Journal: Neoplasia (New York, N.Y.)
Article Title: Inhibition of Epidermal Growth Factor Receptor and Vascular Endothelial Growth Factor Receptor Phosphorylation on Tumor-Associated Endothelial Cells Leads to Treatment of Orthotopic Human Colon Cancer in Nude Mice 1
Figure Lengend Snippet: Double immunofluorescence staining for expression of EGFR, pEGFR, VEGFR, or pVEGFR in tumor-associated endothelial cells. (A) Tissue sections were stained with anti -CD31/PECAM1 antibody (red) and with anti-EGFR, pEGFR, VEGFR, or pVEGFR (green) as described in the Materials and Methods section. Colocalization of CD31 and EGFR, pEGFR, VEGFR, or pVEGFR appears as yellow fluorescence. Expression of EGFR and VEGFR by tumor-associated endothelial cells was found in tumors from all treatment groups. Phosphorylation of EGFR and VEGFR on endothelial cells was inhibited by treatment with AEE788 or AEE788 plus CPT-11. Note that treatment with AEE788 alone decreased the number and diameter of blood vessels and that this effect was even more pronounced with combined AEE788 and CPT-11.
Article Snippet: The following primary antibodies were purchased: rabbit anti-EGFR (SC03; Santa Cruz Biotechnology, Santa Cruz, CA); rabbit anti-phosphorylated EGFR (pEGFR) (Tyr1173; Biosource, Camarillo, CA); rabbit anti-VEGFR2 (Flk-1, C1158; Santa Cruz Biotechnology); rabbit anti-phosphorylated VEGFR2 (pVEGFR2) (Flk-1, PC460; Oncogene, Boston, MA); rabbit anti-VEGFR1 (C17; Santa Cruz Biotechnology); rabbit anti-mouse F4/80 (MCAP497; Serotec, Raleigh, NC) and rat anti-mouse CD31 (BD PharMingen, San Diego, CA) for frozen samples; mouse anti-human TGF-α (GF10; Oncogene); mouse anti-human VEGF (23071D; BD Phar-Mingen) and Ki-67 (MIB-1; DakoCytomation, Carpinteria, CA) for paraffin-embedded samples; and rabbit anti-EGFR (SC03; Santa Cruz Biotechnology), rabbit anti-pEGFR (Tyr1068; Cell Signaling Technology, Danvers, MA), and rabbit anti-VEGFR2 (Flk-1, C1158; Santa Cruz Biotechnology) for Western blot analysis.
Techniques: Double Immunofluorescence Staining, Expressing, Staining, Fluorescence, Cycling Probe Technology