4.4. Endothelial Immunofluorescence Staining

NDPK-B depleted and control HUVECs were seeded in 24 well plates on sterilized coverslips coated with 1% gelatin. After 24 h, serum starvation was performed with ECGM + 0.5% FCS for 24 h. The cells were further stimulated with high glucose (30 mM) in ECGM + 0.5% FCS for 24 h. On the day of staining, cells were washed with PBS and fixed with 4% formaldehyde. After permeabilization and blocking with 2.5% BSA and 0.3% Triton X-100 for 1 h, the cells were incubated with the primary antibody anti-Tie2 (Tie2 clone AB33, Merck Millipore, 1:100) overnight at 4 °C. Post-incubation, the cells were washed and incubated with the secondary antibody (Donkey anti-mouse Cy3, Jackson Labs 715-166-150, 1:200). Finally, the cells were mounted with Roti-Mount FluorCare (HP19.1, Roth, Karlsruhe, Germany). Photos were taken by confocal laser scanning microscopy (Leica Microsystems, Wetzlar, Germany). Quantification of the membrane protein expression in immunofluorescence was performed using Image J (NIH, Bethesda, MD, USA).