Immunostaining of Endothelial Cells

Immunostaining of ECs was performed as previously described [12 (link)]. In detail, cells were fixed with 4% formaldehyde for 15 min and were blocked and permeabilized for 15 min at room temperature with 3% (v/v) normal goat serum (Sigma-Aldrich, Deisenhofen, Germany) diluted in PBS containing 0.3% (v/v) Triton X-100. Afterward, the cells were incubated with primary antibodies against a myc-tag or p21 (all from Cell Signaling Technologies, Frankfurt, Germany), each 1:100, overnight at 4 °C. The antibody against TIM23 (BD BioSciences, Heidelberg, Germany) was diluted 1:150 and incubated overnight at 4 °C. Subsequently, cells were washed three times with PBS and incubated with an Alexa Fluor^® 594 coupled anti-mouse or anti-rabbit secondary antibody (1:500) (Santa Cruz Biotechnology, Heidelberg, Germany) for 1 h at room temperature. Nuclei were counterstained with DAPI (500 ng/mL) (Carl Roth, Karlsruhe, Germany) in PBS for 5 min at room temperature and the cells were mounted with ProLong™ Diamond Antifade Mountant (Invitrogen, Darmstadt, Germany). Images were taken using Zeiss microscopes (Axio Observer Z or Axio Imager M2, Zeiss, Jena, Germany) using a 400× or 200× magnification. Pixel intensities were measured with Image J [17 (link)].