2.12. Wound Angiogenesis by Immunofluorescence
At 7 and 21 d, wound tissue specimens harvested from wound-healing model rats were used for immunohistochemical fluorescence evaluation. To visualize immunofluorescence, sections were incubated with antibodies against Ki67 (1:300 dilution, GB13030-2, Google Biotechnology) and proliferating cell nuclear antigen (PCNA, 1:200 dilution, GB11010, Google Biotechnology) overnight. After rinsing with PBS, sections were incubated with a fluorescent rabbit secondary antibody for 40 min. After washing four times with PBS, cell nuclei were stained by DAPI, and images were acquired with a confocal laser-scanning microscope (Olympus, LSM 700, Japan).