At the end of drug treatment, the cells were fixed with 3% Formalin for 5 minutes, washed three times with PBS, and then permeabilized with 0.1% saponin (Sigma-Aldrich, St. Louis, MO) for 5 minutes. A mouse-derived monoclonal anti-VE-cadherin antibody (BD Biosciences, Franklin Lakes, NJ) at a concentration of 1:250 was added to each well for 2 hours. The wells were washed twice with PBS and a secondary antibody conjugated with FITC (Thermo Fisher Scientific, Waltham, MA) at a concentration of 1:500 and Hoechst dye (Thermo Fisher Scientific, Waltham, MA) at a concentration of 1:2000 was added together for an additional 30 minutes. When visualized using an inverted epi-fluorescence microscope (DMI 6000B, Leica Inc., Germany, 20x magnification), the FITC-conjugated antibody labels VE-Cadherin expression at the cell-cell junction and the Hoechst dye labels nuclei.
Anti ve cadherin antibody
Manufactured by BD
The Anti-VE-cadherin antibody is a lab equipment product developed for research purposes. It is a reagent used to detect and study the expression of VE-cadherin, a protein involved in the formation and maintenance of endothelial cell-cell junctions. This antibody can be used in various laboratory techniques, such as Western blotting, immunohistochemistry, and flow cytometry, to investigate the role of VE-cadherin in cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst dye, by Thermo Fisher Scientific (2 mentions)
Fluorescein isothiocyanate (fitc), by Thermo Fisher Scientific (2 mentions)
Saponin, by Merck Group (2 mentions)
Dnase 1, by Thermo Fisher Scientific (2 mentions)
Collagenase type 4, by Thermo Fisher Scientific (1 mentions)
Pe rat anti mouse cd144, by BD (1 mentions)
Liberase dl, by Merck Group (1 mentions)
Collagenase d, by Merck Group (1 mentions)
Anti cd68, by Bio-Rad (1 mentions)
Alexa 488 anti rat, by Thermo Fisher Scientific (1 mentions)
Anti cd45, by BD (1 mentions)
5 protocols using anti ve cadherin antibody
1
Immunofluorescent Labeling of VE-Cadherin
2
Isolation and Purification of Bone Marrow Endothelial Cells
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Bone marrow endothelial cells (ECs) were stained by intravenous injection of 10 μg anti-VE-cadherin antibody (BD Biosciences, PE Rat Anti-Mouse CD144, #562243) 5-10 min before euthanizing the mice. Tibias and femurs were gently crushed using a mortar and pestle and then digested with DNase I (200 U/mL; ThermoFisher, # EN0521), LiberaseDL (250 mg/mL; Sigma, # 5401160001), type IV collagenase (1 mg/mL; ThermoFisher, # 17104019), and collagenase D (500 μg/mL; Sigma, # 11088858001) with agitation for 30 min at 37 °C. The cells were dissociated to a single-cell suspension by passing through a 25G needle several times and filtered with a 70-µm nylon mesh to generate a single-cell suspension. Cells were sorted in two successive rounds to ensure high purity using a FACS Aria II flow cytometer.
3
Comprehensive Immunostaining of Retinal Vasculature
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Retinal vasculature and CNV lesions were immunostained with directly conjugated Alexa Fluor 488-Isolectin B4, Alexa Fluor 594-Isolectin B4, or Alexa Fluor 647-Isolectin B4 (Sigma). Endothelial cell junctions and phosphorylated VE-cadherin were stained with anti-VE-cadherin antibody (1:100; BD Rat 555289) and affinity purified rabbit antibodies against VE-cadherin pY658 and pY685 (Orsenigo et al., 2012 (link)). Phosphorylated c-Src was assessed using anti-phospho-Src (Tyr418) Antibody (1:100; Invitrogen Rabbit 44–660G). Secondary antibodies used were Alexa488 anti-Rat (1:500; Invitrogen Donkey A-21208) and Alexa555 anti-Rabbit (1:500; Donkey A-31572). Inflammatory cells were stained with anti-CD45 (1:300; BD Biosciences Goat 553076) and anti-CD68 (1:300; BioRad Rat MCA1957). Secondary antibodies used were Alexa488 anti-Rat (1:500; Invitrogen Donkey A-21208). Alexa555 anti-Goat (1:500; Invitrogen Donkey A-21432).
4
Immunofluorescent Labeling of VE-Cadherin
5
In Vivo Imaging of Extracellular Vesicles
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1 µl of labeled EVs was injected into mouse dorsal ear dermis. After 30 min, the mouse was sacrificed and perfused with Ringer’s solution followed by zinc fixative (4.5 mM CaCl2, 52 mM ZnCl2, 32 mM Zn(CF3COO)2, 2 mM Tris, and 38 mM glycine, pH 6.5, 340 mOsm/liter). Whole mount staining of the ear was performed as described previously (Kilarski et al., 2013 (link)). Ears were cut and fixed for 24 h in zinc fixative and 1% Triton X-100. The dorsal skin was separated from the rest of the ear, washed in TBS, and blocked for 1 h in TBS 0.5% casein. The dorsal skin was incubated with anti-VE-cadherin antibody (550548; BD Biosciences) and anti-podoplanin antibody (AF3244; R&D Systems) for 24 h, washed in TBS 0.1% Tween, and incubated with secondary antibodies for 24 h. After washing in TBS Tween 0.1%, the tissue was dehydrated with 70% ethanol and 100% ethanol and cleared and mounted on a glass slide in 2:1 benzyl benzoate/benzyl alcohol solution. Fluorescence images were acquired with an Olympus IX2-DSU fluorescence microscope and a 63× lens. Image stacks were processed with ImageJ (National Institutes of Health).
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