To assess the function of the isolated CCs, CCs were characterised based on its capability to form cardiomyocytes, smooth muscle cells and vascular endothelial cells through the formation of CardioStem Spheres (CSps). Briefly, approximately 2 × 104 CCs were plated onto a 100 mm bacteriological-grade petri dish in 5 mL leukemic inhibitory factor- (LIF) deprived CGM to generate CSps. The generated CSps were then plated onto laminin-coated (1 µg/mL) Lab-Tek 8 well-chamber slides and incubated in 5% CO2 incubator at 37oC for 7 days and the medium was changed every three days. Samples were collected and fixed with 4% PFA on ice at day 7 and stained for cardiac troponin I (cTnI) (Santa Cruz Biotechnology, Dallas, TX, USA), smooth muscle actinin (SMA) (Sigma-Aldrich, St. Louis, MO, USA), and von Willebrand factor (vWF) (Dako, Denmark) to detect successful differentiation.
Von willebrand factor vwf
Manufactured by Agilent Technologies
Sourced in United States
The Von Willebrand factor (vWF:) is a plasma glycoprotein that plays a crucial role in hemostasis. It facilitates the adhesion of platelets to the site of vascular injury, promoting the formation of a platelet plug. vWF also binds to and stabilizes the blood coagulation factor VIII, preventing its rapid degradation.
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Lab products found in correlation
Biotinylated anti mouse igg, by Vector Laboratories (2 mentions)
Fr180204, by Santa Cruz Biotechnology (2 mentions)
Goat anti rabbit igg peroxidase, by Merck Group (2 mentions)
Hrp conjugated donkey anti goat igg, by Jackson ImmunoResearch (2 mentions)
Recombinant scf, by Thermo Fisher Scientific (2 mentions)
α smooth muscle actin α sma, by Merck Group (2 mentions)
Goat anti mouse igg alkaline phosphatase, by Merck Group (2 mentions)
Wbb6f1 kit w v, by Jackson ImmunoResearch (2 mentions)
Mouse c kit neutralizing antibody ack2, by Thermo Fisher Scientific (2 mentions)
Stem cell factor (scf), by Abcam (2 mentions)
Cardiac troponin 1 ctni, by Santa Cruz Biotechnology (1 mentions)
α sma, by Merck Group (1 mentions)
Methanol, by Thermo Fisher Scientific (1 mentions)
Prolong gold with 4 6 diamidino 2 phenylindole dapi, by Thermo Fisher Scientific (1 mentions)
Horse serum, by Vector Laboratories (1 mentions)
Anti cd31, by Agilent Technologies (1 mentions)
Phase contrast microscope, by Zeiss (1 mentions)
C2 confocal microscope, by Nikon (1 mentions)
Prox1, by R&D Systems (1 mentions)
Ve cadherin, by Santa Cruz Biotechnology (1 mentions)
7 protocols using von willebrand factor vwf
1
Characterization of Cardiac Cell Differentiation
2
Immunocytochemical Characterization of Cell Monolayers
3
Histological Analysis of Tumor Samples
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For histology and immunohistochemistry, following euthanasia animals were perfused with PBS and then 3% paraformaldehyde (Acros Organics, New Jersey, USA), tumor was collected, and fixed in 3% paraformaldehyde containing 3% sucrose (Fisher Chemical, Waltham, MA, USA). Tissues collected from radioactive animals were kept in a shielded area for decay, and then tissue sections were prepared for paraffin blocks and sectioning. Standard immunohistochemical staining procedures were performed as recommended by the suppliers of primary antibodies. The following antibodies were used to delineate the expression of corresponding antigens: Von Willebrand factor (vWF) (Dako, Carpinteria, CA, USA) and Ki67 (Millipore, Billerica, MA, USA).
4
Immunohistochemical Staining Protocol
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ThermoScript RNase H−reverse transcriptase and RNase inhibitor RNaseOut were obtained from Invitrogen (Carlsbad, CA). HotStart Taq DNA polymerase was purchased from Qiagen (Valencia, CA). The following rabbit polyclonal antibodies were used: anti-rat CCL2 (1 μg/mL) from Antigenix America (Huntington Station, NY); anti-human myeloperoxidase (MPO; 13.2 μg/mL) and von Willebrand factor (vWF; 10 μg/ml) from Dako (Glostrup, Denmark). The following mouse monoclonal antibodies were used: anti-rat CD68 (clone ED1; 1 μg/mL), CD11b (clone MRC OX-42; 1 μg/mL), and RECA-1 (clone HIS52; 5 μg/mL) from Serotec (Oxford, UK); anti-porcine glial fibrillary acidic protein (GFAP; clone GA5; 0.1 μg/mL) from Chemicon International (Temecula, CA); anti-bovine S100B (clone 4C4.9; diluted 1:200) and anti-human intercellular adhesion molecule 1 (ICAM1; clone MEM-111; 10 μg/mL) from Abcam (Cambridge, MA); anti-rat P-selectin (SELP; clone RP-2; 50 μg/mL) [16 (link)], a generous gift from Dr. Andrew Issekutz (Department of Pediatrics, Dalhousie University, Halifax, Nova Scotia, Canada). Secondary antibodies were obtained from Molecular Probes (Eugene, OR). These were goat anti-rabbit and anti-mouse antibodies conjugated with Alexa Fluor 488 or 594. They were used at a concentration of 2 μg/mL.
5
Regorafenib and PDGF-BB Signaling
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Regorafenib (BAY 73-4506) and dimethyl sulfoxide (DMSO) were purchased from Selleckchem (Houston, TX, USA) and Sigma–Aldrich (St. Louis, MO, USA). Platelet-derived growth factor-beta (PDGF-BB) was purchased from R&D Systems (Minneapolis, MN, USA). Crystal violet and 4′,6′-diamidino-2-phenylindole (DAPI) were from Sigma–Aldrich (Munich, Germany). The following antibodies were used in this study: Phospho-ERK 1/2 (Thr 202/Tyr 204), ERK (Santa Cruz Biotechnology, Dallas, TX, USA), Phospho-SAPK/JNK (Thr183/Tyr185), SAPK/JNK (Cell Signaling, Danvers, MA, USA), von Willebrand factor (vWF, Dako, Hamburg, Germany), alpha smooth muscle actin (α-SMA, Sigma–Aldrich, Munich, Germany) and vinculin (Sigma–Aldrich, MO, USA).
6
Examining Placental Angiogenesis in Mice
7
Examining Placental Angiogenesis in Mice
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Pregnant FVB/NJ mice, WBB6F1- Kit W− v/ + were obtained from Jackson Laboratories (Bal Harbor, ME). Primary antibodies utilized were: c-kit (1:50, DAKO Carpinteria, CA), SCF (1:500; Abcam, Cambridge, MA), α-Smooth Muscle Actin (α-sma: 1:500, Sigma-Aldrich; St. Louis, MO), von Willebrand factor (vWF: 1:200; DAKO), and Ki67 (1:100; Abcam). Secondary antibodies utilized were: Biotinylated anti-mouse IgG (1:200, Vector, CA), HRP Conjugated Donkey Anti-Goat IgG (1:2000, Jackson Immunoresearch, PA), Goat Anti-Mouse IgG Alkaline Phosphatase (1:100, Sigma-Aldrich), and Goat Anti-Rabbit IgG-Peroxidase (1:100, Sigma-Aldrich). Mouse c-kit neutralizing antibody (ACK2; 50μg/kg) and recombinant SCF were both obtained from EBioscience (San Diego, CA). FR180204 (a selective ERK1/2 antagonist) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
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