Jain Rakesh K, Munn Lance L, Fukumura Dai
Cold Spring Harb Protoc. 2013 Apr 1;2013(4):354-8. doi: 10.1101/pdb.prot073916.
This protocol outlines methods to measure vascular parameters, including angiogenesis (e.g., vascular density, length, diameter) and hemodynamics (e.g., erythrocyte velocity), in tumors and normal vascular networks in mice. Fluorescein-isothiocyanate (FITC)-dextran is injected into the tail vein of mice to visualize microvessels within 150 μm (using single-photon microscopy) or ~600 μm (using multiphoton laser-scanning microscopy [MPLSM]) of a tumor/window interface. Randomly selected areas (three to six locations/tumor or animal) are investigated using long-working-distance objectives with appropriate magnification. During each observation period, FITC-fluorescence images are recorded for 60 sec, and the videotapes are analyzed off-line for single-photon microscopy; or a three-dimensional (3D) image stack of the vessel network is generated, and vessel properties are measured for MPLSM. If desired, red blood cell (RBC) flux can be measured on a vessel-by-vessel basis using fluorescent tracer RBCs.
本方案概述了测量小鼠肿瘤和正常血管网络中血管参数的方法，包括血管生成（如血管密度、长度、直径）和血流动力学（如红细胞速度）。将异硫氰酸荧光素（FITC）-葡聚糖注入小鼠尾静脉，以可视化肿瘤/窗口界面150μm内（使用单光子显微镜）或约600μm内（使用多光子激光扫描显微镜[MPLSM]）的微血管。使用具有适当放大倍数的长工作距离物镜对随机选择的区域（每个肿瘤或动物三到六个位置）进行研究。在每个观察期内，单光子显微镜记录FITC荧光图像60秒，并离线分析录像带；或生成血管网络的三维（3D）图像堆栈，并测量MPLSM的血管特性。如果需要，可以使用荧光示踪红细胞逐个血管测量红细胞（RBC）通量。
