Anti vegf c 1 antibody

Polystyrene 96-well plates were sensitized (Maxisorp Nunc) with 50 µl of protein (10 µg), serum (1:2 dilution), or VEGF (Santa Cruz Biotechnology) standard (0.001 ng-1ng) in bicarbonate buffer (in duplicate) and incubated at 4°C overnight following manufacturer instructions. Subsequently, the plates were washed three times with a wash solution. 200 µl of the blocking solution (1%) was added and incubated for one hour at 4°C. Next, 50 µl of anti-VEGF C-1 antibody (sc-7269, Santa Cruz Biotechnology) (1:200) was incubated for one hour at 4°C. For recognition of anti-VEGF IgG, 50 µl of m-IgGκ BP-HRP (sc-516102 Santa Cruz Biotechnology) was added (1:400) for 2 h at room temperature. Finally, after washing, the enzyme-substrate reaction was performed with 50 µl of the chromogen solution. The reaction was stopped after 20 min with 50 µl of 2 N sulfuric acid. The absorbances were determined at 492 nm in the Stat Fax 4200 microplate reader (Awareness Technology). All cytokine and VEGF concentrations were calculated with interpolation from a standard curve. The determination of TES concentration, dilutions of sera, and antibodies was developed after the standardization of the respective ELISAs.

Mycoplasma tested human umbilical vein endothelial cells (HUVECs; Commercially obtained from Lonza #CC‐2935) were cultured in EBM‐2 Basal Media (#00190860, #cc‐4176, Lonza, mycoplasma free) with EGM‐2 Single Quots supplements (Lonza #cc‐4176) on 0.2% gelatin (Sigma Aldrich #G1393)‐coated 96‐well plates (5,000 cells/well) or 6‐well plates (150,000 cells/well). After obtaining stable culture conditions, HUVECs were incubated with culture supernatants collected from three groups of mouse myofibroblasts: (i) untreated myofibroblasts (TurboFect), (ii) control siRNA‐treated myofibroblasts, and (iii) myofibroblasts treated with siRNA against PDGF‐Rα. For caspase activity assay, incubation with 40 μg/ml anti‐VEGF (C‐1) antibody (#sc‐7269, Santa Cruz) served as a positive control. Caspase activity was assessed after 6 h using the Caspase‐Glo 3/7 assay kit (#G8091, Promega). After 6 h of incubation, caspase activity was assessed using the Caspase‐Glo 3/7 assay kit (Promega GmbH, Germany #G8091) according to the manufacturer's instructions. Cells plated in 6‐well plate were lysed after 6‐h incubation, and lysate was processed for immunoblot analysis with cleaved caspase‐9 (Cell Signaling Technologies #9509) and eNOS (Cell Signaling Technologies #880) protein.

The VEGF concentrations were calculated by interpolation from a standard curve (0.001–1 ng/mL) performed with the mouse VEGF protein, VEGF mBA-165 (Cat# sc-4571, Santa Cruz Biotechnology, Santa Cruz, CA USA). Tumor protein was obtained and quantified through the same protocol implemented for cytokine determination.
The polystyrene wells (96-well plate, MaxiSorp Nunc Cat# NNC#442404) were coated with 50 µL of tumor protein (10 µg) or with the different concentrations of the standard curve, all diluted in bicarbonate buffer (pH 9.6), coated per duplicate, and incubated at 4 °C overnight. The plate was washed and blocked with 200 µL of PBS/bovine serum albumin (BSA) 1%/Tween 20 0.05% for 1 h at 4 °C and washed again. Furthermore, 50 µL of anti-VEGF/C-1 antibody (Cat# sc-7269, RRID:AB_628430, Santa Cruz Biotechnology) in a 1:200 dilution was added and incubated for 1 h at 4 °C. After washing, 50 µL of m-IgGκ/BP-HRP (Cat# sc-516102, RRID:AB_2687626, Santa Cruz Biotechnology) (1:400) was added and maintained for 2 h at room temperature. An enzyme-substrate reaction was developed with 50 µL of substrate solution and stopped after 15 min with 50 µL 2N sulfuric acid. The plates were read at a wavelength of 492 nm in a Stat Fax 4200 microplate reader (Awareness Technology). Cytokine and VEGF concentrations were calculated by interpolation from a standard curve.