For flow cytometry analysis, EC cells generated via induction of hETV2 were dissociated with Accutase. Single cells were incubated with CD31 (Abcam, Ab119339, 0.5 μq/5*105 cells) for 30 min on ice. The cells were washed and subsequently incubated with secondary FITC-labelled antibody for 30 min on ice. Finally, the cells were washed, filtered and analyzed on the BD LSRII instrument.
Ab119339
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab119339 is a recombinant monoclonal antibody targeting the human Ki-67 protein. The antibody is produced in HEK293 cells and purified by protein A affinity chromatography.
Automatically generated - may contain errors
Lab products found in correlation
Lsr 2, by BD (2 mentions)
Ab46154, by Abcam (2 mentions)
Ab18197, by Abcam (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Ab8822, by Abcam (2 mentions)
Ab185732, by Abcam (2 mentions)
Ab6586, by Abcam (2 mentions)
Ab76442, by Abcam (1 mentions)
Mouse and rabbit specific hrp dab detection ihc kit, by Abcam (1 mentions)
Image pro plus software, by Media Cybernetics (1 mentions)
Ab104139, by Abcam (1 mentions)
Cy3 conjugated goat anti mouse igg h l, by Jackson ImmunoResearch (1 mentions)
4 6 diamidino 2 phenylindole dapi, by Abcam (1 mentions)
Axio scan z1, by Zeiss (1 mentions)
Osteocalcin, by Santa Cruz Biotechnology (1 mentions)
Ab8448, by Abcam (1 mentions)
Ix83 microscope, by Olympus (1 mentions)
Ab34710, by Abcam (1 mentions)
Prolong gold antifade mountant containing 4 6 diamidino 2 phenylindole dapi, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
17 protocols using ab119339
1
Flow Cytometry Analysis of hETV2-induced EC Cells
2
Immunofluorescent Staining of Periodontal Tissues
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Frozen sections of periodontal tissues were prepared for immunofluorescent staining. The gingiva and periodontal ligament were tested by immunofluorescence against CD31 (ab119339) and VEGF (ab46154) (Abcam, USA) 30 days after induction of periodontitis. Sections were stained using primary antibodies at 4℃ overnight. The nuclei were then stained using DAPI at 37℃ for 30 min after incubation with secondary antibodies of Alexa Fluor 488 and Alexa Fluor 594.
3
Immunohistochemical Analysis of Ovarian Tissues
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Cleared ovaries were transferred to 10 mL 1× PBST (PBS with 0.1% Triton-X100) for 1 day to remove the residual SDS. Samples were then incubated with primary antibodies (tyrosine hydroxylase 1:100 dilution, ab76442, Abcam; platelet endothelial cell adhesion molecule 1 (PECAM-1, CD31) 1:20 dilution, ab119339, Abcam; and proliferating cell nuclear antigen (PCNA) 1:50 dilution, ab18197, Abcam) for 3 days, washed in 1× PBST for 1 day, and incubated with secondary antibodies (Alexa Fluor 488 and Alexa Fluor 594 IgG, Sigma–Aldrich, Steinheim, Germany) for another 3 days in the dark. Three hours before scanning, the samples were incubated in sRIM solution (70% sorbitol in 2× PBS with 0.2% sodium azide) to correct the refractive index. All procedures were conducted at 37 °C with shaking.
4
Quantitative Immunohistochemistry Analysis
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Some histological sections were used for immunohistochemistry of PECAM-1, VEGF, and PCNA35 (link), 45 , 49 (link). For PECAM-1, sections were incubated with an anti-CD31 mouse monoclonal antibody (ab119339, abcam, Cambridge, UK) diluted at 1:100 in 3% BSA-PBS and incubated overnight at 4 °C. For VEGF, sections were incubated with an anti-VEGF rabbit polyclonal antibody (ab46154, abcam, Cambridge, UK) diluted at 1:100 in 1% BSA-PBS for 45 minutes. For PCNA, sections were incubated with an anti-PCNA rabbit polyclonal antibody (ab18197, abcam, Cambridge, UK) diluted at 1:4,000 in PBS for 2 hours.
