5 μm fixed sections were incubated with primary antibodies in a hybridization chamber for 1 h at room temperature or overnight at 4°C. The primary antibodies used were DclK1, COX-2, 5-LOX, Ki67, proliferating cell nuclear antigen (PCNA), CD133, CD44, Lgr5, Annexin V and β-catenin procured from Santa Cruz/Abgent/Abcam/Abcam/Cell Signaling. Following primary antibody, sections were incubated for 1 h with anti-mouse/anti-rabbit/anti-goat secondary antibody, then visualized with diaminobenzidine (DAB) and counterstained with H&E for IHC or with DAPI for immunohistofluorescence (IHF). Slides were observed under an Olympus microscope 1X701 and digital computer images were recorded with an Olympus DP70 camera.
Cd133
Manufactured by Santa Cruz Biotechnology
Sourced in United States, United Kingdom
CD133 is a cell surface glycoprotein that is commonly used as a marker for stem and progenitor cells. It is expressed on the surface of various cell types, including hematopoietic stem cells, endothelial progenitor cells, and certain types of cancer stem cells. CD133 plays a role in cell adhesion and signaling processes, and its expression can be used to identify and isolate these cell populations for research and clinical applications.
Automatically generated - may contain errors
Lab products found in correlation
β catenin, by Santa Cruz Biotechnology (4 mentions)
E cadherin, by Santa Cruz Biotechnology (3 mentions)
Aldh1, by Santa Cruz Biotechnology (3 mentions)
Proliferating cell nuclear antigen (pcna), by Santa Cruz Biotechnology (2 mentions)
Nanog, by Santa Cruz Biotechnology (2 mentions)
Vimentin, by Santa Cruz Biotechnology (2 mentions)
Mmp 9, by Santa Cruz Biotechnology (2 mentions)
Stat3, by Santa Cruz Biotechnology (2 mentions)
Egm 2 mv medium, by Cambrex (2 mentions)
Histopaque 1077, by Merck Group (2 mentions)
Fibronectin, by Santa Cruz Biotechnology (2 mentions)
Bcl 2, by Santa Cruz Biotechnology (2 mentions)
Dp70 camera, by Olympus (1 mentions)
5 lox, by Santa Cruz Biotechnology (1 mentions)
Annexin 5, by Santa Cruz Biotechnology (1 mentions)
Microscope 1x701, by Olympus (1 mentions)
Cox 2, by Santa Cruz Biotechnology (1 mentions)
Facscalibur flow cytometry system, by BD (1 mentions)
Cd105, by Santa Cruz Biotechnology (1 mentions)
Cd166, by Santa Cruz Biotechnology (1 mentions)
21 protocols using cd133
1
Immunohistochemical Profiling of Stem Cell Markers
2
Mesenchymal Stem Cell Characterization
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Isolated hTGSCs, hDPSCs, and hPDLSCs (passage 3) were characterized for their mesenchymal cell surface profiles, as described previously.16 (link)–18 (link) The hTGSCs and hDPSCs were trypsinized and incubated with the following conjugated antibodies: CD29, CD34, CD45, CD73, CD90, CD105, CD133, and CD166 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). The hPDLSCs were then incubated with primary antibodies raised against STRO-1, CD146, CD90, CD44, CD19, or CD14. The cells were washed with phosphate-buffered saline (PBS) to remove the excess primary antibodies. The cells were analyzed by flow cytometry using a FACSCalibur flow cytometry system (Becton Dickinson, San Jose, CA, USA).
3
Immunohistochemical Analysis of Pancreatic Cancer Markers
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Tissue arrays of pancreatic cancer specimen were purchased from Shanghai Outdo Biotech Co., Ltd (Shanghai, China). Sections were cut from paraffin embedded pancreatic tumour tissues. Immunostaining was performed with primary antibodies specific for DKK3 13 , β‐catenin, E‐cadherin (Epitomics Inc., Burlingame, CA, USA) 16 and CD133 (Santa Cruz Biotech) with appropriate dilutions and using normal host serum for negative controls, followed by staining with appropriate horse radish peroxidase(HRP)‐conjugated secondary antibodies. The slides were developed in diaminobenzidine and counterstained with a weak solution of haematoxylin solution stain. The stained slides were dehydrated and mounted in permount and visualized on an Olympus microscope (Olympus, Shinjuku, Tokyo, Japan).
