Cellular distribution of PDI in HUVECs. Intracellular localization of PDI was detected by immunostaining of fixed cultured HUVECs with monoclonal antibody RL90. (A) Simultaneous immunostaining of cells for PDI and SERCA2b in HUVECs indicates that these 2 proteins are colocalized in the endoplasmic reticulum. In addition, PDI is observed in granules distinct from the endoplasmic reticulum. Alexa 647–labeled anti-SERCA2b, red; Alexa 488–labeled RL90, green; colocalization, yellow. (B) Simultaneous immunostaining of cells for PDI and VWF indicates that PDI is not stored in Weibel Palade bodies. Alexa 647–labeled goat anti–rabbit IgG was used as a secondary antibody to detect VWF, red; Alexa 488–labeled RL90, green; colocalization, yellow. (C) Simultaneous immunostaining of chemokine Gro-α and PDI shows partial colocalization in small cytoplasmic granules. Original magnification in all panels ×60. Insets show high magnification (×100) of framed areas. (D) Immunogold labeling for PDI (10-nm gold particles) in HUVECs showed PDI in endoplasmic reticulum–related tubulovesicular structures and in small moderately electron-dense granules of approximately 100-150-nm diameter. WPB, Weibel-Palade bodies; ER, endoplasmic reticulum, SG, moderately electron-dense secretory granules (×99 000). (E) PDI and SERCA2b are colocalized in the endoplasmic reticulum, as indicated by immunogold staining. Only PDI is detected in secretory granules (×99 000). F) No plasma membrane–associated signal is detected in resting HUVECs (×119 000). (G) PDI, but not SERCA2b, is bound to the plasma membrane in activated HUVECs (×119 000). PM, plasma membrane. Panels E-G: PDI, 5-nm gold particles; SERCA2b, 15-nm gold particles.