Anti vegfr 3

Paraffin-embedded sections were deparaffinized, permeabilized, and incubated with goat polyclonal anti-VEGFR-3 (1:100, R&D Systems) or anti VEGFR-2 (1:100, R&D Systems) followed by biotin-streptavidin-HRP amplification using the Vectastain-ABC kit (Vector Lab), and post-stained with eosin.
For whole-mount staining, tissues were fixed overnight in 4% PFA and blocked overnight in blocking buffer (PBS, 5% goat serum, 0.3% Triton X-100, and 0.2% BSA). Tissues were incubated overnight at 4°C with biotinylated anti–mouse LYVE-1 (1:100, R&D Systems) or PECAM-1 (1:100, BD Biosciences) in blocking buffer followed by biotin-streptavidin-HRP amplification using the Vectastain-ABC kit.

HUVECs and HLECs were suspended in RIPA buffer [50 mΜ Tris-HCl (pH 8.0), 150 mΜ NaCl, 1% Nonidet P-40, 0.5% deoxycholic acid, 0.1% sodium dodecyl sulfate (SDS)] and incubated on ice for 30 min for complete cell lysis. After centrifugation at 12,000 × g for 10 min, lysates (25 μg of protein) were separated by SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane, followed by Western blotting with antibodies against target proteins. Antibodies against mTOR (#2972), p-mTOR (#2971), S6K (#9202), p-S6K (#9205), 4E-BP1 (#9644), p-4E-BP1 (#2855), ERK (#9102), p-ERK (#9106), Akt (#9272), p-Akt (#9271), VEGFR-1 (#2893), VEGFR-2 (#2479), p-VEGFR-2 (#2478), EGFR (#4267), and IGF-1Rβ (#3027) were purchased from Cell Signaling Technology (Denver, CO). Other antibodies used included anti-β-actin (#A5441; Sigma-Aldrich, Burlington, MA), anti-REDD1 (#10638-1-AP; Proteintech, Rosement, IL), anti-VEGFR-3 (#AF349; R&D Systems), anti-p-VEGFR-3 (#CY111; Cell Applications, Inc., San Diego, CA), and goat anti-mouse IgG (#31430; Invitrogen), goat anti-rabbit IgG (#31460; Invitrogen), and rabbit anti-goat IgG (#31402; Invitrogen) secondary antibodies. Relative protein levels were quantified using ImageJ.