Formalin-fixed, paraffin-embedded specimens of OSCC and normal oral mucosal tissues were sectioned to a 5-μm thickness. The sections were deparaffinized in xylene and rehydrated through graded alcohol solutions. Heat-induced antigen retrieval was performed in a microwave oven for 10 min in Tris/EDTA buffer (pH 9.0). Endogenous peroxidase activity was inactivated by treating with 3% H2O2 in PBS for 15 min. After blocking nonspecific binding (0.75% BSA in PBS), the sections were incubated with the anti-TFPI2 (Abcam, dilution 1:100), anti-SOX17 (Abcam, dilution 1:100), and anti-GATA4 antibodies (Abcam, dilution 1:100) overnight at 4 °C. The staining was visualized by a peroxidase-conjugated secondary antibody and diaminobenzidine (Vector labs, Burlingame, CA, USA). Finally, the sections were counterstained by Mayer’s hematoxylin and were mounted and photographed with an Axioplan microscope (Carl Zeiss, Germany). Primary antibodies were omitted for the negative controls.
Anti sox17
Manufactured by Abcam
Sourced in United States
Anti-SOX17 is a primary antibody that targets the SOX17 protein. SOX17 is a transcription factor involved in the regulation of embryonic development and cell fate determination. This antibody can be used in various research applications, such as Western blotting, immunohistochemistry, and immunocytochemistry, to detect and study the SOX17 protein.
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Lab products found in correlation
Anti notch1, by Abcam (2 mentions)
Anti notch4, by Santa Cruz Biotechnology (2 mentions)
Anti notch3, by Abcam (2 mentions)
Axioplan microscope, by Zeiss (2 mentions)
Anti β actin, by Abcam (2 mentions)
Anti tfpi2, by Abcam (1 mentions)
Bicinchoninic acid protein assay kit, by Thermo Fisher Scientific (1 mentions)
Anti n cadherin, by Abcam (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Complete protease inhibitor, by Roche (1 mentions)
Anti e cadherin, by Abcam (1 mentions)
Anti vimentin, by Abcam (1 mentions)
Anti zeb1, by Cell Signaling Technology (1 mentions)
Anti n cadherin, by Cell Signaling Technology (1 mentions)
Anti e cadherin, by Cell Signaling Technology (1 mentions)
Anti ar, by Cell Signaling Technology (1 mentions)
Anti vimentin, by Cell Signaling Technology (1 mentions)
Anti notch2, by Cell Signaling Technology (1 mentions)
Gapdh, by Immunoway (1 mentions)
Anti ssea4, by Merck Group (1 mentions)
8 protocols using anti sox17
1
Immunohistochemical Analysis of OSCC
2
Western Blot Analysis for Protein Expression
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Western blot analysis was performed as described previously (18 (link)). Total protein was prepared using extraction buffer comprising NaCl/Pi supplemented with 0.5% Triton X-100, 1 mM EDTA, 1 mM phenylmethyl sulfonyl fluoride and complete protease inhibitors (Roche Diagnostics). The concentration of each protein lysate was determined using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Inc.). Equal amounts of total protein (50 µg/lane) were separated by 12% SDS/PAGE. Subsequently, samples were transferred to nitrocellulose membranes and blocked for 60 min at room temperature in NaCl/Pi containing 5% skim milk powder (w/v). The membranes were immunoblotted using the following primary antibodies: Anti-fibronectin (cat. no. ab2413; 1:500; Abcam), anti-N-cadherin (cat. no. ab18203; 1:500; Abcam), anti-vimentin (cat. no. ab92547; 1:500; Abcam), anti-E-cadherin (cat. no. ab40772; 1:500; Abcam), anti-SOX17 (cat. no. ab224637; 1:500; Abcam) and anti-β-actin (cat. no. ab8227 1:500; Abcam) overnight at 4°C. Subsequently, they were incubated with IRDye-800-conjugated anti-rabbit secondary antibodies (cat. no. ab6940, 1:10,000; Abcam) for 30 min at room temperature. The specific proteins were visualized using the Odyssey™ Infrared Imaging system (LI-COR Biosciences). β-actin expression was used as an internal control to confirm equal loading of the protein samples.
3
Western Blot Analysis of EMT Markers
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Protein samples (50 µg) were transferred to PVDF membranes. After blocking with 5% non-fat milk for 2 h at room temperature, the membranes were incubated with primary antibodies overnight at 4°C, anti-E-Cadherin (1:2,000, Cell Signaling Technology, cat.no.14472), anti-N-cadherin (1:2,000, Cell Signaling Technology, cat.no.13116s), anti-Vimentin (1:1,000, Cell Signaling Technology, cat. no.5741s), anti-Zeb-1(1:2,000, Cell Signaling Technology, cat.no.70512s), anti-AR (1:2,000, Cell Signaling Technology, cat.no.19672s), anti-SOX17 (1:2,000, Abcam), anti-Notch1(1:2000, Abcam); anti-Notch2 (1:2,000, CST); anti-Notch3 (1:1,000, Abcam); anti-Notch4 (1:1000, Santa Cruze), and anti-GAPDH (1:1,000, CST, cat. no.5174s) was used as a loading control. The intensity of the protein bands was determined using Image-Pro plus 6.0.