All sections were incubated with a Mouse and Rabbit Specific HRP/DAB Detection IHC kit (abcam, Cambridge, UK) before counterstained with hematoxylin. Images were captured with a microscope imaging system. Images of the sections were quantified using ImageJ software (1.48 v, Wayne Rasband, National Institutes of Health, USA) with color threshold selection. The common values used for all samples in terms of Hue, Saturation, and Brightness are in the range between 50–235, 0–255, and 0–155, respectively. Once the area of the intensity with common values was selected, ImageJ calculated the area of intensity over background in percentage.
All sections were incubated with a Mouse and Rabbit Specific HRP/DAB Detection IHC kit (abcam, Cambridge, UK) before counterstained with hematoxylin. Images were captured with a microscope imaging system. Images of the sections were quantified using ImageJ software (1.48 v, Wayne Rasband, National Institutes of Health, USA) with color threshold selection. The common values used for all samples in terms of Hue, Saturation, and Brightness are in the range between 50–235, 0–255, and 0–155, respectively. Once the area of the intensity with common values was selected, ImageJ calculated the area of intensity over background in percentage.
5
Rat Angiogenesis Quantification Protocol
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The assessment of angiogenesis in the rat liver and lung was conducted on three different rats from each group. Paraffin-embedded lung and liver sections (4 µm) were dewaxed and hydrated, and antigen was repaired by EDTA. After blocking with 10% bovine serum albumin for 1 h at room temperature, the sections were incubated with anti-CD31 (ab119339, 1:100; Abcam, Cambridge, UK) antibody overnight. The next day, after washing with PBS, sections were incubated with Cy3-conjugated goat anti-mouse IgG (H+L) (115165003, 1:500; Jackson ImmunoResearch, West Grove, PA, USA) and anti-mouse secondary antibody for 1 h at room temperature. After washing with PBS three times, sections were fixed with 4′,6-diamino-2-phenylindole (DAPI) (ab104139; Abcam, Cambridge, UK) for 10 m. For each section, five randomly selected fields were observed with a fluorescence microscope (Pannoramic DESK, P-MIDI, P250; 3DHISTECH Inc, Budapest, Hungary), and the microvessel density (MVD) was calculated as the number of CD31 positive cells by Image-Pro Plus software (version 6.0; Media Cybernetics Inc, Rockville, MD, USA).
6
Immunostaining of Bone Markers
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For immunohistochemistry and immunofluorescence, the sections were incubated with primary antibodies against osteocalcin (1:3200; Santa Cruz Biotechnology, Dallas, TX, USA), CD-31 (1:200; Abcam, ab119339; Cambridge, MA, USA), and osteopontin (1:200; Abcam, ab8448) for 24 h at 4 °C. After 24 h, the sections were washed with Dulbecco’s PBS(DPBS) and incubated with the following secondary antibodies: DISCOVERY UltraMap anti-Rb HRP (Roche Diagnostics Ltd., Basel, Switzerland) and goat anti-rabbit Alexa Fluor 488 (Invitrogen; A11034; 1:200) at room temperature for 1 h. After washing with DPBS, the sections were stained with 4,6-diamidino-2-phenylindole (DAPI) (1:500, Abcam) for 10 minutes. The sections were then mounted and examined using a fluorescence microscope (Zeiss Axioscan Z1, Germany).
