Immunofluorescent double staining analysis was performed on ECDHCC and HUVECs which were seeded on sterile slice cover slips in six well plates overnight. Following several washes with PBS, cells were fixed with 4% paraformaldehyde for 30 min at room temperature and permeabilized with 0.1% Triton X-100 in PBS for 5 min. The cells were overlaid with 5% BSA for 30 min, rinsed with PBS and incubated with a mixture of rabbit-anti-human CD105 (1:20 dilutions; Abcam, Cambridge, UK) and mouse-anti-human CD31 (1:40 dilutions; DAKO) or a mixture of rabbit-anti-human CD105 (1:20 dilutions) and mouse-anti-human VEGFR-2 (1:200 dilutions) (both from Abcam) overnight at 4°C. Cells were washed three times with PBS and then incubated with Alex-488-conjugated donkey-anti-rabbit IgG and Dylight-649-conjugated donkey-anti-mouse IgG (1:200 dilutions, Jackson ImmunoResearch, West Grove, PA, USA) secondary antibodies. The slices were incubated at 37°C for 45 min, and nuclear staining was performed with DAPI. Coverslips were mounted with fluorescent mounting medium onto glass slides, and examined with confocal microscopy (Leica TCS SP5II; Leica Microsystems, Wetzlar, Germany). Images were collected from at least three independent experiments and processed for presentation in figures using Adobe Photoshop 6.0 (Adobe Systems, San Jose, CA, USA).
Mouse anti human cd31
Manufactured by Agilent Technologies
Sourced in United Kingdom, Denmark
The Mouse anti-human CD31 is a laboratory reagent used for the identification and characterization of cells expressing the CD31 antigen, also known as PECAM-1 (Platelet Endothelial Cell Adhesion Molecule-1). CD31 is a transmembrane glycoprotein expressed on the surface of endothelial cells, platelets, and certain leukocyte subsets. This antibody can be used in various immunological techniques, such as flow cytometry and immunohistochemistry, to detect and analyze CD31-positive cells.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (4 mentions)
Rabbit anti human vwf, by Agilent Technologies (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (2 mentions)
Lsm 880 confocal microscope, by Zeiss (2 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Rabbit anti human cd105, by Abcam (1 mentions)
Dylight 649 conjugated donkey anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Tcs sp5 2, by Leica (1 mentions)
Fitc donkey anti chicken, by Jackson ImmunoResearch (1 mentions)
Donkey anti mouse cy3, by Jackson ImmunoResearch (1 mentions)
Alexa 555 conjugated goat anti rat igg, by Thermo Fisher Scientific (1 mentions)
Axiovision v4, by Zeiss (1 mentions)
Axiovert 200 microscope, by Zeiss (1 mentions)
Rat anti mouse cd31, by BD (1 mentions)
Biotinylated secondary antibody, by Vector Laboratories (1 mentions)
Streptavidin hrp, by Vector Laboratories (1 mentions)
Entellan, by Merck Group (1 mentions)
Formalin, by Merck Group (1 mentions)
22 protocols using mouse anti human cd31
1
Immunofluorescent Staining of Endothelial Cells
2
Immunofluorescent and Immunohistochemical Analysis of LOX-1 in Sickle Cell Disease
3
Immunofluorescence Staining of Vascular Markers
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Immunostaining was performed using the following primary antibodies: rabbit anti-human VWF (1:150; Dako), mouse anti-Human TSP (1:75, Laboratory Vision/ Neomarkers) rat anti-mouse CD31 (1:50; BD Biosciences), rat anti-mouse GPIbα/CD42 (1:100; EMFRET Analytics), mouse anti-human CD31 (1:50; Dako), and rabbit anti-mouse vascular endothelial growth factor-A (VEGF-A) (1:150; Santa Cruz). Following secondary antibodies were used: fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (1:200; BD Pharmingen), Alexa 555-conjugated goat anti-rat IgG (1:200; Invitrogen). Nuclei were counterstained with DAPI (1:1000). Fluorescence images were acquired with a Zeiss Axiovert 200 microscope and were analyzed using AxioVision v4.8 (Zeiss) and ImageJ v1.47c.
