The opsonization of conidia with PTX3 increases the colocalization of CD11b and CD32 in the neutrophil phagocytic cup. (A-C) Confocal microscopy analysis (FluoView FV1000; Olympus) of PTX3, CD11b, and CD32 in resting cells (A), CD11b and PTX3 (B), or CD32 and PTX3 (C) colocalization by double staining. After phagocytosis of nonopsonized conidia (B-C left) or PTX3-opsonized conidia (B-C right), cells were fixed with 4% PFA and stained for PTX3 (green) and CD11b (red; B) or PTX3 and CD32 (red; C). DNA labeling is also shown (DAPI [4,6 diamidino-2-phenylindole]). Panels from top to bottom show single staining for CD11b (A left, B) or CD32 (A right, C); for PTX3, double fluorescence for PTX3 and CD11b (A left, B) or PTX3 and CD32 (A right, C); double fluorescence and differential interference contrast (Nomarski; inset, Normaski and DAPI). (D) Triple staining for CD11b, CD32, and PTX3 in cytospun neutrophils by confocal microscopy. After phagocytosis of nonopsonized conidia (left) or PTX3-opsonized conidia (right), cells were cytospun and fixed with 4% PFA and stained for human PTX3 (green), CD11b (blue), and CD32 (red). Panels from top to bottom show double fluorescence for PTX3 and CD11b; CD11b and CD32; PTX3 and CD32; triple staining for PTX3, CD11b, and CD32; triple fluorescence and differential interference contrast (Nomarski). Images (1024 × 1024 pixels) were acquired with an oil immersion objective (100× 1.4 NA Plan-Apochromat; Olympus). One or more internalized conidia per cell are indicated by asterisks or arrows. Bars indicate magnification.