Cells were cultured in slide flasks, washed twice in phosphate-buffered saline (PBS), and fixed for 10 minutes using 90% ice-cold acetone, or 2% paraformaldehyde for plasmalemma vesicle-associated protein (PLVAP), human leukocyte antigen (HLA)-ABC, and carbonic anhydrase IV staining. Cell monolayers were blocked for 1 hour at room temperature (RT) with 1% (wt/vol) bovine serum albumin (BSA, Sigma-Aldrich Chemie) in PBS. Cells were stained using goat anti-vascular endothelial cadherin antibody (Santa Cruz Biotechnology), rabbit anti-von Willebrand factor antibody (DAKO, Glostrup, Denmark), rabbit anti-PLVAP (Atlas Antibodies, Bromma, Sweden), mouse anti-HLA-ABC (ITK Diagnostics, Uithoorn, The Netherlands), or rabbit anti-carbonic anhydrase IV (Life Technologies) in 1% BSA in PBS for 1 hour at RT. Antibodies were detected using fluorescently labeled donkey anti-goat IgG:Alexa 594 or goat anti-rabbit IgG:Alexa 488 (Life Technologies) in 1% BSA in PBS for 1 hour at RT. For internalization of acetylated low-density lipoprotein (LDL), unfixed ciChEnCs or ARPE-19 cells were incubated with Alexa 488–labeled acetylated LDL (Life Technologies) in EGM-2 MV for 4 hours at 37°C. Stained cells were postfixed using 1% paraformaldehyde in PBS for 15 minutes at RT and embedded in Vectashield H-1000 mounting medium (Brunschwig Chemie, Amsterdam, The Netherlands) containing 4′,6-diamidino-2-phenylindole. Pictures were taken at ×200 total magnification, or ×400 total magnification for PLVAP, HLA-ABC, and carbonic anhydrase IV using an Axio Imager M1 microscope (Zeiss, Jena, Germany).