Formalin-fixed paraffin embedded patient tissue from 5 invasive ductal carcinomas was collected under the Montefiore-Einstein IRB approval. Paraffin embedded human breast cancer tumors were cut to 5 μm sections, deparaffinized and stained for H&E or TMEM. The sequence was anti-CD31 (clone JC70A, DAKO, Carpinteria, CA) and Vector Blue chromogen (for endothelial cells); anti CD-68 (clone PG-M1; DAKO) with and DAB chromogen (for macrophages); and anti-pan-Mena with Fast Red chromogen (for carcinoma cells) (8 (link), 11 (link)). Sequential sections were cut for tyramide signal amplification (TSA) for quantitative immunofluorescence using the Opal 3-plex Kit (Perkin Elmer) according to manufacturer’s directions. The sequence was rabbit anti-VEGF (1:2000, Rb 9031-P0-A, Thermo) with TSA Plus Cy3; rabbit anti-Tie2 (1:3000, clone C-20, Santa Cruz Biotech) with TSA Plus Cy5; goat anti-VE-cadherin (1:200, clone C-19, Santa Cruz Biotech) with TSA Plus fluorescein and nuclei stained with 4,6-diamidino-2-phenylindole (DAPI). All quantitative analysis was performed on the raw 16-bit TIFF images and images of TMEM were validated independently by a pathologist.