Anti-FN rabbit polyclonal antibody was purchased from Abcam (Cambridge, UK); Anti-F-actin rhodamine conjugated antibody, Hoechst 33342 solution, Alexa Fluor 488 goat anti-rabbit IgG, Alexa Fluor 594 goat anti-rabbit IgG, and Alexa Fluor 488 donkey anti-goat IgG were purchased from Thermo Fisher Scientific (Waltham, MA); Anti-elastin antibody (RA75) was purchased from Elastin Products Co. Inc. (Owensville, MI); Anti-α SMA (A2547) were purchased from Sigma (St. Louis, MO); Anti-von Willebrand factor antibody was purchased from Dako Cytomation (Glostrup, Denmark); Anti-fibrillin-1 was kindly provided by Professor T. Nakamura (Kansai Medical University, Japan); Y27632 and C3 were purchased from Cayman Chemical (Ann Arbor, MI) and Cytoskeleton, Inc. (Denver, CO), respectively.
Anti von willebrand factor antibody
Manufactured by Agilent Technologies
Sourced in Denmark
The Anti-von Willebrand factor antibody is a laboratory reagent used to detect and measure the presence of von Willebrand factor, a protein involved in blood clotting. This antibody can be used in various immunoassay techniques to quantify von Willebrand factor levels in samples.
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Lab products found in correlation
Alexa fluor 488 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 donkey anti goat igg, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342 solution, by Thermo Fisher Scientific (1 mentions)
Anti α sma a2547, by Merck Group (1 mentions)
Alexa fluor 594 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Y 27632, by Cayman Chemical (1 mentions)
Infinite 200 plate reader, by Tecan (1 mentions)
96 well plate, by Thermo Fisher Scientific (1 mentions)
O phenylenediamine, by Merck Group (1 mentions)
Bovine serum albumin solution bsa, by Merck Group (1 mentions)
Alexa fluor 488 conjugate, by Thermo Fisher Scientific (1 mentions)
Metamorph software, by Molecular Devices (1 mentions)
4 protocols using anti von willebrand factor antibody
1
Immunofluorescence Staining of Cytoskeletal Proteins
2
Quantification of von Willebrand Factor
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The von Willebrand factor is released into the blood in a continuous and controlled manner by factors that stimulate the endothelium or platelets. The content of the von Willebrand factor antigen in citrated porcine plasma was determined in an ELISA test. A 96-well plate (Nunc, Roskilde, Denmark) was coated with the anti-von Willebrand factor antibody (DAKO, Glostrup, Denmark) for 12 h and blocked with 1% bovine serum albumin solution (BSA) (Sigma Aldrich, Saint Louis, MO, USA) in phosphate buffer saline (PBS; 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4) for 2 h at 37 °C. Samples diluted in 1% solution of BSA in PBS (1:200 v/v) were incubated for 1 h at 37 °C, and detection was carried out with labeled antibodies conjugated to horseradish peroxidase and diluted in 1% BSA in PBS (DAKO, Glostrup, Denmark). O-phenylenediamine was used as a substrate (Sigma Aldrich, Saint Louis, MO, USA). Samples were incubated with the substrate solution for 30 min at 37 °C. Absorbance was read at λ = 492 nm wavelength using a Tecan Infinite 200 plate reader (Tecan, Grödig, Austria). Pooled normal plasma in sodium citrate was the reference, and the results were expressed as a percentage of the reference standard. Specimens were analyzed in duplicate.
3
In vitro Angiogenesis Coculture Assay
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In vitro
angiogenesis was determined by using
a coculture assay. Briefly, HDF cells were grown to confluence in
24-well plates in 10% DMEM, 1% penicillin/streptomycin. Medium was
replaced, and 10000 HUVECs were plated on top of the fibroblast layer
cultured in complete endothelial growth medium supplemented with 1%
FBS. HUVECs, or medium with 0.1% DMSO, VEGF 25 ng/mL,1 (30 μM), and 1 (30 μM) + VEGF 25 ng/mL
and fibroblasts were propagated in coculture for 4 days at 37 °C
and 5% CO2. Cells in coculture were fixed in absolute ethanol
for 1 h at room temperature, washed twice in PBS, and blocked using
PBS 5% milk. HUVECs were identified by incubating with anti-von Willebrand
factor antibody (Dako, 1:1000) in PBS 5% milk overnight at 4 °C.
Antibody was removed and cells washed with PBS. The secondary antibody,
goat anti-rabbit IgG, Alexa Fluor 488 conjugate (Life Technologies,
1:1000), was added on cells and left for 1 h in the dark. Solution
was removed and plate was scanned using IncuCyte.
Photomicrographs
of von Willebrand factor-stained cocultures were analyzed using ImageJ
software. The Network area, length of all tubular structures, and
the number of branching points were measured in four representative
microscopic fields per well.
angiogenesis was determined by using
a coculture assay. Briefly, HDF cells were grown to confluence in
24-well plates in 10% DMEM, 1% penicillin/streptomycin. Medium was
replaced, and 10000 HUVECs were plated on top of the fibroblast layer
cultured in complete endothelial growth medium supplemented with 1%
FBS. HUVECs, or medium with 0.1% DMSO, VEGF 25 ng/mL,
and fibroblasts were propagated in coculture for 4 days at 37 °C
and 5% CO2. Cells in coculture were fixed in absolute ethanol
for 1 h at room temperature, washed twice in PBS, and blocked using
PBS 5% milk. HUVECs were identified by incubating with anti-von Willebrand
factor antibody (Dako, 1:1000) in PBS 5% milk overnight at 4 °C.
Antibody was removed and cells washed with PBS. The secondary antibody,
goat anti-rabbit IgG, Alexa Fluor 488 conjugate (Life Technologies,
1:1000), was added on cells and left for 1 h in the dark. Solution
was removed and plate was scanned using IncuCyte.
Photomicrographs
of von Willebrand factor-stained cocultures were analyzed using ImageJ
software. The Network area, length of all tubular structures, and
the number of branching points were measured in four representative
microscopic fields per well.
4
Histological Analysis of Rat Cardiac Tissue
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Cell sheets and heart ventricles of nude rats were immersion-fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 5-μm sections using a microtome. The sections were stained with HE or SR and assessed by optical microscopy [36 (link), 41 (link)]. Metamorph software (Molecular Devices, Sunnyvale, CA, USA) was used to separate the SR-stained and non-stained myocardium and to quantitatively measure each area. The sections were immunohistologically labeled with polyclonal anti-von Willebrand factor antibody (1:100) (DAKO, Glostrup, Denmark) at 4 °C overnight, and visualized using the Universal LSAB2 System HRP Kit (DAKO), which is an automated immunostaining system based on the LSAB Lepto-streptavidin-biotin-peroxidase method [36 (link), 41 (link), 42 (link)].
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