7
Visualizing Melanoma Tumor Microenvironment
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Human melanoma cryosections were obtained from OriGene Technologies. Murine melanoma sections were prepared from B16F10 melanoma tumors embedded in optimal cutting temperature (OCT) compound following fixation with 10% formalin and cryopreservation in 30% sucrose. Tissue sections were blocked for 1 hour with 2% bovine serum albumin in PBS with 0.05% Tween 20 (PBS-T) at room temperature (RT), after which samples were incubated with WT CCL4 (25 μg/ml) or equimolar CBD-CCL4 in PBS-T for 2 hours at RT. Tissues were then stained with mouse anti-human CD31 (ab119339, Abcam), goat anti-mouse CCL4 (AF-451-NA, R&D Systems), and rabbit anti–collagen I antibody (ab34710, Abcam) for 1 hour at RT. After staining with the appropriate fluorescent secondary antibodies, sections were covered with ProLong Gold Antifade Mountant containing 4′,6-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific) and sealed with a coverslip. Imaging was done with an IX83 microscope (Olympus), and images were analyzed using ImageJ software (National Institutes of Health).
8
Immunofluorescence Characterization of ADPs
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The ADPs (passage 3) were grown on a four-well chambered cell culture slide. After fixation with 4% paraformaldehyde and washing with PBS, the cells were incubated with primary antibody against CD146 (dilution 1:100; ab210072), PDGFR-β (dilution 1:100; ab69506), NG2 (dilution 1:100; ab50009), CD45 (dilution 1:100; ab10558), CD34 (dilution 1:100; ab81289), CD31 (dilution 1:100; ab119339), CD73 (dilution 1:100; ab175396), CD90 (dilution 1:100; ab225), CD105 (dilution 1:100; ab11414), Nestin (dilution 1:100; ab11306), and MAP2 (dilution 1:100; ab36447) (all from Abcam, see Supplemental Table 1) in PBS/0.1%Tween 20 and 10% normal goat serum overnight at 4°C. The cells were washed with PBS and incubated with secondary antibodies: goat anti-rabbit IgG Alexa Fluor 594 (dilution 1:500; A-11012), goat anti-rabbit IgG Alexa Fluor 488 (dilution 1:500; A-11008), and goat anti-mouse IgG Alexa Fluor 488 (dilution 1:500; A-11029) (all from Thermo Fisher Scientific, Waltham, MA, USA) in PBS for 45 min at room temperature. After washing in PBS, the cells were mounted in antifade reagent with 4’,6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific) for 1 min and visualized with an inverted fluorescence microscope Axio Observer A1 (Carl Zeiss, Jena, Germany).
9
Immunofluorescence Staining of Tissue Sections
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Sections (8 μm thickness) were cut using the cryostat (Microm HM 525, Thermo Scientific). Immunofluorescence staining was performed using the following primary antibodies: mouse anti-CD31 (ab119339, Abcam, Cambridge, UK), rabbit anti-CD34 (ab185732, Abcam), mouse anti-collagen type I (ab6308, Abcam), rabbit anti-collagen type IV (ab6586, Abcam) or FITC-conjugated von Willebrand factor (ab8822, Abcam). The following secondary antibodies were used: goat anti-mouse Alexa Fluor 568 conjugate (ab175473, Abcam) and donkey anti-rabbit Alexa Fluor 488 conjugate (ab150073, Abcam). Autofluorescence was minimized with the Autofluorescence Eliminator Reagent (Millipore) according to the manufacturer’s instructions. All sections were counterstained with DAPI (Sigma) and mounted with ProLong Gold Antifade (Life Technologies, Carlsbad, CA, USA). Staining was visualized by confocal microscopy (LSM 700, Carl Zeiss, Oberkochen, Germany).
10
Flow Cytometry Analysis of hETV2-induced EC Cells
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For flow cytometry analysis, EC cells generated via induction of hETV2 were dissociated with Accutase. Single cells were incubated with CD31 (Abcam, Ab119339, 0.5 μq/5*105 cells) for 30 min on ice. The cells were washed and subsequently incubated with secondary FITC-labelled antibody for 30 min on ice. Finally, the cells were washed, filtered and analyzed on the BD LSRII instrument.
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