The 4T1 tumors were fixed in 4% paraformaldehyde solution and then embedded in paraffin. A commercially available TUNEL kit (Promega, Madison, WI, USA) was used to identify apoptotic cells in the tumors. This analysis was performed following the manufacturer’s protocol, and the samples were then examined using a fluorescence microscope (×400).
The proliferation of tumor cells was detected by Ki-67 staining method. The primary antibody was rat antimouse monoclonal antibody Ki-67 (GeneTex Inc., Irvine, CA), and the secondary antibody was biotinylated goat antimice immunoglobulin (BD Biosciences). The Ki-67 labeling index (LI) was calculated as the number of Ki-67-positive cells/total number of cells counted under magnification (×400). The data were counted in five randomly selected areas in each tumor sample by two independent investigators.
For blood vessel staining, the tumor tissues were immunostained with epithelial cell marker goat antimouse CD31 antibody (dilution 1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and rabbit antigoat FITC (dilution 1:100; Santa Cruz Biotechnology) secondary antibody. Microvessel density was determined as the average number of small CD31-positive vessels in a high-power (×400) field.
The proliferation of tumor cells was detected by Ki-67 staining method. The primary antibody was rat antimouse monoclonal antibody Ki-67 (GeneTex Inc., Irvine, CA), and the secondary antibody was biotinylated goat antimice immunoglobulin (BD Biosciences). The Ki-67 labeling index (LI) was calculated as the number of Ki-67-positive cells/total number of cells counted under magnification (×400). The data were counted in five randomly selected areas in each tumor sample by two independent investigators.
For blood vessel staining, the tumor tissues were immunostained with epithelial cell marker goat antimouse CD31 antibody (dilution 1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and rabbit antigoat FITC (dilution 1:100; Santa Cruz Biotechnology) secondary antibody. Microvessel density was determined as the average number of small CD31-positive vessels in a high-power (×400) field.