4
Immunoreactivity of Notch Signaling Pathway
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All the embedded PCa and CRPC samples were cut into 5-µm-thick sections. The immunoreactivities of SOX17, Notch1, Notch2, Notch3, Notch4 were detected by an immunoperoxidase staining procedure with the primary antibodies (anti-SOX17, 1:200, Abcam, cat.no.ab192453; anti-Notch1, 1:200, Abcam, cat. no.ab8925; anti-Notch2, 1:200, Cell Signaling Technology, cat.no. D76A6; anti-Notch3, 1:200, Abcam, cat.no. ab23426; anti-Notch4, 1:200, Santa Cruz Biotechnology, cat.no. sc-377399 ). Staining scoring, according to staining intensity, was defined as 0, no staining; 1, weak staining; 2, light staining; 3, moderate staining; and 4, strong staining. Staining scores of ≤1 were defined as negative expression, while staining scores of ≥2 were defined as positive expression.
5
Chromatin Immunoprecipitation (ChIP) Assay for SOX17 Binding
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For the ChIP Assay, cells were lysed using SDS lysis buffer and DNA was sheared optimized for about 500bp fragments by sonication. Protein-DNA complexes were precipitated by anti-SOX17 (Abcam) or anti-IgG antibody, then recovered using protein G agarose beads, washed, and eluted. Crosslinks were then reversed at 65°C overnight. The immunoprecipitated DNA was amplified by PCR for specific sequences (R1, R2 and R3) containing putative SOX17 binding sites. Primers are listed in Supplementary Table 3 .
6
Western Blot Analysis of SOX17 and SDF1
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Western blotting was performed as described previously [16 (link)]. In brief, we used radioimmunoprecipitation assay (RIPA) buffer to extract the proteins needed for western blotting. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis was then performed to separate the proteins, which were transferred to poly (vinylidene fluoride) membranes. The following antibodies were used: anti-SOX17 (Abcam, USA), anti-SDF1/CXCL12 (Abcam), and GAPDH (ImmunoWay, USA). Corresponding Alexa Fluor dyes were used for fluorescent detection. DAPI was used for nuclear counter staining. Images were captured on a Zeiss LSM780 laser scanning confocal microscope.
7
Immunofluorescence Staining of Stem Cells
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Cells were fixed with 3.7% formaldehyde in phosphate-buffered saline (PBS). After permeablisation with 0.1% to 0.2% Triton X-100/PBS, cells were incubated with a primary antibody, followed by staining with a secondary antibody conjugated with AlexaFluor488 (1:500) or AlexaFluor555 (1:500). The following primary antibodies were used in this study: anti-SSEA4 (1:250; Merck Millipore, Darmstadt, Germany), anti-OCT4 (1:400; Abcam, Cambridge, UK), anti-NANOG (1:20; R&D Systems, Minneapolis, MN, USA), anti-TRA-1-60 (1:250; e-Bioscience, Santa Clara, CA, USA), anti-β-III Tubulin (1:1,000; BioLegend, San Diego, CA, USA), anti-SOX17 (1:200; Abcam), anti-smooth muscle actin (1:250; Dako, Santa Clara, CA, USA), anti-Desmin (1:200; Thermo Fisher Scientific), anti-MAP2 (1:500; Santa Cruz, Dallas, TX, USA), and anti-Synapsin I (1:500; Abcam). Nuclei were counterstained with DAPI using VECTASHIELD mounting medium with DAPI (Vector Laboratories, Burlingame, CA, USA).
8
Characterization of Stem Cell Differentiation
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The following primary antibodies were used: 12G10 (anti-active β1 integrin), P5D2 (anti-β1 integrin, blocking antibody), anti-E-cadherin, anti-protein 4.1B (all from Santa Cruz Biotechnology), anti-α6 integrin antibody (LSB Biotech), anti-TSC2, anti-RhoA, anti-phosphorylated myosin light chain (all from Cell Signaling Technology), anti-SOX17 and anti-beta-actin (both from Abcam). The secondary antibodies were used as shown in Table S1 . Anti-NANOG, anti-CD184 (PE conjugate), anti-nestin (Alexa-647 conjugate) antibodies and their isotype control antibodies were purchased from BD Biosciences. Anti-brachyury and anti-SOX1 antibodies were purchased from R&D Systems (Abingdon, UK). The reagent used in mesodermal lineage differentiation (CHIR99021) was purchased from Sigma-Aldrich Chemicals.
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