CD8+ T lymphocytes were purified from splenocytes using a CD8+ isolation kit (Miltenyi Biotec). Purified CD8+ T lymphocytes were cultured in the presence of plate-bound anti-CD3 (10 µg/ml) with or without recombinant murine VEGF-A (50 ng/ml; Miltenyi Biotec). After 48 h of culture, cells were harvested and analyzed by cytometry or used to extract mRNA. In some experiments, anti–VEGF-R1 (20 µg/ml; R&D Systems) or anti–VEGF-R2 (10 µg/ml; clone 91202; R&D Systems) antibodies or isotype control were added to the culture medium. In some experiments, 11R-VIVIT (Merck Millipore) was added 1 h at 5 µM before the addition of VEGF-A and during the stimulation with VEGF-A.
Anti vegfr2
Manufactured by R&D Systems
Sourced in United States
Anti-VEGFR2 is a laboratory reagent used for research purposes. It is an antibody that specifically binds to the VEGFR2 receptor, which is a key component in the regulation of angiogenesis. The primary function of Anti-VEGFR2 is to facilitate the study of VEGFR2 and its role in various biological processes.
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Lab products found in correlation
Anti cd45, by BD (2 mentions)
Vegf a, by Merck Group (1 mentions)
Anti vegfr1, by R&D Systems (1 mentions)
11r vivit, by Merck Group (1 mentions)
Anti cd3, by Miltenyi Biotec (1 mentions)
Pecam 1, by BD (1 mentions)
Vectastain abc kit, by Vector Laboratories (1 mentions)
Anti vegfr 3, by R&D Systems (1 mentions)
Anti cxcr1, by R&D Systems (1 mentions)
Fluorescein isothiocyanate (fitc), by Jackson ImmunoResearch (1 mentions)
Anti lyve 1, by R&D Systems (1 mentions)
Anti prox1, by R&D Systems (1 mentions)
Anti nrp2, by R&D Systems (1 mentions)
Goat anti vegfr3, by R&D Systems (1 mentions)
Anti itga9, by R&D Systems (1 mentions)
Anti gfp, by R&D Systems (1 mentions)
Dylight 488, by Jackson ImmunoResearch (1 mentions)
Dylight 549, by Jackson ImmunoResearch (1 mentions)
Anti podocalyxin, by R&D Systems (1 mentions)
Lsm 800, by Zeiss (1 mentions)
11 protocols using anti vegfr2
1
VEGF-A Modulation of CD8+ T Cell Activity
2
Immunohistochemical Analysis of Vascular Markers
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Paraffin-embedded sections were deparaffinized, permeabilized, and incubated with goat polyclonal anti-VEGFR-3 (1:100, R&D Systems) or anti VEGFR-2 (1:100, R&D Systems) followed by biotin-streptavidin-HRP amplification using the Vectastain-ABC kit (Vector Lab), and post-stained with eosin.
For whole-mount staining, tissues were fixed overnight in 4% PFA and blocked overnight in blocking buffer (PBS, 5% goat serum, 0.3% Triton X-100, and 0.2% BSA). Tissues were incubated overnight at 4°C with biotinylated anti–mouse LYVE-1 (1:100, R&D Systems) or PECAM-1 (1:100, BD Biosciences) in blocking buffer followed by biotin-streptavidin-HRP amplification using the Vectastain-ABC kit.
For whole-mount staining, tissues were fixed overnight in 4% PFA and blocked overnight in blocking buffer (PBS, 5% goat serum, 0.3% Triton X-100, and 0.2% BSA). Tissues were incubated overnight at 4°C with biotinylated anti–mouse LYVE-1 (1:100, R&D Systems) or PECAM-1 (1:100, BD Biosciences) in blocking buffer followed by biotin-streptavidin-HRP amplification using the Vectastain-ABC kit.
3
IL-11 Modulates Endothelial Tube Formation
4
Immunohistochemical Analysis of Lymphatic Markers
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The following primary anti-human antibodies were used: sheep anti-podoplanin, goat anti- VEGFR-3, anti-LYVE-1, anti-PROX1, anti-ITGA9, anti-VEGFR-2, anti-NRP2, anti-podocalyxin and anti-GFP, all from R&D Systems, Minneapolis, MN. Mouse anti-human CD14 and anti-CD68 antibodies were from Santa Cruz Biotechnology, Dallas, TX and Thermo Fisher, Waltham, MA, respectively. Rabbit anti-acetylated histone H3 and anti-Ki-67 were from Upstate, Billerica, MA and Cell Signaling Technologies, Danvers, MA, respectively. Rabbit anti-mouse Lyve-1 and rat anti-Meca-32 antibodies were from AngioBio, Del Mar, CA, and BioXCell, West Lebanon, NH, respectively. Secondary antibodies conjugated to FITC, Cy3, DyLight 488, DyLight 549, and APC donkey anti-rabbit, anti-sheep, anti-rat, anti-mouse and anti-goat IgG were all from Jackson ImmunoResearch Laboratories (West Grove, PA).
