Anti fade media

To observe the expression of WA-25, VEGF and VEGFR2 on WA-25-treated endothelial cells, immunofluorescence staining was performed as described previously [41 (link)]. After treatment with WA-25 (20 μM) for 48 h, the fixed HUVECs were permeabilized using buffer containing 0.1% normal goat serum and 0.1% Triton X-100 in PBS, and incubated with the VEGF or VEGFR2 antibody (1:100 dilution) at 4 °C overnight. The cells were then washed three times with PBS and incubated with the corresponding Alexa-488-conjugated (or Alexa-546-conjugated) secondary antibody (1:1000 dilution; Molecular Probes) for 1 h at room temperature. Finally, the cells were rinsed twice with PBS and incubated with DAPI for 5 min. After mounting in anti-Fade media (Invitrogen, Carlsbad, CA, USA), the fluorescence images of cells were captured using a ZEISS LSM PASCAL multiphoton confocal microscope image system (Carl Zeiss, Jena, Germany).

The protocols for immunofluorescence staining of eNOS, phospho-Ser1177 eNOS, iNOS, and NF-κB in HUVECs were performed, as described previously [21 (link)]. HUVECs were treated alone or in combination with PBS, 10 nM α-MSH, 10 μM H89, 5 mM l-arginine, 10 μg/mL MC1-R antibody, and 10 μg/mL MC2-R antibody for 24 h. Cell cultures were fixed, permeabilized with 0.1% Triton X-100, blocked in 0.1% normal goat serum and followed by incubating with respective antibodies (1:1000 dilution) at 4 °C for 16 h. After rinsing, the specimens were further incubated with Alexa-488- or Alexa-546-conjugated secondary antibody (1:1000 dilution; Molecular Probes, Thermo Fisher Scientific, Waltham, MA, USA) for 1 h, counterstained with DAPI, and then mounted in anti-Fade media (Invitrogen, Carlsbad, CA, USA). Immunofluorescence images were captured with ZEISS LSM PASCAL multiphoton confocal microscope image system (Carl Zeiss, Oberkochen, Germany).