PDI expression in HUVECs is strictly intracellular. HUVECs were grown in 48-well plates. Second- or third-passage cells were fixed with 3% paraformaldehyde (IF) or 2% paraformaldehyde + 0.2% glutaraldehyde (IEM) using standard procedures. Electron micrographs were obtained with a JEOL 1200EX electron microscope. (A) Nonstimulated HUVECs. Immunogold labeling with monoclonal PDI-1. (B-C) HUVECs stimulated with 0.5 U/mL thrombin for 60 minutes. Immunogold labeling with monoclonal anti-PDI-1 (B) and polyclonal anti-PDI (C). indicate PDI localization within the cisternal lumen of the endoplasmic reticulum. (D-E) IF staining of thrombin-stimulated HUVECs using monoclonal anti–PDI-2. IF images were recorded on a Leitz DMIRB fluorescence microscope, interfaced with a Leica TCSNT confocal laser scanning microscope, using a 63×/1.4 Plan-Apo objective. (D) Nonpermeabilized cells. (E) Permeabilized cells. PM indicates plasma membrane; and M, mitochondria. Scale bars represent 200 nm.