5
Immunofluorescence Staining of Wound Tissues
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For immunofluorescence staining, wound tissues were fixed overnight in 4% paraformaldehyde, dehydrated in 20% sucrose solution overnight, and embedded in tissue-freezing medium (Leica, Wetzlar, Germany). Frozen blocks were cut into 20 μm thick sections. Tissue sections were blocked in PBST (0.2% Tween-20 in PBS) with 5% goat or donkey serum and incubated with the following primary antibodies: anti-CD31 (hamster, 2H8; Millipore, Billerica, MA; or rat, MEC13.3; BD Biosciences, Franklin Lakes, NJ), anti-Sox7 (goat; R&D Systems, Minneapolis, MN), anti-Sox17 (goat; R&D Systems), and anti-VEGFR2 (goat; R&D Systems) antibodies. After several washes, the sections were incubated with the following secondary antibodies: Alexa Fluor 488- or Alexa Fluor 596-conjugated anti-hamster IgG, Alexa Fluor 488- or Alexa Fluor 596-conjugated anti-rat IgG, or Alexa Fluor 488- or Alexa Fluor 596-conjugated anti–goat IgG (Jackson ImmunoResearch, West Grove, PA) antibodies. Nuclei were stained with DAPI (Invitrogen, Carlsbad, CA). The samples were mounted with fluorescent mounting medium (DAKO, Glostrup, Denmark), and immunofluorescence images were obtained using a confocal microscope (LSM800; Zeiss, Jena, Germany).
6
Immunohistochemical Analysis of Fibrovascular Tissues
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Paraffin sections of fibrovascular tissues were deparaffinized and hydrated through exposure with xylene and graded alcohols followed by water. As a pretreatment, microwave-based antigen retrieval was performed in 10 mM citrate buffer (pH 6). Sections were probed with the following primary antibodies: anti-Galectin-1 (Abcam), anti-VEGFR2 (R&D systems, Minneapolis, MN), anti-CD31 (DAKO, Tokyo, Japan), and anti-GFAP (glial fibrillary acidic protein; Leica, Exton, PA) antibodies. The secondary antibodies for fluorescent detection were AlexaFluor 488 and 546 (Life Technologies). Nuclei were counterstained with DAPI (diamidino-2-phenylindole), and sections were visualized under a Biorevo microscope (Keyence, Tokyo, Japan).
7
Characterization of Mechanically Activated hADSCs
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Cultures of mechanically activated hADSCs at different passages were phenotypically characterized by Fluorescence-Activated Cell Sorting (FACS). As previously reported by us for hADScs obtained from lipoaspirate [36 (link)], after trypsinization, cells were washed with PBS and 1 × 105 cells were re-suspended in 250 uL of PBS w/o Ca2+ and Mg+ (Euroclone, Pero, Italy) and incubated with antibodies directed against specific surface markers. The following antibodies were used: anti-CD44 (BD Biosciences, Milano, Italy), anti-CD90 (Merck Millipore, Vimodrone, Milano, Italy), anti-CD34 (Mylteni Biotec, Bologna, Italy), anti-CD45 (BD Biosciences), anti-CD146 (Biocytex, Marseille, France), anti-CD31 (Mylteni Biotec), anti-CD56 (Mylteni Biotec), anti-CD105 (Biorad, Milano, Italy), anti-CD144 (R&D System, Minneapolis, MN, USA), anti-CD166 (BD Biosciences), anti-CD133/2 (Mylteni Biotec), anti-CD73 (BD Biosciences), and anti-VEGFR2 (R&D System). Samples were fixed with paraformaldehyde 4% and then studied by flow cytometry (FACS Vantage, BD Bioscience, Milano, Italy) using a specific software (CellQuest Pro, BD Bioscience). Results were expressed as mean ± SD.
8
Multimarker Immunohistochemistry for Cardiovascular Tissues
9
Rat Liver Protein Expression Analysis
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Rat liver tissues and cells were lysed in RIPA buffer (Sigma-Aldrich) supplemented with protease inhibitor cocktail (Roche) and phosphatase inhibitor (Sigma-Aldrich). A total of 45 μg protein was separated in sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE). The separated proteins were transferred onto PVDF membranes (Bio-Rad). The membranes were incubated with anti-CRP (1：1,000, Abcam Inc.), anti-VEGF (R&D system), anti-VEGFR2 (R&D system), anti-β-catenin (active form, Cell Signaling), anti-pGSK3 (Cell Signaling), anti-β-catenin (active form, Cell Signaling), anti-albumin (Novus), anti-HNF1α (Abcam Inc.), anti-cyclinD1 (AbFrontier), anti-actin (Sigma-Aldrich), anti-tubulin (Abcam Inc.), and anti-GAPDH (AbFrontier) antibodies at 4℃ over-night. The membranes were then incubated with horseradish peroxidase-conjugated secondary anti-mouse IgG (Cell Signaling), anti-rabbit IgG (Cell Signaling) or anti-goat IgG (Santa Cruz) antibody for 1 hour at RT. Bands were detected using Clarity Western ECL kit (Bio-Rad).
10
Immunostaining of Adipose Tissue
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Fresh sWAT was fixed overnight and cut with a scalpel into smaller slices. Following a protocol adopted from a previous report,47 (link) the sWAT slices were incubated with 20 μg/mL proteinase K in 10 mmol/L Tris-HCl buffer for 5 min at room temperature, followed by methanol for 30 min, blocked with serum and stained with antibodies including: anti-CD31 (Abcam), anti-VEGFR2 (R&D system), anti-αSMA (Abcam), all at 1:200 dilution. After overnight incubation, donkey anti-rabbit AlexaFluor 546 and anti-goat AlexaFluor 488 secondary antibodies (ThermoFisher) were used. For visualising adipocytes, some slides were then incubated with BODIDY (1:200 from ThermoFisher) for 30 min. Images were taken by Olympus FV1200 confocal microscope.
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