Capturing the spatial dynamics of the EET by applying a fluorescent time-lag double stain. (A) Experimental design of the fluorescent time-lag double stain experiment. CMFDA (green) was subsequently applied, whereas CMTPX (red) was applied 24 h after wounding, and the predicted outcomes hypothesized by the leap-frog and sliding theory were compared with the results of the experiment. (B) Sections of the EET showing the initial migration of basal CMFDA-labeled keratinocytes 0 and 12 h after wounding and the spatiotemporal dynamic behavior of CMFDA, CMTPX–labeled double-positive cells 24 and 48 h after wounding. The 0- and 24-h images shown here are presented again in Fig. 8 B to represent 1.5 and 53.3% reepithelialized wounds, respectively. The white arrows indicate CMFDA, CMTPX–negative keratinocytes, which have entered the EET from unstained regions. (C) Displays a representative EET 36 h after wounding, which can be separated into three different zones. Zone 1 shows the wound margin where labeled cells had started migration. Zone 2 displays the unstained migrating basal keratinocytes originated from tissue adjacent to the wound entering the EET. Zone 3 shows the collision of two EETs and the formation of a bulk. Arrowheads indicate the wound margin. Broken lines denote dermal–epidermal junction. The images shown are from a single representative experiment out of three repeats; 60 wound-healing models were analyzed in total. Bars, 100 µm.