Mouse vegf

Competition ELISA was conducted to measure mAb inhibition of VEGF binding to sFlt-1 using plates coated with 250 ng/mL mouse VEGF (R&D Systems) or human VEGF (R&D Systems) in 0.2 M carbonate-bicarbonate buffer. Anti-sFlt-1 Fabs from screening selections and full IgG candidates (series of dilutions from 20 μg/mL) were added to an equal volume of 20 ng/mL mouse or 80 ng/mL human recombinant VEGFR-1/Flt-1 Fc chimera (R&D Systems) for 1 h and then added to the plate for 30 min. For both competition assays, a mouse anti-histidine TAG biotin-conjugated Ab (Bio-Connect, Huissen, the Netherlands) was added for 1 h, followed by washing and a 1-h incubation with streptavidin-HRP (R&D Systems). The plate was developed for 10–20 min with tetramethylbenzidine substrate, stopped with 0.5 M H₂SO₄, and then read at 450 nm.

Thin blood smears of cultured blood were fixed in a mixture of 25% acetone (Merck Chemicals, Germany) and 75% absolute ethanol (Bie & Berntsen A/S, Denmark). After fixation, slides were permeabilized with 0.1% triton x-100 (Sigma-Aldrich, Denmark) and blocked with 5% bovine serum albumin (Sigma-Aldrich, Denmark) for one hour. Incubation for one hour with primary antibody (rabbit anti-VEGF Ab-1, ThermoScientific, Denmark) at a concentration of 0.01 mg/mL, and secondary antibody (goat anti-rabbit Alexa Fluor 488, Life Technologies, Denmark) at a concentration of 0.5 μg/mL, were followed by a nuclear stain (DAPI, Life Technologies). Samples without primary antibody served as negative controls. The negative control was further verified by pre-incubation where an excess (1.5x) of mouse VEGF (R&D Systems, UK) was added to the primary antibody 0.5 hours prior to incubating on the thin blood smears. Slides were covered with anti fade reagent (Slow Fade Gold anti fade reagent, Life Technologies) and cover slipped.

Rabbit polyclonal, anti-PKM1, PKM2, HK-1 and PDK-1 antibodies were purchased from Cell Signaling Technology (Danvers, MA). A mouse monoclonal anti-α-SMA antibody was purchased from Life Span Biosciences, Inc Seattle WA. Mouse monoclonal anti-HIF-1α, rabbit polyclonal anti-GLUT1 and PDK4, mouse monoclonal O-linked N-acetyl glucosamine, rabbit PKC (phosphoT497) and rabbit PKC (ab19031) were purchased from Abcam (Cambridge, UK). A rabbit polyclonal anti-phospho smad3 (s423 and s425) antibody was purchased from Rockland Immunochemicals (Gilbertsville, PA). Mouse VEGF was purchased from RD System. A rabbit polyclonal anti-αSMA antibody was purchased from GeneTex (Irvine, CA). Rabbit polyclonal anti-TGF-βR1 antibody, mouse monoclonal anti-β-actin antibody, and rabbit polyclonal anti-GAPDH antibodies were obtained from Sigma (St Louis, MO). A mouse specific rabbit polyclonal anti-SIRT1 and goat polyclonal anti-SIRT3 antibody, anti-Ki67 was purchased from Santa Cruz Biotechnology (Dallas, TX). Anti-fibroblast specific proteins (FSP1, sometimes displayed as S100A4) was purchased from Biolegend CA. Fluorescence-, Alexa Fluor 647-, and rhodamine conjugated secondary antibodies were obtained from Jackson ImmunoResearch (West Grove, PA). A horseradish peroxidase-conjugated secondary antibody and TGFβ1 was purchased from PeproTech (Rocky Hill, NJ).