4
Isolation and Characterization of Endothelial Progenitor Cells
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The EPCs isolated from rats were identified using a methodology from a previous study17 (link),18 (link). Mononuclear cells were incubated with Dil-acetyl-low density lipoprotein (LDL, 10
μg/mL; Invitrogen, Carlsbad, CA, USA) and fluorescein isothiocyanate-ulexeuropaeus
agglutinin-1 (UEA-1, 5 μg/mL; Sigma-Aldrich, Saint Louis, MO, USA). EPCs stained with
DiI-acetyl-LDL (absorption wavelength: 555 nm) and UEA-1 (absorption wavelength: 495 nm)
were identified using a confocal microscope. These EPCs were also stained with vascular
endothelial growth factor receptor (VEGFR)-2 (Abcam, Cambridge, UK), CD34, and CD133
(Santa Cruz Biotechnology, Santa Cruz, California, USA), as described in a previous study17 (link),19 (link). Furthermore, in order to characterize the subtype of EPCs, cells with antibodies
against FITC-CD14 (Santa Cruz Biotechnology) and PE-CD45 (Biolegend, San Diego, CA, USA)
were analyzed using a flow cytometer. CD14-/CD45- cells were
considered as advanced EPCs (endothelial colony-forming cells), while
CD14+/CD45+ cells were considered as early EPCs20 (link).
μg/mL; Invitrogen, Carlsbad, CA, USA) and fluorescein isothiocyanate-ulexeuropaeus
agglutinin-1 (UEA-1, 5 μg/mL; Sigma-Aldrich, Saint Louis, MO, USA). EPCs stained with
DiI-acetyl-LDL (absorption wavelength: 555 nm) and UEA-1 (absorption wavelength: 495 nm)
were identified using a confocal microscope. These EPCs were also stained with vascular
endothelial growth factor receptor (VEGFR)-2 (Abcam, Cambridge, UK), CD34, and CD133
(Santa Cruz Biotechnology, Santa Cruz, California, USA), as described in a previous study17 (link),19 (link). Furthermore, in order to characterize the subtype of EPCs, cells with antibodies
against FITC-CD14 (Santa Cruz Biotechnology) and PE-CD45 (Biolegend, San Diego, CA, USA)
were analyzed using a flow cytometer. CD14-/CD45- cells were
considered as advanced EPCs (endothelial colony-forming cells), while
CD14+/CD45+ cells were considered as early EPCs20 (link).
5
Western Blotting of Stem Cell Markers
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Western blotting was performed as previously described using total protein obtained from hDPCs. Antibodies for phosphorylated-AKT (1:1000, cat. no. #9271, Cell Signaling Technology, Inc., Danvers, MA, USA), AKT (1:1000, cat. no. #4685, Cell Signaling Technology, Inc.), phopholyrated-GSK3β (1:1000, cat. no. #9323, Cell Signaling Technology, Inc), GSK3β (1:1000, cat. no. #12456, Cell Signaling Technology, Inc.), β-catenin (1:1000, cat. no. 610153, BD Biosciences, Franklin Lakes, NJ, USA), proliferating cell nuclear antigen (1:1000, cat. no. #13110, Cell Signaling Technology, Inc.), β-actin (1:1000, cat. no. sc-4778, Santa Cruz Biotechnology, Inc., Dallas, TX, USA), ALP (1:100, cat. no. SC-15065, Santa Cruz), CD 133 (cat. no. NB120-16518, Novus Biologicals, CO, USA) were used as the primary antibodies, and goat anti-rabbit (cat. no. BA-1000, VECTOR LABORATORIES, INC.) and goat anti-mouse (HRP) (cat. no. BA-9200, VECTOR LABORATORIES, INC.) were used as the secondary antibodies (1:10000). The blots were analyzed using densitometry with ImageJ 1.44 software (National Institutes of Health).
