Ab51874

After the extraction of total protein with lysis buffer, the protein concentration was measured by Bicinchoninic acid (BCA) assay. Later, the extracted protein (50 μg) got electrophoresis under constant voltage in 10% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAG), followed by wet-transfer into a polyvinylidene fluoride (PVDF) membrane. The transferred membrane was blocked with 5% skim milk powder at room temperature, followed by the addition of primary antibodies (VEGF-C: ab135506, 1:500, Abcam, Cambridge, UK; VEGFR-3: ab51874, 1:800, Abcam, Cambridge, UK; LYVE-1: ab33682, Abcam, 1:100, Cambridge, UK). Then this sample was incubated overnight at 4°C on a shaking table and washed for 3 times, after which goat-anti-rabbit horseradish peroxidase conjugated antibodies were added (ab6721, Abcam, 1:2000, Cambridge, UK) as secondary antibodies. Subsequently, the sample was incubated at room temperature for 1 hour, followed by coloration with enhanced chemiluminescence (ECL) plus reagent and imaging. The calculation of the grey level of target bands was conducted with Synegene gel imaging system and GeneTools was employed for analyses on protein bands. β-actin was used as internal reference (ab194952, 1:1000, Abcam, Cambridge, UK) for adjustment.

Mouse anti-human CD31 (PECAM) monoclonal antibody (MEM-5) was purchased from Thermo, mouse anti-human FLT1 (ab9540) and rat anti-mouse FLT4 (ab51874) monoclonal antibodies were purchased from AbCam, rabbit anti-KDR monoclonal antibody (D5B1) was purchased from Cell Signaling and α-tubulin (DM1A) was purchased from Millipore. Fluorescently labelled secondary antibodies were purchased from Invitrogen: goat anti-mouse/rat/rabbit coupled with Alexa Fluor 488, Cy3, Alexa 555, Cy5 or Alexa 647. HRP-conjugated secondary antibodies were purchased form Jackson Immunoresearch.