Immunofluorescent Staining of Tumor Vasculature

Sections from the same tumors and samples from normal oral mucosa biopsies used for LCM were deparaffinized and rehydrated, and incubated in low pH antigen-retrieval solution (Dakocytomation; Dako, Carpinteria, CA) for 20 minutes at 95°C. Double staining was performed using antibodies against CD31 (Clone JC70A, Dako, Carpinteria, CA) and Grp78 (clone H-129, SantaCruz Biotechnology, Santa Cruz, CA). Alexa Fluor 488 or Alexa Fluor 647 (Molecular Probes, Eugene, OR) conjugated sencondary antibodies were used. Slides were counterstained with 3.5 µM DAPI (Sigma-Aldrich, St. Louis, MO), and mounted with Fluorescent Mounting Media (Dako, Carpinteria, CA). Images were taken with Olympus IX70 microscope (Tokyo, Japan) and analyzed by ImageJ software (National Institute of Health, Bethesda, MD, USA). Only CD31 positive cells lining tubular structures were considered blood vessels. Tissue samples were obtained from the University of Michigan School of Dentistry tissue core [19] (link).