6
Immunoblotting Analysis of Signaling Pathways
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Immunoblotting was performed using standard methods. After treatment with the
indicated drugs, cells were washed with cold PBS and lysed in buffer containing 20 mM
Tris pH 7.5, 150 mM NaCl, 100 mM MgCl2, 1% Nonidet P-40 and 10% glycerol
supplemented with HALT protease and phosphatase inhibitor cocktail (Thermo Fisher
Scientific) using a Q800R sonicator (Qsonica, Newtown, CT). Lysates were centrifuged
at 16,000×g for 5 min at 4°C. Protein concentrations
were determined by BCA assay (Thermo Fisher Scientific). Proteins were resolved by
SDS-PAGE and transferred to a polyvinylidenes difluoride membranes (Biorad) using the
Transblot Turbo Transfer System (Biorad, Hercules, CA). Immunoblotting was performed
per each antibody manufacturer's specifications. Antibodies used were pEGFR
(Y1068), EGFR, pAKT (S473), AKT, pERK (T202/Y204), ERK, cleaved PARP, cleaved
caspase-3, SOX2, BIM, phospho-FOXO1 (T24)/FoxO3a (T32), FOXO1, FOXO3 (Cell Signaling
Technology, Beverly, MA), GAPDH (EMD Millipore, Billerica, MA), Ki67 (Epitomics,
Burlingame, CA), phospho-FOXO6 (S184) (Abcam, Cambridge, MA), FOXO6 (Proteintech,
Chicago, IL), CD133, GKLF, OCT4, MYC (Santa Cruz, Dallas, TX), CD44 and CD24 (BD
Biosciences, San Jose, CA), and TY1 (Diagenode, Denville, NJ).
indicated drugs, cells were washed with cold PBS and lysed in buffer containing 20 mM
Tris pH 7.5, 150 mM NaCl, 100 mM MgCl2, 1% Nonidet P-40 and 10% glycerol
supplemented with HALT protease and phosphatase inhibitor cocktail (Thermo Fisher
Scientific) using a Q800R sonicator (Qsonica, Newtown, CT). Lysates were centrifuged
at 16,000×g for 5 min at 4°C. Protein concentrations
were determined by BCA assay (Thermo Fisher Scientific). Proteins were resolved by
SDS-PAGE and transferred to a polyvinylidenes difluoride membranes (Biorad) using the
Transblot Turbo Transfer System (Biorad, Hercules, CA). Immunoblotting was performed
per each antibody manufacturer's specifications. Antibodies used were pEGFR
(Y1068), EGFR, pAKT (S473), AKT, pERK (T202/Y204), ERK, cleaved PARP, cleaved
caspase-3, SOX2, BIM, phospho-FOXO1 (T24)/FoxO3a (T32), FOXO1, FOXO3 (Cell Signaling
Technology, Beverly, MA), GAPDH (EMD Millipore, Billerica, MA), Ki67 (Epitomics,
Burlingame, CA), phospho-FOXO6 (S184) (Abcam, Cambridge, MA), FOXO6 (Proteintech,
Chicago, IL), CD133, GKLF, OCT4, MYC (Santa Cruz, Dallas, TX), CD44 and CD24 (BD
Biosciences, San Jose, CA), and TY1 (Diagenode, Denville, NJ).
7
Comprehensive Protein Expression Analysis
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Cells and homogenised tumour tissue were lysed, and total protein (30 μg) resolved by SDS-PAGE and transferred to PVDF membranes, which were probed with following antibodies: EpCAM, erbB2 (both from Sigma-Aldrich), caspase-9, caspase-8, cleaved caspase-3, VDAC, COX IV, SDHA (all from Cell Signaling), CD44, HSP60, actin (all from Abcam), CD133, PARP-1/2 (both from Santa Cruz), and SDHC (Novus Biologicals). ECL western blotting substrate (Thermo Scientific) and ChemiDoc™ XRS+ System (BioRad) were used to visualise and evaluate the blots.
