Evidence of the colocalization of PR3 with MPO in azurophil granules, PR3 with lactoferrin in specific granules, and PR3 with CR1 in secretory vesicles using double immunolabeling electron microscopy. (a) Double immunolabeling with the MoAb anti-PR3 CLB 12.8 coupled with a 10-nm gold particles-conjugated goat antimouse (GAM 10) and the polyclonal anti-MPO coupled with a 5-nm gold particles-conjugated goat antirabbit (GAR 5). The presence of 5-nm gold grains in large empty granules indicates that MPO is localized exclusively in azurophil granules. The colocalization of gold grains of both sizes within these granules indicates that PR3 is located with MPO in these granules. However, in contrast to MPO, PR3 is also localized at the periphery of empty vesicles (v) and in some specific granules (sg1) (original magnification × 54,250). (b) Double immunolabeling with the MoAb anti-PR3 CLB 12.8 coupled with a 10-nm gold particles-conjugated goat antimouse (GAM 10) and the polyclonal antilactoferrin coupled with a 5-nm gold particles-conjugated goat antirabbit (GAR 5) showing the presence of PR3 (arrowheads) along the limiting membrane of an elongated specific granule identified as such thanks to its prominent lactoferrin content (original magnification × 86,800). (c) Visualization of secretory vesicles with CR1 labeling. Immunolabeling of CR1 was performed with the polyclonal anti-CR1 coupled with GAR 5 and shows the localization of CR1 in the membrane of the secretory vesicles, which appear as empty organelles, but not in specific granules (original magnification × 98,700). (d) Double immunolabeling with the MoAb anti-PR3 CLB 12.8 coupled with GAM 10 and the polyclonal anti-CR1 coupled with GAR 5. Both sizes of grains are detected in the membrane of secretory vesicles identified by the presence of CR1 (original magnification × 98,700).