The antibodies against VEGFR3 (goat anti-mouse, R&D Systems (Minneapolis, MN) #AF743) and VEGFR2 (goat anti-mouse, R&D Systems #AF644) were used at a 1:50 dilution; the anti-LYVE (rabbit anti-mouse Abcam,Cambridge, MA) #ab14917) was used at 1:250; the anti-αSMA (mouse anti-mouse Dako,Santa Clara, CA) #M0851) was used at 1:100, and anti-PECAM/CD31 antibody (rat monoclonal, catalog #DIA-310; Dianova, Hamburg, Germany) was used at 1:250. Secondary antibodies were Donkey anti-rabbit 594 (#21207 ThermoFisher, Grand Island, NY); Donkey anti-goat 488 (Abcam #ab150129); Donkey anti-goat 546 (ThermoFisher #A11056); and Donkey anti-mouse 488 (Abcam #ab150105) were all used at a dilution of 1:250. Sections from a total of 3 pairs of A10ΔEC mice and littermate controls were analyzed per antibody, with at least one pair of littermates of each gender.
Af644
Manufactured by R&D Systems
Sourced in China
AF644 is a recombinant human Galectin-3 Binding Protein (LGALS3BP) protein. LGALS3BP is a secreted glycoprotein that binds to galectin-3, a beta-galactoside-binding lectin. The biological function of this interaction is not fully characterized.
Automatically generated - may contain errors
Lab products found in correlation
Af743, by R&D Systems (2 mentions)
Ab150105, by Abcam (1 mentions)
Anti α sma, by Agilent Technologies (1 mentions)
Donkey anti goat 488, by Abcam (1 mentions)
A11056, by Abcam (1 mentions)
Dab horseradish peroxidase color development kit, by Beyotime (1 mentions)
Alexa488 conjugated donkey anti goat, by Thermo Fisher Scientific (1 mentions)
Rat anti mouse cd31, by BD (1 mentions)
Af2727, by R&D Systems (1 mentions)
Rat anti mouse cd41, by BioLegend (1 mentions)
Lsm 710, by Zeiss (1 mentions)
Lsm image browser, by Zeiss (1 mentions)
Mca497r, by Bio-Rad (1 mentions)
L2140, by Merck Group (1 mentions)
6 protocols using af644
1
Immunofluorescence Staining of Vascular Markers
2
Immunofluorescence Staining of CX40 and VEGFR2
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Primary antibodies: Anti-CX40 (1:500, catalog number CX40-A, RRID: AB_1616128; Alpha Diagnostics Int, Inc), Anti-VEGFR2 (1:125, catalog number AF644, RRID: AB_355500; R&D Systems). Secondary antibodies: Alexa fluor-conjugated antibodies (488, 555, and 633) from Invitrogen were used at 1:250 dilutions.
3
Immunohistochemical Evaluation of Angiogenic Markers
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Tumor sections (4 µm) were dewaxed and hydrated as conventional administration. After that, the sections were washed with phosphate-buffered saline (PBS, Baso, Zhuhai, China) three times, 5 min each time. Antigen was renovated at 95 °C, 60 min and cooled to 25 °C, 3min, three times. Primary antibodies CD31 (1:100, Cell signaling technology, Danvers, MA, USA), VEGF (1:400, VC001, R&D systems, Shanghai, China), VEGFR2 (1:400, AF644, R&D systems, Shanghai, China) were incubated at 4 °C, 24 h. Then, the sections were incubated at 37 °C for 30 min before washed with phosphate buffered saline (PBST). Washing 5 min, the sections were incubated with HRP polymer (1:400, R&D systems, Shanghai, China) at 37 °C, 30 min. Washing 5 min with PBST, the slices were cultured with DAB Horseradish Peroxidase Color Development Kit (Beyotime Biotechnology, Shanghai, China) for 3–15 min. Positive expression areas were observed under microscope (DMi8, Feica Microsystems, Germany).
4
Immunohistochemistry Markers for Vascular Imaging
5
Wholemount Fluorescence Staining of Brain
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Samples were fixed in 4% formaldehyde in PBS and processed as wholemounts for fluorescent staining as described previously for wholemount hindbrains (Fantin et al., 2013b) . We used the following antibodies and dilutions: rat anti-CDH5 (1:200; 555289, BD Pharmingen), rat anti-F4/80 (1:500; MCA497R, lot 1605, Serotec), rat anti-KIT (1:500; 553353, lot 30259, BD Pharmingen), rabbit anti-RFP (1:1,000; PM005, lot 045, MBL), goat anti-VEGFR2 (1:200; AF644, lot COA0417021, R&D Systems). Secondary antibodies used included donkey fluorophore-conjugated FAB fragments of anti-goat, -rabbit or -rat IgG (Jackson ImmunoResearch). Biotinylated IB4 (1:200; L2140, lot 085M4032V, Sigma) followed by Alexa-conjugated streptavidin (ThermoFisher) was used to detect brain ECs (Fantin et al., 2013a) . Images were acquired with a LSM710 (Zeiss) or an A1 (Nikon) laser scanning confocal microscope and processed using LSM image browser (Zeiss), Fiji (NIH Bethesda (Schindelin et al., 2012) and Photoshop CS4 (Adobe Inc.) software. Three-dimensional reconstructions including surface rendering and the generation of virtual slices for lateral views of high-resolution confocal z-stacks was performed using Imaris (Bitplane). Z-stack projections of confocal images are shown unless indicated otherwise in the figure legends.
6
Proximity Ligation Assay for ApoER2-VEGFR2 Interaction
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Endothelial cells were fixed with 4% PFA in PBS for 10 min at RT and permeabilized using 0.1% Triton X-100 in PBS for 4 min on ice. Subsequently, the cells were washed with PBS, and blocking solution (2% BSA, 4% NDS in PBS) was applied for 30 min at 37°C in a humidified chamber. Proximity ligation assay was performed as suggested by the manufacturer. All incubation periods were performed at 37°C. In brief, primary antibodies against ApoER2 1:80 (Abcam; ab86548) and VEGFR2 1:80 (R&D Systems; AF644) in blocking solution were added for 30 min. The cells were incubated with the corresponding PLA probes for 60 min. Phalloidin-FITC 1:500 was added to visualize actin filaments. Ligation and amplification of the probes was performed for 30 min and 100 min, respectively. Cells were incubated with DAPI, washed, and mounted. Incubation times with antibodies occurred at 4°C and overnight for the primary antibodies and 1 hour at 37°C for the PLA probe antibodies. For quantification, the number of PLA probe punctae per cell was counted.
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