Comparison of constitutive internalization of VE-cad in HUVECs and MVECs. (A) HUVEC or MVEC monolayers were stained with Alexa Fluor 568–conjugated mAb directed against the extracellular domain of VE-cad at 15°C (time 0). Cells were then transferred to 37°C for 3 hours. The location of VE-cad was examined by immunofluorescence microscopy. (B) Upon 3-hour incubation as in panel A, cells were fixed with 4% formaldehyde and stained for EEA-1. Colocalization of VE-cad and EEA-1 was determined by immunofluorescence microscopy, and quantified by counting merged (yellow) vesicles per field. Data represents the total number of merged vesicles per field using a 60× objective, and are the mean plus or minus SD of 2 different samples and 3 different experiments. ***P < .001.