cellular permeabilization was carried out for 10 min in 0.1M Glycin, 0.3% Triton X-100/PBS. The embryos were in incubated in primary antibody in 10% FCS/PBT for 2h to overnight at room temperature. Secondary antibodies were applied subsequently for 2h. Embryos were stained with DAPI (Invitrogen) and mounted in PBS droplets covered with mineral oil in glass bottom petri dishes. Confocal microscopy was performed using a Leica SP5 microscope and the images were processed using Imaris software (Bitplane). Antibodies: cleaved Caspase-3 (1:200, Cell signaling, 9664), Oct4 (1:200, Santa Cruz, sc-5279), Sox2 (1:200, Santa Cruz, sc-17320), aPKC (1:200, Santa Cruz, sc-216), Par6 (1:200, Santa Cruz, sc-67393), E-cad (1:200, (Vestweber and Kemler, 1984 )), gm130 (1:200, Calbiochem, CB1008), Eomes (1:200, Abcam), Nanog (1:200, Abcam, ab80892), Laminin (1:500, Sigma, L9393), Sox17 (1:200, R&D systems, AF1924), pMLC (1:200,Cell signaling, 3674), podocalyxin (1:200, R&D systems, MAB1556), β1-integrin (1:200, BD Pharmigen, 555005).
5
Multimarker Immunofluorescence Staining of Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cultured cells were fixed with 4% paraformaldehyde for 20 min at room temperature, made permeable with 0.4% Triton X-100 in PBS for 15 min and blocked with 3% BSA in PBS for 1 hour. Cells were incubated overnight at 4°C with primary antibodies diluted in 1% BSA in PBS. Primary antibodies used were Oct3/4 (Santa Cruz; rabbit, 1:500), Sox17 (R&D Systems; goat, 1:250), FoxA2 (Novus Biologicals; Mouse, 1:1000), HNF4a (Santa Cruz; goat, 1:250), AFP (Sigma; mouse, 1:1000), and Albumin (DAKO; rabbit, 1:500). Primary antibodies were probed with respective secondary antibodies conjugated to Alexa Fluor 488 or 594 (Molecular Probes; 1:1000) and nuclei were visualized with DAPI.
6
Multilineage Differentiation of iPSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Undirected embryoid bodies (EB) were formed by placing 10,000 iPSCs in multiple wells of a 96-well non-tissue culture treated V-bottom plate (Evergreen) containing HUESM plus 10 μM ROCKi (Stemgent), and underwent brief centrifugation. After 14 d of culturing EBs were transferred into a 6 well low attachment plates (Corning) and cultured for an additional 16 days. Once harvested EBs were fixed in 4% paraformaldehyde for 20 min at room temperature and processed in 15% and 30% sucrose solutions for one day each at 4°C. EBs were then embedded in O.C.T. (Sakura Finetek) and cryosectioned. The sections were blocked in PBS containing 0.1% Triton X-100 and 10% donkey serum for 1 hr at room temperature, followed by an overnight incubation at 4°C with antibodies identifying the 3 germ layers: SMA (1:500, DAKO), AFP (1:500, DAKO) TuJ1 (1:500, Covance), Sox17 (1:500, R&D Systems). Alexa-conjugated anti-mouse or anti-rabbit IgG secondary antibodies were used (Invitrogen) along with Hoechst 33342 counterstain. Sections were set with Vectashield Hard Set Mounting Media (Vector Laboratories).
7
Directed Differentiation of Engineered Stem Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In vitro differentiation of H1-RB1(E16)−/+ cells to HPCs, MSCs, and NPCs was performed by well-defined differentiation protocols described previously (Chambers et al., 2009 (link); Qin et al., 2016 (link); Zhao et al., 2015 (link)). For cell characterization, SOX17 (R&D Systems) and HNF4A (Cell Signaling Technology) were used for HPCs, CD105 (Thermo Fisher Scientific) and CD73 (BD Biosciences) were used for MSCs, and PAX6 (BioLegend) and NESTIN (BioLegend) were used for NPCs.
8
Differentiation of Stem Cells into Germ Layers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Embryoid body (EB) formation was performed by plating single cells in AggreWell800 (Stem Cell Technologies, 34811, Vancouver, Canada) in medium containing Knockout DMEM F-12 (Gibco,12660-012, Grand Island, NY, USA), 20% Knockout Serum (Gibco, 10828-028, Grand Island, NY, USA), non-essential amino acids-1x (Gibco, 11140-050) and Glutamax-1x (Gibco, 35050-061). Medium was changed 48 hours later and thereafter till day 7 in an every-other-day mode. On day 7, EB spheres were collected and plated onto plates coated with 0.1% Gelatin (Millipore, ES-006-B) and medium containing Dulbecco’s modified Eagle’s medium (DMEM) (Gibco 11965-092), 20% fetal bovine serum (FBS) (Gibco, SH30071), non-essential amino acids-1x (Gibco, 11140-050) and Glutamax-1x (Gibco, 35050-061). Medium was changed every other day for 7 days. On day 7 post plating, EBs were fixed with 4% paraformaldehyde (Santa Cruz, SC-281692, Dallas, TX, USA), and stained for detection of cells of the three germ layers with antibodies for the following antigens: SOX17 (R&D Systems, AF1924, Minneapolis, MN, USA) for endoderm, PAX6 (BioLegend, PRB-278P, San Diego, CA, USA) for ectoderm and SMA (Millipore, CBL171) for mesoderm.
9
Immunofluorescence Assay for Hepatic Cell Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were fixed with 4% paraformaldehyde (Sigma) for 20 minutes at room temperature, followed by addition of primary antibodies. Primary antibodies against SOX17 (1:200, R&D Systems), Ki67 (1:500, Cell Signaling), AFP (1:200, Dako), ALB (1:200, Dako), CD133 (1:100, Biolegend), and 8-OHdG (1:200, Abcam) were diluted in PBS with 0.3% BSA and 0.1% Triton X-100. Cells were incubated with appropriate primary antibodies at 4oC overnight, and then incubated with Alexa Flour 594 or 488 secondary antibodies (all of the Alexa Fluor Series from Invitrogen) at room temperature for 30 minutes. Finally, cells were counterstained with DAPI. Images were taken using the motorized Nikon Ti-E microscope and NIS-Elements software. Cell counting was performed with ImageJ software (http://imagej.nih.gov/ij /).
10
Histological Characterization of Tumor Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The spleen tumors, liver metastasis tumors, and s.c. tumors were fixated in 4% paraformaldehyde, paraffin-embedded, and sectioned at 4-μm thickness. The sections were stained with H&E or Masson Trichrome. For immunohistochemistry, antibodies against αSMA (Sigma) at 1:800, E-cadherin (R&D Systems) at 1:100, p53 (CM5) (Leica Biosystems) at 1:200, Ki67 (Abcam) at 1:1000 and Sox17 (R&D Systems) at 1:100 were used as the primary antibody. Staining signals were visualized using the Vectastain Elite Kit (Vector Laboratories). For fluorescent immunohistochemistry, Alexa Fluor 594- or Alexa Fluor 488-conjugated antibodies (Molecular Probes) were used as the secondary antibody. The numbers of p53 nuclear-accumulated cells in the liver metastasis foci were scored in 5 microscopic fields, and the ratio was calculated as the mean number of positive cells per tumor gland.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!