Fluorescence immunocytochemistry was performed on cells as previously described (Kacimi et al. 2011 (link)). The wells were washed twice in PBS and then fixed with acetone/methanol (1:1) 5min at −20°C. Alternatively, cells were fixed in 4% paraformaldehyde for 30 min at room temperature. The cells were then washed twice with PBS containing 0.2% Triton X-100 for 15 min. Nonspecific binding sites were blocked in blocking buffer (2% BSA and 0.2% Triton X-100 in PBS) for 2hr. The cells were incubated with primary antibody specific marker for the vascular unit cells as indicated at 1:100 dilution in blocking buffer overnight at 4°C and then washed three times with blocking buffer, 10 min per wash. The cells were incubated with either alexa or FITC-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA) at 1:100 dilution in blocking buffer at RT for 1 h, then washed 2 times in blocking buffer, and one time in PBS, 10 min per wash. Fluorescence was visualized with an epifluorescence microscope (Zeiss Axiovert; Carl Zeiss Inc), and images were obtained on a PC computer using Axiomatic software (Zeiss Inc).