HUVEC and WJ-MSC bilayers were fixed with 4% PFA at 2 and 24 h. Cells were permeabilized with 0.15% triton X-100, blocked with 5% goat serum for 30 min at RT, then incubated overnight at 4°C with mouse anti-human VE-cadherin (10 µg/ml) (BD Biosciences, U.K. catalog# 5556661). After washes, cells were incubated for 2 h in the dark with goat anti-mouse IgG-FITC (Sigma Aldrich, U.K., catalog# F0257) (1:100). Unbiased ten images per coverslip were obtained with Nikon fluorescence microscope with appropriate filters to allow visualization of PKH26 and FITC (duration of co-culture and sample ID were blinded). Images were used to count paracellular junctions and characterised according to VE-cadherin staining pattern at cell-cell borders: continuous or disrupted. A grid was used by ImageJ software so that all the junctional regions had the same chance of being counted, and junctions from every other square which did not cross the ‘forbidden line’ were analysed [23 (link),24 (link)].
Mouse anti human ve cadherin
Manufactured by BD
Sourced in Netherlands
Mouse anti-human VE-Cadherin is a laboratory reagent used in research applications. It is an antibody that specifically binds to the VE-Cadherin protein, which is a cell adhesion molecule expressed on the surface of endothelial cells.
Automatically generated - may contain errors
Lab products found in correlation
Axio observer, by Zeiss (3 mentions)
Vectashield, by Vector Laboratories (3 mentions)
Alexa fluor 488 or 647, by Thermo Fisher Scientific (2 mentions)
Goat anti mouse igg fitc, by Merck Group (1 mentions)
Fluorescence microscope, by Nikon (1 mentions)
Mouse anti human cd31, by Thermo Fisher Scientific (1 mentions)
Mouse anti human vwf, by Agilent Technologies (1 mentions)
6 protocols using mouse anti human ve cadherin
1
Immunostaining of HUVEC-WJ-MSC Bilayers
2
Immunofluorescent Staining of VE-Cadherin
3
Serum-induced Endothelial Cell Responses
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HPMECs (p5) were cultured in eight-well chamber slides (80826, ibiTreat, µ-Slide 8 Well). Confluent monolayers were incubated with 10% pooled serum (healthy control (pooled n=12), non-ICU (pooled n=8) and ICU (pooled n=26)) in no fetal calf serum (FCS) EGM medium for 24 h. Next, cells were fixed with 4% PFA and 0.2% Triton-X100 in Hanks’ buffered salt solution (HBSS) or 4% PFA in HBSS (for HS and LEA staining) for 10 minutes at room temperature, blocked with 3% goat serum in HBSS and incubated with fluorescein isothiocyanate (FITC)-labelled Lycopersicon esculentum (LEA-FITC, L0401; Sigma, Houten, The Netherlands), Mouse Anti-Human VE-cadherin (55-7H1; BD Biosciences, Heidelberg, Germany), Mouse Anti-heparan sulfate (10E4, 370255-1; AMSBIO, Abingdon, UK) or Mouse Anti-TM (MA5-11454; Thermo Fisher) at 4°C overnight, followed by an appropriate secondary antibody and phalloidin-TRITC (VE-cadherin samples) for 1 h. Finally, Hoechst 33528 (1/1000) was added and cells were viewed using a LEICA SP8 WLL confocal microscope (Leica, Rijswijk, The Netherlands). Fluorescent images were analysed using Image J software. Quantification is described in the supplementary material .
4
VE-Cadherin Immunofluorescence Staining
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Cells were fixed in 2% PFA for 15 min at room temperature and incubated with mouse anti-human VE-Cadherin (BD Sciences) at 4 °C overnight48 (link). Secondary antibodies labeled with AlexaFluor 488 were used for detection. Slides were mounted using DAPI-containing Vectashield (Vector Laboratories) and imaged using a Zeiss Axio Observer. Image processing was performed using FIJI.
5
Immunofluorescent Staining of VE-Cadherin
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Cells were fixed in 2% PFA for 15 minutes and incubated with mouse anti-human VE-Cadherin (BD Sciences) at 4 o C overnight. Secondary antibodies conjugated to Alexa Fluor® 488 or 647 were used for detection (Thermo Fisher). Slides were mounted using DAPI (4',6-diamidino-2-phenylindole)-containing Vectashield (Vector Laboratories Inc., Burlingame, CA) and imaged using a Zeiss Axio Observer. Image processing was performed using FIJI (NIH).
6
Immunofluorescence Staining of Endothelial Cells
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Cells were washed with PBS and fixed with 3.7% formaldehyde. For CD31 staining, samples were blocked with 2% BSA and incubated with mouse anti-human CD31 (1:200, eBioscience) overnight at 48C, followed by incubation with the Alexa FluorV R 488 goat anti-mouse secondary antibody (1:500, Molecular Probes) for 30 min. For vonWillebrand factor (vWF) and VE-cadherin staining, samples were permeabilized with 0.2% Triton X-100, blocked with 2% BSA, and incubated with mouse anti-human vWF (1:50, Dako) and mouse anti-human VE-cadherin (1:50, BD Bioscience), respectively, overnight at 48C, followed by incubation with Alexa Fluor V R 555 and Alexa Fluor V R 488 goat anti-mouse secondary antibodies (1:500, Molecular Probes), respectively, for 30 min. Cell nuclei were counterstained with DAPI. HUVECs were stained as positive controls of ECs.
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