Cellosolve

Alcian blue Phloxine Tartrazine staining was performed based on previous reports [55 (link)]. Briefly, slides were stained with Alcian blue (Sigma-Aldrich, St. Louis, MO) for 30 minutes, followed by alum hematoxylin (BioCare, Concord, CA) for 20 seconds, staining with 0.5% phloxine in calcium chloride (Fisher Scientific, Waltham, MA) for 20 minutes, and finally incubate in a saturated solution of tartrazine in Cellosolve (Fisher Scientific, Waltham, MA) for 3-5 minutes. Stained sections were observed using light microscopy.

Fluorescently labeled microspheres were used to estimate tumor blood flow in tumors as described previously^{61 (link)}. In short, 15 μm diameter fluorescent microspheres (Molecular Probes, Eugene, OR) were suspended in saline. 100 μL of this solution was rapidly injected into the animal via the tail vein. Arterial reference samples were simultaneously withdrawn at a constant rate of 100 μL/min for 1 min through an inserted femoral catheter. At the end of the protocol, the mice were euthanized with a lethal dose of sodium pentobarbital. Tumor tissue was then digested in 1 M KOH solution for 24 h. Fluorescent dye was extracted with Cellosolve (Fisher Scientific, Pittsburgh, PA). The fluorescent signal was then measured using an LS 50B luminescence spectrometer (PerkinElmer Corp., Norwalk, CT). Regional blood flow proportional to the fraction of cardiac output was calculated by measuring the number of fluorescent microspheres in the tumor tissue relative to the total in the arterial reference samples.