Immunohistochemical determination of Ki-67 and CD31

Antiproliferation and antiangiogenic effects of Cur/MPEG-PLA micelles were measured by immunochemistry with antibody Ki67 and CD31. Briefly, the tumor tissue frozen sections were fixed in acetone, washed with PBS, and stained with rabbit antirat Ki67 polyclonal antibody (1:50; BD Pharmingen™; BD Biosciences, San Jose, CA, USA) and rabbit antimouse CD31 (1:50; Abcam, Cambridge, UK). Then, it was washed twice with PBS, and stained with secondary antibody conjugating FITC or Rhodamine (Abcam). The positive cells were observed under microscope, and the number of capillaries per high-power field was manually counted.