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Fig. 2
Figure 2. Quantification of the spatial distribution of vascular permeability induced by acute and chronic VEGF in zebrafish (A) Heat-inducible VEGF transgenic zebrafish (pKTol2H70-mC-hVEGF-gcG) (Hoeppner et al., 2012), in which VEGF expression is driven by a heat-inducible HSP70 promoter and preceded by an upstream floxed mCherry gene, were bred with Tg(fli:EGFP) zebrafish (Lawson and Weinstein, 2002) to generate the double transgenic zebrafish Tg(pKTol2H70-mC-hVEGF-gcG);Tg(fli:EGFP). F1 progenies were used to identify true transgenics and the lines with low background but high heat-shock induction of mCherry were selected. (B) In the selected double transgenic zebrafish lines, Cre mRNA was microinjected into F1 single cell embryos to excise the mCherry gene, resulting in expression of hVEGF on induction of the HSP70 promoter. At 3-days postfertilization (3-dpf), zebrafish embryos were anaesthetized and microinjected with Texas Red-dextran (70 kDa) to the pericardium. VEGF-induced vascular hyperpermeability in zebrafish was examined at base line (no exposure to heat shock), upon acute (37°C for 30 min) and chronic (3 times of 37°C for 30 min with 30 min intervals at 28.5°C) exposure to heat shock as demonstrated. (C) An image processing method was developed to analyze the vascular permeability: (1) Maximum intensity projection image was first generated, and then (2) binary mask extracted from green channel was generated. (3) Binary image constituting of red pixels was obtained after masking original image (1) by green channel (2). In addition to vascular leakiness, the resulting red pixels (Texas Red-dextran at 70 kDa) represent leakiness and signals from autofluorescence, caudal veins and arteries, and somites. (4) Pixels corresponding to autofluorescence as well as caudal arteries and veins and (5) somites were highlighted and eliminated. (6) The final processed image containing pixels show the total leakiness. (D and E) Images of whole mount imaging (D) and different regions of zebrafish (E) were acquired immediately after heat-shock using a Zeiss LSM 880 confocal microscope using standard FITC and dsRed filter sets. (F) Using our new algorithm, the vascular permeability of anterior and posterior regions of trunks and tail regions were analyzed and compared. ∗∗∗, p<0.001. Zebrafish numbers in each group are presented in the scatter plot. Data is expressed as mean ± SD. Scale bar, 200 μm in (C, D, and E)