An anti-FAP antibody (1 mg, R&D, MAB3715) was incubated with IR700 (63.5 μg, 32.5 nmol) in 0.3 mol/L Na2HPO4 (pH 8.5) at room temperature (RT) for 2 h in the dark. The mixture was purified with a Sephadex G50 column (PD-10; GE Healthcare). The protein concentration was determined with a Bio-Rad protein assay kit (Bio-Rad, CA) by measuring the absorption at 280 nm for FAP mAb and 689 nm for IR700 with spectroscopy. With this sample, the number of fluorophore molecules per FAP mAb was adjusted to approximately 2 (Supplementary Fig. 5 ). This conjugated antibody was defined as FAP-IR700.
Mab3715
Manufactured by R&D Systems
Sourced in United States
MAB3715 is a Mouse Monoclonal Antibody. It is a laboratory reagent used for research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Sephadex g50 column pd 10, by GE Healthcare (1 mentions)
Protein assay kit, by Bio-Rad (1 mentions)
Ab8211, by Abcam (1 mentions)
Permeabilization buffer, by BioLegend (1 mentions)
Alexa 647, by Thermo Fisher Scientific (1 mentions)
Sc 3877, by Santa Cruz Biotechnology (1 mentions)
Facsuite software, by BD (1 mentions)
Facslyric flow cytometer, by BD (1 mentions)
Zombie aqua, by BioLegend (1 mentions)
Lms780, by Zeiss (1 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
Alexa 568, by Thermo Fisher Scientific (1 mentions)
Ab5694, by Abcam (1 mentions)
Ab53066, by Abcam (1 mentions)
Ab28244, by Abcam (1 mentions)
Ab207178, by Abcam (1 mentions)
Ab227703, by Abcam (1 mentions)
Ab218164, by Abcam (1 mentions)
Ir700, by LI COR (1 mentions)
Af3715, by R&D Systems (1 mentions)
5 protocols using mab3715
1
Fluorescent Labeling of Anti-FAP Antibody
2
Fibroblast Phenotype Characterization
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Fibroblasts were washed with PBS and dissociated from the vessels with trypsin. The cells were fixed with 4% paraformaldehyde at 37 °C for 10 min. The cells were incubated with 1:100 dilution of primary antibody against FAP (MAB3715, R&D Systems) or IgG1 (sc-3877, Santa Cruz Biotechnology, Dallas, TX, USA) for 30 min at room temperature and incubated with 1:200 dilution of secondary antibody Alexa647 (A21237, Thermo Fisher Scientific) for 30 min at room temperature in the dark. The cells were permeabilized with a permeabilization buffer (#421002, BioLegend, San Diego, CA, USA) and a 1:100 dilution of antibody against α-SMA conjugated with FITC (ab8211, Abcam, UK) or IgG2a conjugated with FITC (#400210, BioLegend) for 30 min at room temperature in the dark. The cells were analyzed for two antigens simultaneously using a BD FACSLyric flow cytometer (BD Biosciences, San Jose, CA, USA), and the obtained data were estimated using BD FACSuite software (BD Biosciences). The analyzed cells were confirmed to exclude dead cells using Zombie Aqua (#423101, BioLegend).
3
Immunofluorescent Staining of Activated Fibroblasts
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Fibroblasts were cultured on chambered coverslips for 72 h. For staining with alpha smooth muscle actin (α-SMA), the treated fibroblasts were fixed and permeabilized with methanol for 30 min at 4 °C. Cells were incubated with a 1:300 dilution of primary antibody against α-SMA (ab5694, Abcam, Cambridge, UK) at 4 °C overnight, and then incubated with a 1:200 dilution of secondary antibody Alexa568 (Thermo Fisher Scientific) at room temperature for 2 h. For staining with fibroblast activation protein (FAP), the treated fibroblasts were fixed with 4% paraformaldehyde for 15 min. Cells were incubated with a 1:300 dilution of primary antibody against FAP (MAB3715, R&D Systems, Minneapolis, MN, USA) at 4 °C overnight, and then incubated with a 1:200 dilution of secondary antibody Alexa488 (Thermo Fisher Scientific) at room temperature for 2 h. Cells were incubated with 0.1μg/mL DAPI, and the slides were imaged with a confocal laser scanning microscope (LMS780, ZEISS, Oberkochen, Germany).
4
FAP-α Antibody Sourcing Protocol
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Water soluble phthalocyanine dye IR700 was obtained from Li‐Cor Bioscience. Anti‐FAP‐α polyclonal sheep Ab, AF3715, monoclonal mouse Ab MAB3715, and monoclonal rat Ab MAB9729 were purchased from R&D Systems. Anti‐FAP‐α monoclonal mouse Ab BMS168 was purchased from eBioscience. Anti‐FAP‐α Abs, ab137549, ab218164, ab28244, ab53066, ab207178, and ab227703, were purchased from Abcam. Anti‐FAP‐α polyclonal rabbit Ab PA5‐51057 and sheep IgG isotype control (Cat. No. 31243) were purchased from Thermo Fisher Scientific.
5
Isolation and RNA-seq of Tumor Cells
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Primary tumor tissues were minced, and single-cell suspensions were obtained. A detailed procedure is provided in the supplementary methods. The collected cells were incubated with antibody for 30 min on ice as follows: 10 μL anti-EpCAM (R&D Systems, FAB9601F) per 106 cells in 100 μL buffer; 5 μL anti-CD45 (BD, 557748) per 106 cells in 100 μL buffer; 10 μL anti-CD31 (Miltenyi Biotec, 130-092-652) per 107 cells in 100 μL buffer; and 2.5 μg of FAP (R&D systems, MAB3715) per 106 cells in 100 μL buffer. After washing with Hank’s balanced salt solution (Lonza) twice, the cells were incubated with mouse IgG (H + L) PE as follows: 10 μL per 106 cells in 100 μL buffer for 30 min on ice. After washing away the unstained secondary antibody, the cells were resuspended in 1 mL PBS and then sorted using a BD FACSARIA III (BD Biosciences). Total RNA sequencing libraries were prepared according to the manufacturer’s instructions (Illumina TruSeq RNA Access Library kit). The flow cell was then loaded on a HiSeq 2500 sequencing system (Illumina), and sequencing was performed using 2 × 100 bp read lengths.
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