8
Western Blotting for Stem Cell Markers
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Western blotting was performed as described previously [9 (link), 11 (link), 12 (link)]. The following antibodies were used: CD44 (Cell Signaling), ALDH (BD), ID1 (Santa Cruz), ID2 (Santa Cruz), ID3 (Santa Cruz), CD133 (Santa Cruz Biotechnology), WNT16 (BD) and Ran (BD). Horseradish peroxidase labeled secondary antibodies were from Pierce.
9
Immunocytochemistry and Immunohistochemistry of Prostate Cancer
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For immunocytochemistry, the epithelial cells purified from the tumors and naive DU145 were first grown on a glass coverslip for 48h, and then were fixed in 4% paraformaldehyde. Fixed cells were stained with CD34 (1:400, Santa Cruz Biotech, Santa Cruz, CA), CD44 (1:200, Epitomics, Burlingame, CA), CD133 (1:400, Santa Cruz Biotech., Santa Cruz, CA), and C-KIT(1:200, Epitomics, Burlingame, CA) primary antibodies (mouse anti-human) followed by 1:2000 secondary antibody (anti-mouse IgG, CST, CA).
For immunohistochemistry, tumors were fixed in 4% paraformaldehyde solution and then dehydrated, sealed in wax, and cut. Cut tissue sections were stained with the primary antibodies as described previously 14 (link)
To determine the stromal cell types isolated from the peripheral zone (PZ) and transitional zone (TZ), the cells were stained with primary antibodies against vimentin (a marker for mesenchymal cells, especially fibroblasts) (Epitomics, Burlingame, CA), α-SMA (a marker for smooth muscle cells and myofibroblasts) (Abcam, Cambridge, UK), smoothlin (a marker for smooth muscle cells) (Santa Cruz Biotech, Santa Cruz, CA), and cytokeratin 18 (CK18) (a marker for peripheral cells) ( Abcam, Cambridge,UK) as described previously 14 (link). CK18 was expressed in all four cell types and used to monitor the efficiency of the isolation and purification procedure.
For immunohistochemistry, tumors were fixed in 4% paraformaldehyde solution and then dehydrated, sealed in wax, and cut. Cut tissue sections were stained with the primary antibodies as described previously 14 (link)
To determine the stromal cell types isolated from the peripheral zone (PZ) and transitional zone (TZ), the cells were stained with primary antibodies against vimentin (a marker for mesenchymal cells, especially fibroblasts) (Epitomics, Burlingame, CA), α-SMA (a marker for smooth muscle cells and myofibroblasts) (Abcam, Cambridge, UK), smoothlin (a marker for smooth muscle cells) (Santa Cruz Biotech, Santa Cruz, CA), and cytokeratin 18 (CK18) (a marker for peripheral cells) ( Abcam, Cambridge,UK) as described previously 14 (link). CK18 was expressed in all four cell types and used to monitor the efficiency of the isolation and purification procedure.
10
Investigating Molecular Markers in Stem Cells
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CBD was obtained from Sigma-Aldrich (St. Louis, MO, USA). CBD dissolved in absolute ethanol (EtOH) was stored at −20 °C. Antibodies against HIF-1α, E-cadherin, and N-cadherin were purchased from BD Biosciences (San Jose, CA, USA). Anti-Snai1, Src, VHL, ubiquitin, CD133, ALDH1A1, and Nanog were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-Slug, Snai1, and Vimentin were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-SOX2 was purchased from R&D Systems (Minneapolis, MN, USA). The anti-β-actin antibody was purchased from Sigma-Aldrich. The secondary antibodies anti-mouse-IgG-horseradish peroxidase (HRP) and anti-rabbit-IgG-HRP were purchased from Cell Signaling Technology. Anti-CD24 and CD44 antibodies were purchased from Invitrogen (Carlsbad, CA, USA). Cobalt (II) chloride (CoCl₂) and MG132 (proteasome inhibitor) were purchased from Sigma Aldrich.
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