Multistage angiogenesis, microvascular network patterning, and serial noninvasive imaging of neovascularization by L-PAM in TetON-HIF-1α transgenic mice. (A) Neovascular phenotype of a TetON-HIF-1 transgenic mouse with skin redness and vascular prominence developing 14 days after continuous DOX provision. (B) Comparative determination of TetON-HIF-1 neovascularization using immunofluorescent thin section microvessel markers, MECA32/VWF, with L-PAM. L-PAM was performed in the same transgenic mouse serially imaged for 60 days (red box represents the microvascular domain magnified in panel C). (C) Ear whole-mount images of FITC-L esculentum–perfused TetON-HIF-1 mice over time (red arrowheads in day 14 and day 30 images indicate neocapillaries surrounding each rtTA/HIF-1–expressing hair follicle). The L-PAM image insert (in grayscale) and the day 30 high-power whole-mount micrograph show similar conformation of the perifollicular neocapillaries. The yellow arrowheads indicate the vascular supply of the hair follicle capillary network. (D) Microvascular density as determined by vessel counting of MECA32/VWF double immunofluorescence tissue sections ex vivo, and microvessel volume derived from in vivo L-PAM of serially imaged individual TetON-HIF-1 mice (n = 1-4 for indicated time points) normalized to NTG controls, demonstrating the same trend of rapid elevation and then plateau by day 14. Data for each DOX day were compared with NTG or TetON-HIF-1 day 0 data (data not shown), using unpaired Student t test: *P < .05; **P < .01; ***P < .001. Bars represent 200 μm (B) and 100 μm (C).