4
Immunohistochemical Analysis of CD31 Expression
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CAM sections were deparaffinized and rehydrated in an MICROM HMS740 automatic stainer (MICROM, Walldorf, Germany). Immunohistochemical analysis was performed using a streptavidin–biotin peroxidase complex system. Briefly, after rehydration, slides were subjected to heat-induced antigen-retrieval with 10 mM citrate buffer at pH = 6 for 2 min at 98 °C. The slides were washed with PBS and then incubated with 3% hydrogen peroxide for 10 min to inactivate endogenous peroxidases. Another washing step was performed, and nonspecific binding was blocked with a 2.5% (v/v) horse serum (Vector Labs, Newark, CA, USA) for 30 min, before overnight incubation at 4 °C with mouse anti-human CD31 (1:30) (Dako, Cambridge, UK). Sections were washed with 0.1% tween in PBS and incubated with a secondary biotinylated antibody (Vector Labs, Newark, CA, USA) for 20 min. After thoroughly washed with 0.1% tween in PBS, samples were incubated with streptavidin-HRP (Vector Labs, Newark, CA, USA) for 20 min, followed by 3,3′diaminobenzidine (DAB) incubation (Vector Labs, Newark, CA, USA). Finally, all sections were counterstained with Mayer’s hematoxylin, dehydrated and mounted with resinous mounting medium Entellan® (Merck, Darmstadt, Germany).
5
Immunostaining of Vascular Hydrogels
6
Fluorescent Visualization of 3D Cell Cultures
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Each of the seeded scaffolds was stained using fluorescent antibodies for microscopy. Scaffolds seeded with human dermal fibroblasts were stained with 1:6 phalloidin (Alexa Fluor 488, Thermo Fisher) and scaffolds seeded with human dermal endothelial cells were stained with 1:250 mouse anti-human CD31 (Dako) and 1:1000 rabbit anti-human vWF (Dako). Secondary staining was done using goat anti-mouse (Alexa Fluor 488, Thermo Fisher) and goat anti-rabbit (Alexa Fluor 568, Thermo Fischer). Scaffolds seeded with mesenchymal stem cells were not stained, since they expressed GFP. Z-stacks were taken to visualize cell migration through the scaffold by wide-field imaging at 20× magnification. Images were deconvoluted using AutoQuant X3 (Media Cybernetics) and visualised using Bitplane (Imaris software, Version 9.1).
7
Endothelial Cell Identification for Laser Capture Microdissection
8
Identification of HUVECs in Tissue Aggregates
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CD31 staining was used to identify the presence and spatial distribution of HUVECs located in and/or around the aggregates. The sample sections were incubated for 30 minutes in 10% FBS (diluted in phosphate buffered saline (PBS)) to prevent non-specific background staining (blocking). The samples were then incubated for 1 hour in the primary antibody, mouse anti-human CD31 (Dako, UK), at a dilution of 1:20 in PBS. The samples were then incubated for 1 hour in the secondary antibody, alexa-fluor 594-conjugated goat anti-mouse IgG1 (Life Technologies, UK), diluted 1:200 in PBS. DAPI staining was carried out using a DAPI-conjugated mounting medium without further adjustment. The samples were then ready for imaging.
9
Purification and Application of LYVE-1 Antibody
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Monoclonal mouse anti-human LYVE-1 (LYVE-1 mAb) was affinity-purified from hybridoma cultures as previously described in (Nightingale et al., 2009 (link); Johnson et al., 2007 (link)). Mouse anti-human CD31 was purchased from Dako (#M0823, Dako, UK) and mouse anti-human CD44 were from IGBRL (#9430, IGBRL, UK). Alexa Fluor® 488 Mouse anti-human CD31 (#303109) and Alexa Fluor® 488 rat anti-human CD44 (#103015) was purchased from Biolegend, USA. Secondary antibody goat anti-mouse Oregon Green® 488 was purchased from Life Technologies, UK (#06380). All antibodies were used at a concentration of 10 µg/ml unless otherwise stated.
10
Immunofluorescent Labeling of Endothelial Markers
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Cells were fixed in 4% paraformaldehyde and permeablised with 0.1% TritonX-100 at room temperature. Cells were blocked with donkey serum for 30 minutes to prevent non-specific binding. Cells were then incubated with 1% BSA in PBS containing the following primary antibody dilutions for 1 hr at room temperature; mouse anti-human CD31 (1:300, Dako, Clone JCT0A), mouse anti-human vWF (1:100, Dako), rabbit anti-human VE-Cadherin (1:200, Cell Signaling Technology), rabbit anti-human VEGFR-2 (1:100, Cell Signaling), mouse anti-human eNOS (1:100, BD Biosciences). Following incubation with primary antibody, cells were incubated with species appropriate, fluorophore tagged secondary antibodies for 45 minutes at room temperature. Cells were mounted with Prolong Gold Antifade Reagent (Invitrogen) and visualised using a LSM 880 confocal microscope (Zeiss).
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