Microvessel density measurements

U87MG xenograft tumors from control and treatment groups were excised and rapidly frozen with isopentane cooled with dry ice (−40°C to −50°C). IHC staining with CD31/PECAM-1 antibody was performed, as previously described.²⁴ Briefly, the acetone-fixed 20-μm-thick tumor slices were stained with CD31/PECAM-1 antibody. The number of stained vessels in the digitized images (Aperio Scanscope; Aperio, Vista, CA) were then counted (Analysis Toolbox; Aperio) and the results expressed as the number of vessels per square millimeter of the section. Control and treatment vessel counts were compared with Student's t-test.