Fig. 4.
Confocal images of endogenous H2S detection in live HUVECs using SF7-AM. (A) HUVECs incubated with 5 µM SF7-AM for 30 min at 37 °C, washed, and then imaged. (B) The same field of HUVEC in A was treated on stage with 40 ng/mL VEGF for 30 min at 37 °C and then imaged. (C) Brightfield images of the same field of cells in B overlaid with images of 1 µM Hoechst stain at 37 °C. Images in A and B are the maximum intensity projections of 8 × 2 µm z-stacks. (Scale bar, 100 µm.) (D) Quantification of confocal fluorescence images of H2S signaling in live HUVECs using SF7-AM. HUVECs were incubated with 2.5 µM SF7-AM, washed, and imaged before treatment with 0.1% BSA in H2O as a vehicle control (Cont), n = 8; 40 ng/mL total VEGF stimulation (VEGF), n = 14; 30 µM AAL-993 for 40 min before VEGF stimulation, n = 3; 100 µM PAG for 10 min before treatment with 40 ng/mL VEGF, n = 3. Data were normalized to VEGF-stimulated positive control. Data are expressed as a ratio of final mean fluorescence intensity (Ff) to the initial mean fluorescence intensity (Fi), and error bars are ± SEM.